KR20200034369A - Antibody fragments specifically binding to Her2 and uses thereof - Google Patents

Abstract

The present invention relates to an antibody fragment (Fab) specifically binding to human epidermal growth factor receptor-2 (Her2) and a use thereof. More specifically, by providing an anticancer agent via a drug binding with the antibody fragment (Fab) that the antibody fragment produced by using a microorganism specifically binds to Her2, the intensively drug delivery to target cells is possible by specifically binding to Her2. In addition, by having improved binding affinity to Her2, the antibody fragment can be useful for preventing and treating cancer due to high anticancer activities.

Description

Antibody fragments specifically binding to Her2 and uses thereof}

The present invention relates to an antibody fragment that specifically binds to Her2 and uses thereof, and more specifically, an antibody fragment (Fab) produced using microorganisms specifically binds to Her2 (Human epidermal growth factor receptor-2). Antibody fragments (Fab), and drug binding to anti-cancer drugs.

1. Overview of Antibody Therapeutics

The share of biopharmaceuticals in the global pharmaceutical market is expected to increase to 25% in 2018, especially in the world's top 100 pharmaceuticals, biopharmaceuticals account for 39% in 2004, 17% in 2012, and 51% in 2018. Is expected to increase steeply. In particular, antibody drugs accounted for 52% of biopharmaceutical sales and totaled $ 64 billion in sales. Antibody drugs showed the fastest growth among drugs.

Since the first antibody treatment was released in 1994, more than 30 therapeutic antibodies have been approved by the FDA for treatment of cancer, autoimmune diseases, transplant rejection, infectious diseases, and heart disease.

In particular, monoclonal antibodies such as Rituxan (rituximab), Herceptin (trastuzumab), Campath (alemtuzumab), Erbitux (cetuximab), Avastin (bevacizumab), Vectibix (panitumumab), Prolia / Xgeva (denosumab), and Arzerra (ofatumumab) Monoclonal antibody (mAb) has obtained FDA approval as an anticancer drug, and its sales are increasing exponentially.

Chemotherapy for the treatment of cancer is used close to the maximum allowable amount in order to obtain a great effect in clinical treatment, causing proliferation inhibition and cell death of tumor cells as well as normal cells, and non-tumor specific systemic Toxicity, cytotoxicity, and long-term use of the drug may cause resistance.

On the other hand, antibody therapeutics have the advantage of inhibiting the proliferation of tumor cells and causing cell death without damaging normal cells by binding to specific antigens on the surface of tumor cells, and also have a very long half-life compared to general low molecular drugs. Therefore, the number of administrations can be reduced.

In other words, antibody drugs that are selective for cancer cells, have high efficacy, have fewer side effects, and can reduce the number of administrations have established themselves as next-generation anti-cancer drugs, so their development value is high.

2. Overview of antibody fragment technology

Next-generation antibody-based drugs include antibody-drug conjugates (ADCs), bispecific antibodies, and biobetters, in which antibodies and drugs are combined, and the new concept is a double-antibody-drug conjugate (ADC). As the anti-cancer antibody drug technology, it is spotlighted as the most successful antibody-based drug for treatment. The antibody-drug conjugate (ADC) is composed of three components, including a drug, a monoclonal antibody, and a linker connecting the antibody and the drug, wherein the target specificity of the monoclonal antibody and the cytotoxicity of the drug ( cytotoxicity) is a technique that combines the advantages of two components and focuses on the drug targeting only cancer cells.

That is, antibody-drug conjugate (ADC) technology is a method of delivering a drug to tumor cells using an antibody that specifically binds to a specific antigen expressed on the surface of a cancer cell, wherein the antibody is a selective drug delivery system It is used as a (drug delivery system).

After the antibody recognizes and targets cancer cells, the drug is released in the cell, and the cancer cell has a mechanism of action by which the cancer cell is killed. Thus, such target specific delivery has the advantage of avoiding systemic toxicity caused by exposure of toxic drugs that are released.

Therefore, antibody-drug conjugates (ADCs) are also called immunoconjugates and belong to "targeted chemotherapeutics" anti-cancer drugs.

A number of global pharmaceutical companies are introducing antibody-drug conjugate (ADC) technology from venture companies, and the scale has reached 200-500 billion won.

The market for products with antibody-drug conjugate (ADC) technology has grown explosively, playing a leading role in anti-cancer antibody biobetters.For example, 2014 sales of Adcetris, a drug for non-Hodgkin's lymphoma, and Kadcyla, a breast cancer drug, are about It is expected to exceed KRW 1 trillion with only two types of products at KRW 860 billion.

3. Overview of EGFR targeted drugs

EGFR (HER1) is a receptor HER family that exists on the cell surface and plays an important role in cell growth and death by binding with ligands such as EGF, TGF-α, and Epiregulin.

As a result of investigation through immunostaining, EGFR expression is increased in many types of cancer cells, and it has been reported that an increase in EGFR is closely related to the prognosis of cancer.

Therefore, anticancer drug drugs that treat cancer by suppressing the EGFR signal have been developed.

EGFR blocking antibodies cetuximab (Erbitux) and small molecule EGFR tyrosine kinase inhibitors (EGFR-TK1) gefitinib (iressa®) and erlotinib (tarceva®) are used clinically.

In addition, various therapeutic antibodies against EGFR have been developed and approved overseas, or anti-cancer drugs targeting EGFR are actively being developed.

This status is a result of demonstrating that EGFR targeted drugs can be excellent treatments for various tumors, and suggests the importance of research and development with great marketability.

4. EGFR family Her2 (Human epidermal growth factor receptor-2)

Intracellular Human EGFR2 (HER2) is known as HER2 / neu or ErbB2, and is a tyrosine kinase receptor belonging to the human epidermal growth factor receptor (HER / EGFR / ERBB) family. Induces growth and differentiation. HER2 has very high structural similarity to the other three EGFR families (HER1, HER3, HER4).

However, unlike other anti-cancer targets, in the case of Her2 (Human epidermal growth factor 2), the ligand that binds to it is not known yet, and it forms heterodimers by partnering with other HER receptors that bind the ligand, and thus forms various signaling pathways. It is involved in cell cycle increase, cell proliferation regulation and differentiation and survival. In the case of antibodies targeting Her2, antibody therapeutics have been developed and marketed as IgG2 types, and are not antibodies that are not antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Neutralizing activity due to is the main therapeutic action.

5. Mechanisms of Her2 overexpressing breast cancer

In about 25% of breast cancer patients, attention has been paid to HER2 gene proliferation and overexpression according to cancer cell proliferation, which is classified as HER2-positive cancer, and the risk of recurrence or death is significantly higher than that of HER2-negative cancer. Is becoming.

Trastuzumab is a single antibody that blocks Her2's intracellular signaling pathway tyrosine phosphatase activity by binding to Her2 and the sub-signals RAS-RAF-MEK-MAPK and PTEN-PIK3CA-AKT pathways. It suppresses cell growth and induces cell death, thereby showing the therapeutic effect of Her2 overexpressing breast cancer.

As a result, tumor cell infiltration into normal tissues and tumor spread to new sites are reduced, and tumor cells are inhibited in their ability to repair damage caused by chemotherapy and radiation therapy, thereby suppressing tumor growth. As can be seen from this treatment mechanism, in the case of Her2 overexpressing breast cancer, it is an action to block the sub-mechanism of Her2, which is a possible treatment mechanism even if the Fc portion of the antibody is canceled. You can.

6. Importance of application of antibody fragments as anticancer drugs for breast cancer

In the case of anti-cancer antibodies, not only Fab fragments that bind to antigens, but also effector function functions mediated by the Fc fragment region are important for its activity, but antibody fragments have no Fc and less molecular weight than intact antibodies, so antibody recycling Although there is a disadvantage that the half-life in the blood may be shortened due to inhibition, increased kidney discharge, etc., biopharmaceutical stabilization technologies such as increasing the half-life by directly attaching polyethylene glycol (PEG) to Fab have been developed.

Trastuzumab exhibits strong anticancer activity through inhibition of Her2 function corresponding to the antigen, so the effector function is known to have no significant function, so there is a possibility of high activity as a Fab fragment.

7. Utilization of engineered antibody fragments

The fragmented and improved antibody has a small size compared to an intact antibody, and thus has a good penetration rate into tissues or tumors, and has the advantage of being capable of low-cost production using a bacterial system.

However, since the antibody fragment has no Fc and has a lower molecular weight than the intact antibody, there is a disadvantage that the half-life in the blood may be shortened due to inhibition of antibody recycling, increased kidney discharge, etc., but there are various factors including PEGylation, etc. Biopharmaceutical stabilization technologies are being developed and this is the case with Cimzia, which is FDA approved.

In addition, when applied as a diagnostic agent by attaching a radioisotope, the feature of rapid drug elimination due to the short half-life of the antibody fragment acts as an advantage over an intact antibody.

Accordingly, the antibody fragments currently being developed or used are not only suitable for bio-diagnosis, but also are developed as therapeutic agents by increasing the half-life, developed for drug delivery by conjugating bacterial toxins, or to cytotoxic T cells. Various types of antibody fragments have been developed as therapeutic antibodies, such as preparing bispecific antibodies with binding antibody fragments and developing them as cancer treatment agents.

In particular, in the case of the development of antibody-drug conjugate (ADC) drugs that use antibodies for delivery to targets by attaching cytotoxic drugs to the antibodies, other fragments of immune cells can be obtained by using antibody fragments. In addition to rapid targeting to target cells without interaction with the drug, it is possible to rapidly maximize the effect of the drug by increasing the tumor tissue and cell penetration rate compared to the intact antibody, and reduce the extratumoral outflow of toxic drugs by antibody recycling. There is an advantage.

Republic of Korea Patent Publication No. 10-2010-0129318 Republic of Korea Patent Publication No. 10-2017-0007806

Therefore, the present inventors prepared a SRD110 fragment, an antibody fragment (Fab) that can replace the antibody for anticancer that suppresses the Her2 (Epidermal Growth Factor Receptor2) signal of cancer cells, and bound the drug to the antibody fragment to enhance the anticancer efficacy Thus, a site-specific drug-binding antibody fragment that can be applied as an anticancer agent was prepared. As a result, production through E. coli was possible, an antibody fragment specifically binding to Her2 was prepared, and the site-specific drug-binding antibody SRD110-L1 was completed through the present invention.

In order to achieve the above object, the present invention provides an antibody fragment (Fab) that specifically binds to Her2 (Human epidermal growth factor receptor-2).

In one embodiment of the present invention, the antibody fragment comprises a heavy chain variable region (VH) having an amino acid sequence represented by SEQ ID NO: 13; A heavy chain constant region (CH1) having the amino acid sequence represented by SEQ ID NO: 15; A light chain variable region (VL) having the amino acid sequence represented by SEQ ID NO: 14; And, it may include a light chain constant region (CL) having an amino acid sequence represented by SEQ ID NO: 16.

In one embodiment of the present invention, the antibody fragment may have a cysteine residue at the end of the heavy chain constant region.

In addition, the present invention provides a nucleic acid molecule encoding the antibody fragment.

In one embodiment of the invention, the nucleic acid molecule is a heavy chain variable region (VH) having a nucleotide sequence represented by SEQ ID NO: 7; A heavy chain constant region (CH1) having a nucleotide sequence represented by SEQ ID NO: 9; A light chain variable region (VL) having a nucleotide sequence represented by SEQ ID NO: 8; And, it may include a light chain constant region (CL) having a nucleotide sequence represented by SEQ ID NO: 10.

In addition, the present invention provides a recombinant vector comprising the nucleic acid molecule.

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

In addition, the present invention provides a culture medium or a culture filtrate in which the transformant is cultured.

In addition, the present invention is an antibody fragment; And a drug-linker binding compound.

In one embodiment of the present invention, the drug-linker binding compound may be a compound of maleimide, cathepsin cleavable linker, spacer, and MMAE (Monomethyl auristatin E).

In addition, in one embodiment of the present invention, the linker may be a valine (Val) -citrulline (Cit) peptide linker.

In addition, in one embodiment of the present invention, the spacer may be para-aminobenzylcarbamoyl (PAB).

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, including the antibody fragment, nucleic acid molecule, E. coli culture, culture filtrate, or antibody-drug conjugate.

In one embodiment of the present invention, 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, larynx cancer, head and neck cancer, colon cancer, ovarian cancer, rectal cancer , Colon cancer, vaginal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone marrow cancer.

In addition, the present invention is the antibody fragment; And, it provides a method for preparing an antibody-drug conjugate by reacting a drug-linker binding compound.

In one embodiment of the present invention, the drug-linker binding compound may be a compound of maleimide, cathepsin cleavable linker, spacer, and MMAE (Monomethyl auristatin E).

In addition, in one embodiment of the present invention, the linker may be a valine (Val) -citrulline (Cit) peptide linker.

In addition, in one embodiment of the present invention, the spacer may be para-aminobenzylcarbamoyl (PAB).

According to the present invention as described above, the antibody fragment is capable of intensive drug delivery to target cells by specifically binding to Her2, and has an improved binding affinity for her2, thereby treating cancer with high anticancer activity and It can be useful for prevention.

The effects of the present invention in the above are not limited to the above, and other effects of the present invention are clearly understood by those skilled in the art to which the present invention pertains through the examples described below and the claims. Will be understandable.

1 shows a schematic diagram of an expression vector for preparing SRD110 of the present invention.
Figure 2 shows the production process of the antibody fragment SRD110 of the present invention.
Figure 3 shows the antibody-drug binding degree of SRD110-L1 of the present invention.
Figure 4 shows the GPC results of the antibody fragment SRD110 and antibody-drug conjugate SRD110-L1 of the present invention.
Figure 5 shows the results of RP-HPLC of the antibody fragment SRD110 and antibody-drug conjugate SRD110-L1 of the present invention.
Figure 6 shows the MALS results of the antibody fragment SRD110 and antibody-drug conjugate SRD110-L1 of the present invention.
Figure 7 shows the molecular weight changes of the antibody fragment SRD110 and antibody-drug conjugate SRD110-L1 of the present invention.
Figure 8 shows the change in absorbance of the antibody fragment SRD110 and antibody-drug conjugate SRD110-L1 of the present invention.
9 shows an antibody-drug ratio analysis method of the antibody fragment SRD110 and antibody-drug conjugate SRD110-L1 of the present invention.
Figure 10 shows the results of measuring the antibody-drug ratio of the antibody fragment SRD110 and the antibody-drug conjugate SRD110-L1 of the present invention.
Figure 11 shows the Her2-binding affinity of the antibody fragment SRD110 and antibody-drug conjugate SRD110-L1 of the present invention using ELISA.
Figure 12 shows the Her2-binding affinity of the antibody fragment SRD110 and antibody-drug conjugate SRD110-L1 of the present invention using FACS.
Figure 13 shows the results of cell viability analysis of the antibody fragment SRD110 and antibody-drug conjugate SRD110-L1 of the present invention.
14 shows the results of an animal model administration experiment of the antibody fragment SRD110 and the antibody-drug conjugate SRD110-L1 of the present invention.

Hereinafter, the present invention will be described in more detail.

Antibody fragments specifically binding to Her2 and uses thereof are described for the present invention.

According to the invention it is cleaved under appropriate conditions to provide target cell delivery of the drug. In addition, by providing improved binding affinity to target cells, it can be usefully used for the treatment and prevention of cancer through high anticancer activity. The present invention has desirable properties for the purpose of pharmaceutical compositions for the treatment or prevention of cancer through high specificity for target cells that improve target concentration for drugs and minimize exposure to normal cells.

"Antibody fragment" in the present invention refers to a fragment having an antigen-binding function as a Fab antibody, heavy chain variable region (VH), heavy chain constant region (CH1), light chain variable region (VL) and light chain constant region (CL) ), And has an antigen-binding site. In the present invention, the antibody fragment has a hinge region comprising at least one cysteine residue at the C-terminus of the heavy chain CH1 domain.

The term "heavy chain" in the present invention means both a full-length heavy chain and a fragment thereof comprising a variable region (VH) and a constant region (CH1) comprising an amino acid sequence having sufficient variable region sequences to confer specificity to an antigen. do. The antibody fragment in the present invention is an antibody fragment comprising a heavy chain consisting of the variable region (VH) and the constant region (CH1).

The term "light chain" in the present invention means both a full-length light chain comprising a variable region (VL) and a constant region (CL) comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and fragments thereof. do. In the present invention, the antibody fragment is an antibody fragment comprising a light chain composed of the variable region (VL) and constant region (CL).

Antibody fragments of the present invention may include variants of the nucleotide sequence or amino acid sequence described in the attached sequence list within a range capable of specifically binding to Her2. For example, the amino acid sequence of a Fab fragment can be altered, for example, to improve the binding affinity and / or other biological properties of the Fab fragment. Such modifications include, for example, deletion, insertion and / or substitution of amino acid sequence residues of Fab fragments. 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. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically equivalent functions. In introducing mutations, the hydropathy index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index according to 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 conferring the protein's interactive biological function. It is well known that amino acids with similar hydrophobic indices can be replaced to retain similar biological activity. When introducing a variation with reference to the hydrophobic index, substitution is performed between amino acids showing a hydrophobic index difference, preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that substitution between amino acids having similar hydrophilicity values results in proteins with equivalent 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 variation with reference to a hydrophilicity value, substitution is performed between amino acids showing a difference in hydrophilicity values of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5. Amino acid exchange in proteins that do not entirely alter the activity of the molecule is known in the art (H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The most common 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 / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In view of the above-described variations with biologically equivalent activity, the antibody of the present invention or a nucleic acid molecule encoding the same is interpreted to also include a sequence showing substantial identity with the sequence listed in the sequence listing. The substantial identity above, when aligned with the above-described sequence of the present invention and any other sequence as much as possible, and analyzed the aligned sequence using an algorithm commonly used in the art, a minimum of 61% Refers to a sequence that exhibits homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment can be found in Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), blastp, blasm, It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/. The sequence homology comparison method using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

The term "nucleic acid molecule" in the present invention has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and a nucleotide that is a basic structural unit in a nucleic acid molecule includes not only natural nucleotides, but also sugar or base sites. Modified analogues are also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)). Nucleic acid molecule sequences encoding heavy and light chain variable regions of the antibody fragments of the invention can be modified. Such modifications include addition, deletion or non-conservative substitutions or conservative substitutions of nucleotides.

The nucleic acid molecule encoding the antibody of the present invention is interpreted to include 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 is aligned with any other sequence as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. Means a nucleotide sequence exhibiting homology, more preferably at least 90% homology, and most preferably at least 95% homology.

The expression construct produced in the present invention is constructed to express a desired gene in a host cell. In general, promoters and terminators operatively coupled to the upstream and downstream of the expression construct, respectively, are located.

As used herein, the term "promoter" refers to a DNA sequence that regulates the expression of a coding sequence or functional RNA. The target nucleotide sequence in the recombinant vector of the present invention is operably linked to the promoter.

As used herein, the term “operatively linked” refers to functional binding between a nucleic acid expression control sequence (eg, a promoter sequence, a signal sequence, or an array of transcription regulator binding sites) and another nucleic acid sequence. In this way, the regulatory sequence controls the transcription and / or translation of the other nucleic acid sequence.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). Vectors of the invention can typically be constructed as vectors for cloning or as vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic cells as hosts. Vectors of the invention can typically be constructed as vectors for cloning or as vectors for expression.

For example, when the vector of the present invention is an expression vector, and a prokaryotic cell is a host, a strong promoter capable of progressing 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 sites for transcription initiation and transcription / detox termination sequences (terminators such as T7 terminator, ADH1 terminator, T3 Terminators and TonB terminators). 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 leftward 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.), phages (e.g., λλλλΔ and M13, etc.) Or it can be produced by manipulating a virus (eg SV40, etc.).

Host cells capable of continuously cloning and expressing the vector of the present invention with stability are known in the art and can use any host cell, 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 strains such as Bacillus thuringiensis, and Salmonella typhimurium , Intestinal bacteria and strains such as Ceratia Marcesons and various Pseudomonas species.

The method for transporting the vector of the present invention into a host cell is a heat shock method, a CaCl2 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 method (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)).

On the other hand, an antibody-antibody-drug conjugate that can be cleaved under appropriate conditions from an antibody or antibody fragment disclosed in the present invention and has a better solubility and binding ability of a compound is provided. The increased hydrophobicity of some enzyme-linkers can provide particularly stable binding. The linker described in the present invention can achieve better stability through specific enzyme design.

Antibody-drug conjugates of the present disclosure can be cleaved under appropriate conditions and a targeting region capable of targeting a selected cell population, a drug region providing toxicity to the target cell population, a spacer to provide a distance between the drug region and the targeting region. Linker. Each feature is specifically described below.

The "effective dose" or "effective amount" of an antibody, drug, compound, conjugate, drug conjugate, antibody-drug conjugate or pharmaceutical composition used in the present invention is an amount sufficient to produce a beneficial or desired result. In the case of prophylactic use, beneficial or desired results are presented during or during the onset of the disease, including eliminating or reducing risk, alleviating the severity, or biochemical, histological and behavioral symptoms of the disease. Results such as delaying his complications and intermediate pathologic phenotype.

In the case of therapeutic use, beneficial or desired outcomes include reducing one or more symptoms arising from the disease, increasing the quality of life of a person suffering from the disease, and reducing the dose of other medications needed to treat the disease. Clinical outcomes such as enhancing the effectiveness of other medications, such as through targeting, delaying disease progression, and / or prolonging survival. In the case of cancer or cultivation, an effective amount of a drug can reduce the number of cancer cells, reduce the tumor size, inhibit cancer cell infiltration into peripheral organs, inhibit tumor metastasis, and It may have an effect of alleviating one or more of the related symptoms. The effective dose can be administered in one or more doses. For the purposes of the present invention, an effective dosage of a drug, compound or pharmaceutical composition is an amount sufficient to achieve prophylactic or curative treatment directly or indirectly. As clinically understood, an effective dosage of a drug, compound, or pharmaceutical crude drug may or may not be achieved with another drug, compound, or pharmaceutical composition. Thus, an “effective dosage” can be considered to be given an effective amount if desired results can be achieved or achieved with respect to administration of one or more therapeutic agents.

As used herein, “administration” refers to the general term for the distribution of therapeutic agents to treat a condition. In some embodiments, pharmaceutical compositions of the present disclosure are suitable for orally administering a compound or mixture as a tablet, capsule or pill, or the compound is parenteral, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, Contains a pharmaceutically acceptable carrier or excipient suitable for sublingual, intratracheal, inhalation, ocular, vaginal, rectal, subcutaneous or transdermal administration.

In addition, various routes of administration are available in the present invention. The particular mode chosen will of course depend on the particular agent or agents selected, the particular condition being treated, and the dosage required for therapeutic efficacy. Several modes of administration are described below.

Administration of the compounds or pharmaceutical compositions disclosed in the present invention can be accomplished by any means known to those skilled in the art. Routes of administration include, but are not limited to, oral, parenteral, intravenous, intramuscular, intraperitoneal, intranasal, sublingual, intratracheal, inhalation, subcutaneous, ocular, vaginal and rectal. Systemic routes include oral and parenteral.

For oral administration, the pharmaceutical compositions of the present invention can be readily formulated by combining the active compound (s) with a pharmaceutically acceptable carrier well known in the art. Such carriers can allow the compounds of the present disclosure to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, etc., for oral ingestion by a subject to be treated. Optionally, oral formulations can also be formulated in saline or buffers to neutralize internal acid conditions, or can be administered without any carrier.

If it is desired to deliver the composition systemically, it may be formulated for parenteral administration by injection, for example bolus injection or continuous infusion. Formulations for injection may be presented, for example, in unit dosage form in ampoules or in multi-dose containers, with preservatives added. The composition may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulation agents, such as suspending agents, stabilizers and / or dispersants.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides or liposomes.

Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions. Alternatively, the active compound can be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, prior to use.

For the purposes of the present invention, an effective dosage of a drug, compound or pharmaceutical composition is an amount sufficient to achieve prophylactic or curative treatment directly or indirectly. As understood in the clinical context, an effective dosage of a drug, compound or pharmaceutical composition may or may not be achieved with another drug, compound or pharmaceutical composition. In addition, it can be variously prescribed by factors such as formulation method, administration method, patient's age, weight, sex, morbidity, food, administration time, administration route, excretion rate and response sensitivity. Thus, an “effective dosage” can be considered in relation to the administration of one or more therapeutic agents, and a single agent can be considered to be given in an effective amount if desired results can be achieved or achieved in relation to one or more other agents. . For example, an effective dosage may range from 0.001 μg / kg to 1000 mg / kg on an adult basis.

In addition, the compounds in the present invention may be formulated into rectal or vaginal compositions, such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides.

As used herein, “in conjunction with” refers to administration of another treatment modality in addition to one treatment modality. Thus, “in conjunction with” refers to administration of one treatment modality before, during, or after administration of another treatment modality to an individual.

As used herein, “treatment” or “treating” is preferably an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include reducing or destroying the proliferation of cancerous cells, reducing symptoms arising from the disease, increasing the quality of life of those suffering from the disease, and disease. It includes, but is not limited to, reducing the dose of other medications needed to treat, delaying the progression of the disease, or prolonging the survival of the individual.

As used herein, "delaying the development of a disease" means delaying, obstructing, slowing, inhibiting, stabilizing or delaying the development of the disease (eg, cancer). This delay can be of varying lengths of time, depending on the history of the disease or subject being treated. As is evident to those skilled in the art, a sufficient or significant delay can encompass prevention, which in practice does not cause disease in the individual. For example, it may be meant to include delayed development of late stage cancer, such as metastasis.

The "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, livestock, sports animals, pets (eg cats, dogs, horses), primates, mice and rats. “Treatment of cancer in an individual in need of treatment” has been identified as having the cancer, that is, the individual has had cancer by a doctor (eg, using methods well known in the art) It was diagnosed as being. In some embodiments, the individual in need of treatment is an individual suspected of having or suspected of having cancer. Examples of individuals with or suspected of having cancer include subjects identified as having cancer or mutations associated with the occurrence of cancer, subjects with a family history of cancer, and subjects who have previously had or been cured of cancer ( Including cancer patients in a relaxed state).

As used herein, the terms “specifically recognize” or “specifically bind” to measurable and reproducible interactions, such as targets, that determine the presence of a target in the presence of a heterogeneous population of molecules, including biological molecules. And attraction or binding between antibodies or molecules or regions. For example, an antibody that specifically or preferentially binds to an epitope binds to this epitope with greater affinity, binding capacity, or easier or greater duration than it binds to other epitopes or non-target epitopes of the target. It is a binding antibody. It is also understood that, for example, an antibody that specifically or preferentially binds a first target may or may not specifically bind a second target. Thus, "specific binding" or "preferred binding" does not necessarily require exclusive binding.

In addition, as used herein, the term “site-specific binding” or “site-specific conjugation” refers to the attraction or binding of a target, antibody, molecule or region to a specific location or site. In the present invention, it may mean that the drug selectively binds to a specific site of the antibody, for example, a drug in which a maleimide-based derivative capable of specifically binding only cysteine is introduced by introducing cysteine to the terminal of VH-CH1. (MMAE, etc.) can be designed to be able to bind to the antibody. Thus, cysteine is a specific site for drug binding, and such a drug conjugation method can be understood as a site specific conjugation method.

In the present invention, various immunoassay formats can be used to select antibodies that are specifically immunoreactive with specific proteins. For example, solid-phase ELISA immunoassays are used commercially to select monoclonal antibodies that are specifically immunoreactive with proteins. Descriptions of immunoassay formats and conditions that can be used to determine specific immunoreactivity can be by generally known methods.

The terms "cancer", "tumor", "cancerous" and "malignant" as used herein refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas including adenocarcinoma, lymphoma, blastoma, melanoma, and sarcoma. More specific examples of such cancers include breast cancer, colorectal cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, larynx cancer, head and neck cancer, colon cancer, ovarian cancer, rectal cancer, colon cancer, vaginal cancer , Small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureteral cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone marrow cancer.

The singular form used in the present invention and the appended claims includes a plurality of indications unless the context clearly indicates otherwise. For example, reference to “antibody” refers to one to many antibodies, such as molar amounts, and will include its equivalents and the like known to those skilled in the art.

Reference to “about” a value or parameter in the present invention includes embodiments for the value or parameter itself. For example, description referring to “about X” includes description of “X”. It can be understood that aspects and variations of the present disclosure described in the present invention include “consisting of” and / or “consisting essentially of” aspects and variations.

On the other hand, unless otherwise defined in the present invention, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described in the present invention can be used in practicing or testing the present invention, preferred methods and materials are now described. All publications mentioned in the present invention are incorporated herein by reference to disclose and describe the methods or materials cited in connection with the publication.

Except as otherwise defined, the methods and techniques of embodiments of the present invention are generally according to conventional methods as described in various general and more specific references well known in the art and cited and discussed throughout this specification. Can be performed. The nomenclature used in the present invention to name a target compound is exemplified in the Examples of the present invention. This nomenclature can generally be derived using commercially available AutoNom software (MDL, San Leandro, CA). It is recognized that for clarity, certain features of the present disclosure described in connection with separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure described in connection with a single embodiment for brevity may also be provided individually or in any suitable subcombination. All combinations of embodiments relating to chemical groups represented by variable groups are specifically encompassed by the present disclosure, and such combinations are stable compounds, i.e., isolated, and characterized, as each and every combination is disclosed individually and clearly. It can be understood by the present invention to the extent that it encompasses compounds that can be chemically and tested for biological activity. In addition, all subcombinations of chemical groups listed in the embodiments describing such variable groups are specifically encompassed by the present disclosure, and each and every such subcombination of chemical groups is individually and clearly understood by the present invention. You can.

Antibody fragments that specifically bind to Her2 of the present invention and uses thereof are described in more detail in the Examples below. However, the following examples are only intended to specify the contents of the present invention, and the present invention will not be limited thereby.

<Example 1> Preparation of SRD110 antibody fragment construct

In this example, a novel antibody fragment SRD110 was synthesized by preparing primers at the regions corresponding to the VH-CH1 and VL-CL domains (Table 1). Both chains were expressed using the OmpA signal peptide at the N-terminus and then moved to the periplasmic space to induce correct folding. Through restriction enzyme treatment, OmpA + VH-CH1 and OmpA + VL, respectively. The gene was cloned into the pACYCDuet-1 vector, which is an E. coli expression vector, to express the -CL domain, respectively (FIG. 1). Later, it is internalized into cancer cells using a cathepsin cleavable linker and spacer to release only the drug Monomethyl auristatin E (MMAE), maximizing the effect of the drug and providing a selective drug binding site For this, PCR was performed to have extra free cysteine residue THTCAA at the end of the CH1 domain. The amino acid and nucleotide sequences of the prepared SRD110 construct are shown in Tables 2 and 3.

No Construct primer Sequence (5 '→ 3') One VH Forward primer (SEQ ID NO: 1) CATGCCATGGCAAAAAAGACAGCTATCGCGATTGCA 2 VH Reverse primer (SEQ ID NO: 2) ATAAGAATGCGGCCGCTCACGCCGCGCAGGTATGGG 3 VL Forward primer (SEQ ID NO: 3) CAGCGCTAGCATGAAAAAGACAGCTATCGCGATTGCA 4 VL Reverse primer (SEQ ID NO: 4) CCGCTCGAGTCAGCATTCGCCGCGGTTAAAGC

order OmpA (base)
(SEQ ID NO: 5) AAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCC VH (base)
(SEQ ID NO: 7) GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC CH1 (base)
(SEQ ID NO: 9) GCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGAGCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGTGAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGCGCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGCAACACCAAAGTGGATAAAAAAGTGGAACCG CCG AAAAGCTGCGATAAA ACCCATACCTGCGCGGCG OmpA (amino acid)
(SEQ ID NO: 11) KKTAIAIAVALAGFATVAQA VH (amino acid)
(SEQ ID NO: 13) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS CH1 (amino acid)
(SEQ ID NO: 15) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDK THTCAA

order OmpA (base)
(SEQ ID NO: 6) AAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCC VL (base)
(SEQ ID NO: 8) GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAACACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGACCTTTGGCCAGGGCACCAAAGTGGAAATTAAA CL (base)
(SEQ ID NO: 10) CGCACCGTGGCGGCGCCGAGCGTGTTTATTTTTCCGCCGAGCGATGAACAGCTGAAAAGCGGCACCGCGAGCGTGGTGTGCCTGCTGAACAACTTTTATCCGCGCGAAGCGAAAGTGCAGTGGAAAGTGGATAACGCGCTGCAGAGCGGCAACAGCCAGGAAAGCGTGACCGAACAGGATAGCAAAGATAGCACCTATAGCCTGAGCAGCACCCTGACCCTGAGCAAAGCGGATTATGAAAAACATAAAGTGTATGCGTGCGAAGTGACCCATCAGGGCCTGAGCAGCCCGGTGACCAAAAGCTTTAACCGCGGCGAATGC OmpA (amino acid)
(SEQ ID NO: 12) KKTAIAIAVALAGFATVAQA VL (amino acid)
(SEQ ID NO: 14) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK CL (amino acid)
(SEQ ID NO: 16) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

<Example 2> E. coli expression of SRD110 construct confirmed

E. coli C43 (DE3), pACYCDuet-1 vector was used as the host cell and plasmid of the SRD110 construct prepared in Example 1. For the seed culture, chloramphenicol was added to the LB medium as an antibiotic to a final concentration of 34 μg / ml and cultured overnight at 37 ° C. and 150 rpm in a shaking incubator. For this culture, the cell culture medium grown on the previous day was inoculated to be about 1-2% in TB medium, and the antibiotic chloramphenicol was added and cultured at 37 ° C and 150 rpm in a shaking incubator. About 3-4 hours after inoculation, the degree of culture was confirmed by absorbance (OD ₆₀₀ ). When the absorbance was about OD ₆₀₀ = 2.4, 0.5 mM IPTG was added to the culture medium to induce it, and cultured overnight in a shaking incubator set at 25 ° C and 200 rpm. Thereafter, the culture solution was centrifuged at 4 ° C, 4500 rpm for 20 minutes to obtain pellets. E. coli C43 (DE3) pellets thus obtained were suspended in about 10 ml of cell lysis buffer per 1 g of weight. Cells were suspended with lysis buffer of 50 mM Tris-HCl, 5 mM EDTA, pH 8.0, and 0.2 mM PMSF was added as a protase inhibitor before cell disruption. Thereafter, cells were lysed for 10 minutes (pulse on 3 seconds, pulse off 3 seconds) using an ultrasonic crusher. To separate the antibody fragments and proteins from the aqueous solution and the cells, the cells were centrifuged at 15,000 rpm for 1 hour at a rate of 15,000 rpm.

In order to purify only antibody fragments from the antibody fragments and protein aqueous solution separated from the cells as described above, affinity chromatography, ion chromatography, and size exclusion chromatography were performed in the same steps as in FIG. 2. The open column is filled with kappa resin (GE Healthcare) that binds the CL domain of the antibody fragment, and 10 CV (column volume) of equilibrium buffer (50 mM Tris-HCl, 5 mM EDTA, pH 8.0) is flowed. It was brought to equilibrium. After flowing the antibody fragment and the aqueous protein solution to the column, the resin and the antibody fragment were bound, and then washed with 10 CV (column volume) of washing buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0). Non-specifically bound impurities were removed. To separate the antibody fragment from kappa resin, it was eluted with 5 CV (column volume) of elution buffer (100 mM glycine, 1 mM EDTA, pH 2.5) and confirmed by SDS-PAGE (FIG. 2). The eluted antibody fragment was adjusted to pH of 6.0 using 1 M MES.

Ion chromatography was performed with the eluate. SP Sepharose resin (GE Healthcare) was filled in an open column, and an equilibrium buffer (50 mM MES, 1 mM EDTA, pH 6.0) was flowed to prepare an equilibrium state. After flowing the antibody fragment and the aqueous protein solution to the column to allow the resin to bind to the antibody fragment, 10 CV (column volume) of primary washing buffer (50 mM MES, 1 mM EDTA, pH 6.0) was flowed, and 10 CV ( Non-specifically bound impurities were removed by flowing a second washing buffer (pH 6.0 / 50 mM MES, 100 mM NaCl, 1 mM EDTA, pH 6.0) of column volume). To separate antibody fragments from SP Sepharose resin, eluted with an elution buffer (50 mM MES, 500 mM NaCl, 1 mM EDTA, pH 6.0) and confirmed by SDS-PAGE (FIG. 2).

Size exclusion chromatography was performed to confirm the homogeneity of the antibody fragments. The HiLoad 16/600 Superdex 200 column (GE Healthcare) was allowed to equilibrate by flowing 1X PBS buffer (137 mM Sodium Chloride, 10 mM phosphate, 2.7 mM Potassium Chloride; pH is 7.4). The antibody fragment aqueous solution eluted in the purification process was flowed into the column. Only the fraction containing the antibody was recovered and the final concentration was quantified, and as shown in FIG. 2, only a single peak was shown, and it was confirmed that an antibody fragment having a single component of 95% or more was obtained. Stored at 4 ° C until use.

<Example 3> Preparation of antibody-drug conjugate SRD110-L1 using SRD110-MMAE

In this example, the antibody-drug conjugate SRD110-L1 using the SRD110 antibody fragment was mixed with the maleimide-VC-PAB-MMAE drug-linker binding compound and bound using a maleimide reaction. In order to maximize the effect of the drug, only cathepsin cleavable linker and spacer were used to internalize the cancer cells, and only the drug Monomethyl auristatin E (MMAE) was released (FIG. 3).

In the present invention, SRD110-L1, SRD110-MMAE conjugate, was prepared by conjugating Thr-His-Thr-Cys-Ala-Ala of antibody fragment SRD110 with Monomethyl auristatin E (MMAE), which is known to have much higher cytotoxicity than doxorubicin. After reducing the thiol group by adding 2-10 equivalents of reducing agent TCEP (tris (2-carboxyethyl) phosphine) per 1 equivalent of purified SRD110 antibody fragment at 4 ° C., 2-10 equivalents of MC-vc-PAB-MMAE It is added and reacted at room temperature for 4 hours. The reaction was terminated by adding an excess of cysteine, and excess MC-vc-PAB-MMAE and TCEP (tris (2-carboxyethyl) phosphine) were removed using a centrifugal filtration filter and size exclusion chromatography. The final purified antibody fragment SRD110-MC-vc-PAB-MMAE, SRD110-L1, was prepared.

<Example 4> Characterization of novel antibody fragments (SRD110) and conjugates (SRD110-L1)

4-1. Analysis of SRD110 and SRD110-L1 using GPC

Purified protein SRD110 (VL-VH) and Monomethyl auristatin E (MMAE) binding reaction and homogeneity of fusion SRD110-L1 (VL-VH-MMAE) and Monomethyl auristatin E (MMAE) binding to determine whether GPC (Gel Permeation Chromatography) ). AKTA prime HPLC system was connected to a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) and flowed into an equilibration buffer (PBS, pH 7.4) to prepare an equilibrium state. The aqueous solution of SRD110-L1, which is a fusion of SRD110 and Monomethyl auristatin E (MMAE) purified in Example 2, was flowed to a column, respectively, and the absorbance was measured at 280 nm in a chromatogram. According to this method, the antibody fragment-MMAE fusion SRD110-L1 with increased molecular weight was confirmed by conjugation of the drug Monomethyl auristatin E (MMAE) to SRD110 with high homogeneity (FIG. 4).

4-2. Analysis of SRD110 and SRD110-L1 using RP-HPLC

Purified protein SRD110 (VL-VH) and Monomethyl auristatin E (MMAE) binding reaction and homogeneity of fusion SRD110-L1 (VL-VH-MMAE) and Monomethyl auristatin E (MMAE) binding RP-HPLC (Reversed) phase-High performance liquid chromatography). 40 μg of antibody fragment conjugate samples were analyzed using PLRP-S (4.6 × 150 mm, 300 mm 3, 8 μm, Agilent Technologies). The flow rate was set at 1 mL per minute and the column temperature was kept constant at 65 ° C. during the analysis time. Mobile phase A was 0.1% (v / v) trifluoroacetic acid in water, and mobile phase B was 0.1% (v / v) trifluoroacetic acid in ACN. : Mobile phase B 30% 0-1 minutes, mobile phase B 30% -45% 1-14 minutes, mobile phase B 45% -95% 14-17 minutes, mobile phase B 95% -30% 17-19 minutes, Mobile phase B 30% 19-25 minutes. Analysis time was 25 minutes per sample. By this method, it was confirmed that the MMAE fusion (SRD110-L1) with increased hydrophobicity was obtained by conjugating the drug Monomethyl auristatin E (MMAE) to SRD110 with high purity with each separated peak (FIG. 5).

4-3. SRD110 and SRD110-L1 analysis using MALS

The purified antibody fragment SRD110 from Example 2 and the purified antibody fragment drug conjugate SRD110-L1 from Example 4-1 were measured for multi-angle light scattering (MALS) (FIG. 6). As shown in FIG. 7, the molecular weight of 46.7 kDa was confirmed, and it was confirmed that the molecular weight was increased due to the drug conjugation to SRD110 (molecular weight when measuring MALS: 45.9 kDa) along with homogeneity.

4-4. DAR analysis of SRD110-L1 using UV measurement

SRD110 antibody fragment and the drug Monomethyl auristatin E (MMAE) after the binding reaction to purify the protein to determine whether the drug is still bound to the protein is a commonly applied method that utilizes the maximum UV-VIS absorbance of the protein and drug . Proteins usually exhibit maximum absorbance in the UV region of 280 nm wavelength and the drug used in the present invention has maximum absorbance in the visible region at 248 nm wavelength. Since both the antibody fragment and drug have intrinsic extinction coefficients, absorbance was measured at around 280 nm and 250 nm. As shown in Fig. 8, the absorbance increased as the equivalent of the mixed drug per antibody fragment molecule increased. The absorption changes for the antibody-drug conjugates tested were analyzed and an assay for drug-antibody ratio (DAR) was established based on the absorption ratio at two wavelengths (280 nm, 250 nm) (FIG. 9). ).

For the SRD110-L1 separated by antibody fragments and GPC (Gel permeation chromatography), after measuring the absorption coefficient of each material through the peak area on GPC (Gel permeation chromatography), DAR using the absorbance ratios at two wavelengths of 250 and 280 nm Drug-antibody ratio (Drug-antibody ratio) value was calculated, and as a result, the DAR (Drug-antibody ratio) analysis result of the antibody-drug conjugate SRD110-L1 was found to be 1.2 (FIG. 10).

<Example 5> Confirmation of activity and efficacy of new antibody fragments and conjugates (SRD110-L1)

5-1. Measurement of Her2-binding affinity using ELISA

Add antigen Her2 (100 ng / well) to a 96-well ELISA (enzyme-linked immunosorbent assay, enzyme-linked immunospecific assay) plate and react at 4 ° C for 17 hours or more to immobilize the antigen on the plate surface and remove the supernatant And 200 μl of the blocking solution was dispensed into each well and blocked at room temperature for 1 hour. Herceptin (Trastuzumab) and purified antibody fragments, which are standard materials for obtaining an assay curve, were diluted to a concentration of 0 to 125 ng / ml using 1X PBS. After dispensing 100 ul each, the mixture was reacted for 1 hour at room temperature to bind to the antigen, and washed 3 times with 100 μl / well of 0.05% PBS-T (PBS, 0.05% tween 20, pH 7.4) after the reaction was completed. Anti-human IgG (Fab specific) (Sigma, I5260) was diluted 1 / 1,000, dispensed into each well at 100 μl, reacted for 1 hour at room temperature, the supernatant was removed, and washed 3 times with 0.05% PBS-T. . Secondary antibody anti-goat IgG-peroxidase (Sigma, A5420) was diluted 1 / 3,000, dispensed in 100 μl of each well, and reacted at room temperature for 1 hour. After removing the supernatant and washing three times with PBS-T, 100 µl of TMB (color reagent) was dispensed. The reaction was stopped by adding 100 μl of 1 M HCl to the developed well, and the absorbance was measured at a wavelength of 450 nm using a microplate reader. As a result of measuring the binding strength to Her2 using ELISA (enzyme-linked immunosorbent assay, enzyme-linked immunospecific assay), the activity of SRD110 compared to Herceptin (88.4%) was 92.58%, and the activity of SRD110-L1 was 54.51%. Became. Through this result, it was confirmed that the purified new antibody fragment SRD110 has a higher binding strength than the control drug Herceptin, but the drug conjugated antibody fragment SRD110-L1 has a lower binding strength than the control drug Herceptin (FIG. 11).

5-2. Measurement of Her2-binding affinity using FACS

The binding affinity of Her2 and antibody fragments endogenously expressed in SK-OV-3 cells was confirmed using a FACS (Fluorescence activated cell sorter). Prepare two 100 pie culture dishes with SK-OV-3 cells spread at a density of 90% or more. After checking the cells, cells were separated using 0.25% Trypsin-EDTA and washed once with DPBS. After washing the cells, the DPBS is completely removed, then 1.2 ml of DPBS is added to the pipette, and 100 μl is added to the e-tube. After further adding 400 μl of DPBS, the buffer was completely removed by centrifugation, and 100 ul of a substance diluted with 1.9 nM to 500 nM was added to each mixture, followed by reaction at 4 ° C. for 15 minutes. The reaction was stopped by adding 400 μl DPBS to each tube, and then washed twice with DPBS. After completely removing the buffer, 100 μl of FITC-conjugated secondary antibody anti-human IgG was added to each tube, and after sufficiently taping, the light was blocked and reacted at 4 ° C. for 15 minutes. After the reaction, 400 μl DPBS was added to each tube to stop the reaction, followed by washing twice with DPBS, and then binding affinity was measured using FACS. As a result of measuring Her2 binding strength using FACS (Fluorescence activated cell sorter), it was found that the dissociation constant (Kd) for Her2 expressed is about 2.6 times higher than Herceptin and SRD110 about 3.6 times higher than that of Herceptin. (Figure 12).

Based on the in vitro Her2 binding experiment by the ELISA (enzyme-linked immunosorbent assay, enzyme-linked immunospecific assay) method and the FACS (Fluorescence activated cell sorter) analysis result of Her2 (+) SK-OV-3 cell binding It was confirmed that it maintains a sufficiently effective target binding ability, although it is inferior to the drug Herceptin.

<Example 6> Cell viability analysis

Using SK-OV-3 cells, the cells were dispensed to 6X10 ³ cells / well in each well of a 96-well plate, and cultured at 5% CO2 in a 37 ° C incubator for one day. The next day, Herceptin, SRD110, and SRD110-L1 were treated in each well so as to have a concentration of 0 to 10000 nM in serum-free DMEM medium and cultured for 72 hours. After 72 hours, the EZ-Cytox reagent was diluted to 10% in serum-free medium, treated in each well, and then incubated for 2 hours at 5% CO2 in a 37 ° C incubator. After 2 hours, the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. As a result of performing cell viability assay by treatment with concentrations of the control drugs Herceptin and SRD110 and SRD110-L1 in SK-OV-3 cells, EC50 values of each material are about 5000 nM, 133 nM, 7.6 nM, and SRD110-L1 It was confirmed to have the highest anticancer activity compared to the control drug, and reproducibility was confirmed (FIG. 13).

<Example 7> Anti-cancer efficacy confirmed in vivo

Since it is a substance having both target binding capacity and high cytotoxicity, good efficacy in the animal model was expected. Therefore, it was analyzed in vivo by administering to the SK-OV-3 (Her2 +) animal model, and as a result, it was more effective than the control drug Herceptin. It was confirmed that it showed high efficacy (Fig. 14).

As described above, specific embodiments of the present invention have been described in detail, but those skilled in the art who understand the spirit of the present invention may add other elements within the scope of the same spirit, change, delete, etc., and degenerate other inventions However, other embodiments included within the scope of the inventive concept may be easily proposed. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the scope of the claims, which will be described later, rather than the detailed description above, and all the changed or modified forms derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. It should be interpreted as.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation          SINIL PHARMACEUTICAL CO., LTD. <120> Antibody fragments specifically binding to Her2 and uses thereof <130> PN1807-221 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> VH Forward primer <400> 1 catgccatgg caaaaaagac agctatcgcg attgca 36 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> VH Reverse primer <400> 2 ataagaatgc ggccgctcac gccgcgcagg tatggg 36 <210> 3 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> VL Forward primer <400> 3 cagcgctagc atgaaaaaga cagctatcgc gattgca 37 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> VL Reverse primer <400> 4 ccgctcgagt cagcattcgc cgcggttaaa gc 32 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> VH-OmpA <400> 5 aaaaagacag ctatcgcgat tgcagtggca ctggctggtt tcgctaccgt agcgcaggcc 60                                                                           60 <210> 6 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> VL-OmpA <400> 6 aaaaagacag ctatcgcgat tgcagtggca ctggctggtt tcgctaccgt agcgcaggcc 60                                                                           60 <210> 7 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 7 gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggctt taacattaaa gatacctata ttcattgggt gcgccaggcg 120 ccgggcaaag gcctggaatg ggtggcgcgc atttatccga ccaacggcta tacccgctat 180 gcggatagcg tgaaaggccg ctttaccatt agcgcggata ccagcaaaaa caccgcgtat 240 ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcag ccgctggggc 300 ggcgatggct tttatgcgat ggattattgg ggccagggca ccctggtgac cgtgagcagc 360                                                                          360 <210> 8 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> VL <400> 8 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 ctttggccag 300 ggcaccaaag tggaaattaa a 321 <210> 9 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> CH1 <400> 9 gcgagcacca aaggcccgag cgtgtttccg ctggcgccga gcagcaaaag caccagcggc 60 ggcaccgcgg cgctgggctg cctggtgaaa gattattttc cggaaccggt gaccgtgagc 120 tggaacagcg gcgcgctgac cagcggcgtg catacctttc cggcggtgct gcagagcagc 180 ggcctgtata gcctgagcag cgtggtgacc gtgccgagca gcagcctggg cacccagacc 240 tatatttgca acgtgaacca taaaccgagc aacaccaaag tggataaaaa agtggaaccg 300 ccgaaaagct gcgataaaac ccatacctgc gcggcg 336 <210> 10 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> CL <400> 10 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 gcggcgaatg c 321 <210> 11 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> VH-OmpA <400> 11 Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala Thr   1 5 10 15 Val Ala Gln Ala              20 <210> 12 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> VL-OmpA <400> 12 Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala Thr   1 5 10 15 Val Ala Gln Ala              20 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 13 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> 14 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala              20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 15 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CH1 <400> 15 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 Cys Asp Lys Thr His Thr Cys Ala Ala             100 105 110 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CL <400> 16 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu   1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe              20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln          35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser      50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu  65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                  85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105

Claims (20)

Antibody fragment (Fab) that specifically binds to Her2 (Human epidermal growth factor receptor-2).
According to claim 1,
The antibody fragment is characterized in that it comprises the following region.
A heavy chain variable region (VH) having an amino acid sequence represented by SEQ ID NO: 13;
A heavy chain constant region (CH1) having the amino acid sequence represented by SEQ ID NO: 15;
A light chain variable region (VL) having the amino acid sequence represented by SEQ ID NO: 14; And,
Light chain constant region (CL) having the amino acid sequence represented by SEQ ID NO: 16.
According to claim 2,
The antibody fragment, wherein the antibody fragment has a cysteine residue at the end of the heavy chain constant region.
A nucleic acid molecule encoding the antibody fragment of claim 1.
According to claim 4,
The nucleic acid molecule is a nucleic acid molecule comprising the following regions.
A heavy chain variable region (VH) having a nucleotide sequence represented by SEQ ID NO: 7;
A heavy chain constant region (CH1) having a nucleotide sequence represented by SEQ ID NO: 9;
A light chain variable region (VL) having a nucleotide sequence represented by SEQ ID NO: 8; And,
Light chain constant region (CL) having the nucleotide sequence represented by SEQ ID NO: 10.
A recombinant vector comprising the nucleic acid molecule of claim 4.
E. coli transformed with the recombinant vector of claim 6.
The culture medium or culture filtrate of E. coli of claim 7.
The antibody fragment of claim 1; And a drug-linker binding compound.
The method of claim 9,
The drug-linker binding compound is a compound comprising maleimide, cathepsin cleavable linker, spacer, and MMAE (Monomethyl auristatin E), an antibody-drug conjugate.
The method of claim 10,
The linker is a valine (Val) -citrulline (Cit) peptide linker, antibody-drug conjugate.
The method of claim 10,
The spacer is para-aminobenzylcarbamoyl (PAB), an antibody-drug conjugate.
A pharmaceutical composition for the prevention or treatment of cancer, comprising the antibody fragment of any one of claims 1 to 12, a nucleic acid molecule, an E. coli culture, a culture filtrate, or an antibody-drug conjugate.
The method of claim 13
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, larynx cancer, head and neck cancer, colon cancer, ovarian cancer, rectal cancer, colon cancer, vaginal cancer, small intestine cancer, A pharmaceutical composition characterized by being selected from the group consisting of endocrine cancer, thyroid cancer, parathyroid cancer, ureteral cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone marrow cancer.
The antibody fragment of claim 1; And,
A method of preparing an antibody-drug conjugate by reacting a drug-linker binding compound.
The method of claim 15,
The drug-linker binding compound is a compound comprising maleimide, cathepsin cleavable linker, spacer, and monomethyl auristatin E (MMAE).
The method of claim 16,
The linker is a valine (Val) -citrulline (Cit) peptide linker.
The method of claim 16,
Wherein the spacer is para-aminobenzylcarbamoyl (PAB).
An antibody-drug conjugate produced by the method of claims 15-18.
The antibody fragment, nucleic acid molecule, E. coli culture medium, culture filtrate, or antibody-drug conjugate of any one of claims 1 to 12; And, Pharmaceutically acceptable carrier; Pharmaceutical composition for the prevention or treatment of cancer comprising a.

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