WO2017176007A1 - Antibody-drug conjugate comprising modified antibody - Google Patents

Abstract

The present invention relates to: an antibody-drug conjugate in which a modified antibody, containing a motif of a specific structure at an end of the antibody, is coupled to a drug through a linker; and a composition containing the same and, specifically, to: a modified antibody-drug conjugate (mADC) comprising a modified antibody coupled to the C-terminus of heavy chain or light chain so as to have a remarkably increased drug conjugation yield; and a composition containing the same.

Description

Antibody-Drug Conjugates Including Modified Antibodies

The present invention relates to an antibody-drug conjugate in which a modified antibody comprising a motif of a specific structure at the terminal of the antibody is bound to a drug through a linker, and specifically to a drug linked to the heavy or light chain C-terminus. A modified Antibody-Drug Conjugate (mADC) conjugate comprising a modified antibody having a significantly increased conjugation yield and a composition comprising the same.

Drugs used for chemotherapy often show toxicity, especially bone marrow, mucosa, and neurotoxicity. Therefore, there is a need for the development of an anticancer agent that is safer while showing strong anticancer activity and specificity to cancer cells. Anti-cancer drugs that act only on cancer cells and reduce side effects are being developed in various ways.

In this regard, therapies using antibodies that are specifically expressed in specific diseases, that is, antibodies that specifically bind to antigens, are currently being actively studied among biopharmaceuticals. In particular, methods for diagnosing and treating tumors using antibodies, such as anti-cancer antibodies, which identify tumor-associated antigens specifically expressed on the surface of cancer cells, bind to them, and inhibit cell growth or induce death, are now widely used. Is also a very bright technology field.

However, these anticancer antibodies have very high target specificities, but the killing effect of cancer cells is often lower than that of conventional cytotoxic drugs (anticancer drugs), so they may be used in combination with cytotoxic drugs and other cell proliferation inhibitors. It is often used as a combination therapy. Anticancer drugs show significantly higher cytotoxicity than anticancer antibodies, but have very high side effects compared to antibody therapeutics because of their low target specificity to cancer cells. Therefore, the combination therapy of anticancer antibody and anticancer drug shows a higher therapeutic effect than when each drug is administered separately, but has the fundamental limitation that side effects of anticancer drug are always accompanied.

In addition, due to its very high cytotoxicity, among the anticancer drugs, drugs that can be used alone as anticancer drugs are limited to relatively low toxicity Taxol or cisplatin-based drugs. Most anticancer drugs with high cytotoxicity are virtually impossible to prescribe as single drugs because of their very high cytotoxicity. By binding an anticancer drug that cannot be used as a prescription alone to an antibody having a very high target specificity for cancer cells, the anticancer drug can be delivered only to the target cancer cells without the side effects of normal cells. Therefore, antibody-drug conjugates have been in the spotlight as a method for improving the therapeutic efficacy of anti-cancer drugs that cannot be used conventionally.

Antibody-drug conjugates currently on the market include Adcetris®, a treatment for Hodgkin's lymphoma, and Kadcyla®, a treatment for metastatic breast cancer. These antibody-drug conjugates are conjugated to tubulin inhibitors that inhibit the growth and division of cancer by conjugating to the tubule, an intracellular microtubule involved in cell division, thereby inhibiting cell division. Has These antibody-drug conjugates bind the drug to the lysine originally possessed by the antibody (cadcyla), or to the cysteine group that reduced the disulfide group linking the heavy chain-heavy chain or heavy-light chain that maintains the structural stability of the antibody. (Adsetless). The anticancer drug conjugation method used in the first-generation antibody-drug conjugate does not control the number of conjugated drugs per antibody, and even in antibody-drug conjugates having the same number of drug conjugates, the positional conjugations of the drugs are different from each other. isomer) is generated. The number of conjugates of drugs per antibody affects not only the cytotoxicity of antibody-drug conjugates but also the stability of antibody-drug conjugates and the possibility of aggregate formation.In general, the more conjugated drugs, the less stable the antibody itself and the formation of aggregates. The chances are high.

In addition, since the first generation antibody-drug conjugate does not control the number of drugs conjugated per antibody, the number of conjugated drugs per antibody has an average value. For example, in the case of cascaila, the number of drugs conjugated to the antibody may range from 1 to 8, and on average 3.5 drug conjugates. Even in the same number of drug conjugates, the properties of the antibody-drug conjugate may vary depending on the location of the drug conjugate. When the drug is conjugated near the Fab or near the hinge of the Fc, there is a possibility that the stability of the antibody or antigen-antibody reactivity may be impaired due to a difference in the binding force between the antigen and the Fcγ or FcRn receptor. As the number of conjugated drugs per antibody increases, the number of positional isomers with different conjugated positions of the drug increases proportionally, and this result may have a great influence on maintaining the consistent characteristics of the antibody-drug conjugate according to the production batch. .

Therefore, in recent years, various conjugation techniques for site-specific drug conjugation have been developed to ameliorate the disadvantages of the first generation of antibody-drug conjugates. Genentech has developed a technique for conjugating drugs using ThioMab technology, which introduces a cysteine group by substituting an amino acid of an antibody, and then conjugates the drug in a specific position to the introduced cysteine group. This demonstrates that the site-specific drug conjugated antibody-drug conjugate has superior activity in vivo than that produced by the first-generation antibody-drug conjugate (Junutula et al., Nature Biotechnology, 2008, 26, 925-932). Site specific conjugation has also been tried by several companies in a drug conjugation manner using protein enzymes with very high substrate specificity.

Site-specific conjugation using formylglycine-generating enzyme (Drake et al., Bioconjugate Chemistry, 2014, 25, 1331-1341), conjugation using glutamine transferase (Strop et al. , Chemistry & Biology, 2013, 20, 161-167) and the like have been reported that the desired number of drugs can be selectively conjugated to the antibody. However, the conjugation using such a protein enzyme has disadvantages such as conjugation in the presence of an excess of drug at 37 ° C. for 72 hours, or the need for a high concentration of conjugation enzyme.

Conjugation using non-natural amino acid, another site-specific conjugation method, is a natural amino acid that synthesizes tRNA that can introduce artificial amino acids into proteins through mutants of tRNA synthase. It is based on a technique that can introduce side-chains into proteins (Wang et al., Proc. Natl. Acad. Sci. USA, 2003, 100, 56-61). This allows the drug to be conjugated at a desired position in such a way as to perform site-specific conjugation to the residues of the artificial amino acids introduced (zimmerman et. Al, Bioconjugate Chem. 2014, 25, 351-361). However, these methods require high-level tasks that require high levels of genetic engineering to regulate the translation pathway.

Such site-specific conjugation is very time-consuming and costly for site-specific conjugation reactions, such as modification of transcriptional systems using complex genetic engineering methods or addition of excess conjugation enzymes. Antibodies act as carriers for the specific delivery of anticancer drugs to cancer cells, and the drug is a basic concept of an antibody-drug conjugate that needs to be stably conjugated to an antibody until it is delivered to cancer cells. The necessity of producing antibody-drug conjugates using the same complex and time-consuming and expensive conjugation method is questionable. The location-specific conjugation in the production of antibody-drug conjugates, and the economic conjugation method, which is excellent in economical efficiency, overcome the limitations of the existing method of conjugation of the first generation antibody-drug conjugates as well as the newly proposed location-specific conjugation method. It can be a great alternative.

Antibody-drug conjugates show superior in vitro and in vivo efficacy over existing antibody pharmaceuticals. However, some clinical results for using antibody-drug conjugates as primary therapeutics failed to show significant clinical utility differences compared to the combination therapy of monoclonal antibody and chemosynthetic drugs (http: // www.roche.com/media/store/releases/med-cor-2014-12-19.htm). These results can be a significant constraint on the economic utility of antibody-drug conjugates, given that antibody-drug conjugates require significantly higher therapeutic costs than synthetic drugs as well as monoclonal antibody therapeutics. In this respect, the above-mentioned location-specific conjugation methods have serious problems in providing economic utility that can be used as a primary therapeutic agent.

Under this technical background, the inventors of the present application recognize that there is an urgent need for the development of antibody-drug conjugates that perform site-specific conjugation reactions, and which are superior in economic efficiency over the proposed conjugation reactions, and to solve these problems. Invented by a modified antibody comprising a peptide motif containing a metal ion binding motif, a modified antibody having a position-specific conjugation and at the same time superior drug conjugation yield. Through this invention, as well as the superior in vivo anticancer effect of the antibody-drug conjugate according to the site-specific conjugation method, it is to provide an antibody-drug conjugate that can be produced more economically.

Summary of the Invention

In order to solve the above problems, the present invention provides a antibody (hereinafter modified antibody) of the type having a specific binding motif to the parent antibody having a drug binding site. These modified antibodies modify modified antibodies comprising existing metal ion binding peptide motifs to provide modified antibodies with higher drug conjugate yields while maintaining the properties of site specific conjugation. The present invention also provides an antibody-drug conjugate using the modified antibody and a method for producing an anticancer drug.

In order to achieve the above object, the present invention provides an antibody-drug conjugate wherein a modified antibody comprising a motif represented by the following structural formula (1) at the terminal of the antibody is bound to the drug via a linker:

Structural Formula (1)

_{X a - [M motif1] n1} -X b - [M motif2] n2

_Wherein M _motif1 or M _motif2 each independently include any one of a sequence consisting of ACGHA (SEQ ID NO: 1), AHGCA (SEQ ID NO: 2), AXGHA (SEQ ID NO: 3), and AHGXA (SEQ ID NO: 4), wherein X in SEQ ID NO: 3 or 4 includes amino acid residues other than cysteine,

X _a And X _b is a peptide consisting of 0 to 20 amino acid residues each independently selected from the group consisting of A (alanine), S (serine), G (glycine),

n1 and n2 are integers of 1-10, respectively.

The present invention also provides a composition for preventing or treating cancer comprising the antibody-drug conjugate.

Figure 1. Samples conjugated to MC-vc-PAB-MMAE with FM2, FM2b (lot no: 4390f), FM2b (lot no: 5698f), FM2a, FM2L, and FM1 antibody variants of the antibody targeting the Folate receptor SDS-PAGE. The drug is conjugated to a motif containing a cysteine residue introduced into the heavy chain of the antibody, and appears on the SDS-PAGE as two bands in the heavy chain near 50 kDa.

Figure 2. SDS-PAGE of a sample conjugated with the antibody variants of FM2, FM2b, FM2a, FM2L, FM1 targeting the Folate receptor to br-vc-PAB-MMAE, a drug-linker conjugate linked to MMAE via bromoacetamide . The drug is conjugated to a motif containing a cysteine residue introduced into the heavy chain of the antibody, and appears on the SDS-PAGE as two bands in the heavy chain near 50 kDa.

Figure 3. Cell growth inhibition test using KB-cells overexpressed Folate receptor. Parent antibody Fwt and modified antibody-drug conjugate FM2-D2 (modified antibody-drug conjugate with DAR 2 in modified antibody-drug conjugate of modified antibody FM2 and MMAE), FM2b-D2 (or FM2b-S-D2) Comparison of cell growth inhibitory ability of (modified antibody-drug conjugate having DAR 2 in modified antibody-drug conjugate of FM2b-S and MMAE modified antibody). FM2-D2 and FM2b-D2 show almost the same intracellular activity.

Figure 4. Cell growth inhibition test using KB-cells overexpressed Folate receptor. Cell growth inhibition of FM2b-S-D2, FM2b-F-D2, and FM2b-Y-D2. FM2b-S-D2, FM2b-F-D2, and FM2b-Y-D2 show almost the same intracellular activity.

Figure 5. FM2b-S-D2, FM2b-F-D2, FM2b-K-D2, FM2b-Y-D2 after storage for 0, 1, 3, 5, 7, 14 days at 25 ℃ and 50 ℃, drug Determination of the change in the number of joints DAR and aggregate. Almost no changes of DAR and monomer were observed at 25 ℃, but at 50 ℃, the content of DAR2 was decreased and aggregates formed over time. However, no differences in DAR changes and aggregate formation were observed for each variant.

Detailed description of the invention and preferred Embodiment

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The antibody-drug conjugate must be stably bound to the antibody until the anti-cancer drug is delivered to the target cancer cell. Anticancer drugs delivered to target cancer cells must be released from the antibody to induce the death of cancer cells. To this end, the anti-cancer drug must be stably deficient in the antibody by using a linker and have a structure of a drug-linker having sufficient cytotoxicity to induce cancer cell death when released from cancer cells. In addition, the drug should show the anti-potency of the antibody-drug conjugate and its uniformity in the manufacturing process by site-specific conjugation to the antibody.

To this end, the inventors of the present application in Korea Patent No. 1541764 introduced a peptide containing a metal ion binding motif at the C-terminus of the antibody can be drug-specifically conjugated, the constant antibody to drug conjugation ratio Showed that it can achieve. This demonstrates that the drug can be site-specifically conjugated to the modified antibody and at the same time expect high target specificity and drug effect while maintaining the parental antibody characteristics. However, the significantly higher production cost compared to synthetic drugs or monoclonal antibody therapeutics is urgently needed to expand the economic efficiency through improved drug yield.

Accordingly, in the present invention, the modified antibody comprising a peptide motif containing a metal ion binding motif is improved, that is, the ratio of conjugation of the drug conjugated to the antibody according to the arrangement and primary structure of the metal ion binding motif introduced into the terminal of the parent antibody. We tried to prove that this could increase significantly.

In one aspect, the present invention relates to an antibody-drug conjugate wherein a modified antibody comprising a motif represented by the following structural formula (1) at the terminal of the antibody is bound to a drug via a linker.

Structural Formula (1)

_{X a - [M motif1] n1} -X b - [M motif2] n2

_Wherein M _motif1 or M _motif2 each independently include any one of a sequence consisting of ACGHA (SEQ ID NO: 1), AHGCA (SEQ ID NO: 2), AXGHA (SEQ ID NO: 3), and AHGXA (SEQ ID NO: 4), wherein X in SEQ ID NO: 3 or 4 includes amino acid residues other than cysteine,

X _a and X _b are each independently a peptide consisting of 0 to 20 amino acid residues selected from the group consisting of A (alanine), S (serine) and G (glycine),

n1 and n2 are integers of 1-10, respectively.

The motif represented by Structural Formula (1) is a peptide including a CGH motif which is a metal ion binding motif, and the CGH motif has the structure of Formula 1 below.

In Chemical Formula 1, M means a metal ion, and R is an amino acid residue except cysteine, particularly alanine is preferable.

In the motif according to the present invention, M _motif1 or M _motif2 includes ACGHA (SEQ ID NO: 1) in which alanine is located at the C-terminal and N-terminal, respectively, or AXGHA (SEQ ID NO: 3) in which a cysteine is substituted with an amino acid residue other than cysteine. do. N-terminus and C-terminus in ACGHA (SEQ ID NO: 1) or AXGHA (SEQ ID NO: 3) for M _motif1 or M _motif2 in the motif according to the present invention, even if the N-terminus and C-terminus are still changed. Also included are AHGCA (SEQ ID NO: 2) or AHGXA (SEQ ID NO: 4), which have been terminated.

In the motif according to the present invention, M _motif1 or M _motif2 may each include the same sequence and may include different sequences from each other.

In one embodiment according to the invention, when M _{motif 1} or M _{motif 2} corresponds to AXGHA or AHGXA, wherein X is serine (S), alanine (A), threonine (T), tyrosine (Y), aspartic acid It may be an amino acid residue selected from the group consisting of (D), lysine (K) and phenylalanine (F).

In one embodiment according to the present invention, it was _confirmed that the M _motif2 includes AXGHA (SEQ ID NO: 3), and X may show higher drug conjugation ability than the case of including ACGHA when amino acid residues other than cysteine are included. . The M _motif2 is AXGHA, where X is, for the amino acid residue Examples of the non-cysteine, serine (S), alanine (A), threonine (T), tyrosine (Y), aspartic acid (D), lysine (K) and It may be an amino acid residue selected from the group consisting of phenylalanine (F).

X _a is an amino acid residue present at the 5 'end of the motif M _motif1 , which may be a peptide positioned to connect with the terminal of the antibody, and may be composed of A (alanine), S (serine), and G (glycine). The amino acid residue selected from the group is a peptide consisting of 0-20. When the amino acid residue of X _a is 0, the motif does not include X _a and may be in a form in which M _motif1 of the motif directly bonds to the antibody. May be one or more, two or more, three or more, four or more, five or more amino acid residues of X _a , for example, 2-20, 2-18, 2-16, 2-14 , 2-12, 2-10, 2-8, 2-6, 2, 3, 4, 5, 6, 7, 8, 9, 10.

X _b is a linker for linking M _motif1 and M _motif2 and is a peptide consisting of 0 to 20 amino acid residues selected from the group consisting of A (alanine), S (serine) and G (glycine). If the amino acid residue of X _b is 0, the motif does not include X _b and may be in a form in which M _{motif 1} and M _{motif 2} are directly connected. X _b of the amino acid residues in one or more, two or more, three or more, four or more, may be more than five, e.g., 2-20, 2-18, 2-16, 2-14 , 2-12, 2-10, 2-8, 2-6, 2, 3, 4, 5, 6, 7, 8, 9, 10.

n1 and n2 represent the number of repetitions of M _motif1 and M _motif2 , respectively, and are an integer of 1 to 10. N1 and n2 may each be 1, wherein M _motif1 And M _motif2 to ACGHA (SEQ ID NO: 1), AHGCA (SEQ ID NO: 2), AXGHA (SEQ ID NO: 3) and AHGXA (SEQ ID NO: 4) any of the sequences consisting of comprises a linker for X _b without a linker or X _b Can be connected. For _{example, [M motif1] n1 -X b} - [M motif2] n2 structure is the absence of a linker X _b ACGHAACGHA (SEQ ID NO: 5), ACGHAAHGCA (SEQ ID NO: 6), ACGHAAXGHA (SEQ ID NO: 7), ACGHAAHGXA ( SEQ ID NO: 8), AHGCAAHGCA (SEQ ID NO: 9), AHGCAACGHA (SEQ ID NO: 10), AHGCAAXGHA (SEQ ID NO: 11), AHGCAAHGXA (SEQ ID NO: 12), AXGHAAXGHA (SEQ ID NO: 13), AXGHAACGHA (SEQ ID NO: 14), AXGHAAHGCA Number 15), AXGHAAHGXA (SEQ ID NO: 16), AHGXAAHGXA (SEQ ID NO: 17), AHGXAACGHA (SEQ ID NO: 18), AHGXAAHGCA (SEQ ID NO: 19), or AHGXAAXGHA (SEQ ID NO: 20), and C ( Motifs (essentially cysteine) may be selected as M _motif1 and M _motif2 .

When X _b is present, a peptide consisting of 1 to 20 amino acid residues at the 5 'end of the amino acid sequences may be further included. In this case, X may be selected from the group consisting of serine (S), alanine (A), threonine (T), tyrosine (Y), aspartic acid (D), lysine (K) and phenylalanine (F).

[M _motif1 ] _n1 -X _b- [M _motif2 ] The _n2 structure contains a serine at position X in SEQ ID NOs 7, 8,11 to 20, including X if there is no linker of X _b , for example if X is serine ACGHAASGHA (SEQ ID NO: 21), ACGHAAHGSA (SEQ ID NO: 22), AHGCAASGHA (SEQ ID NO: 23), AHGCAAHGSA (SEQ ID NO: 24), ASGHAASGHA (SEQ ID NO: 25), ASGHAACGHA (SEQ ID NO: 26), ASGHAAHGCA (SEQ ID NO: 27), ASGHAAHGSA (SEQ ID NO: 28), AHGSAAHGSA (SEQ ID NO: 29), AHGSAACGHA (SEQ ID NO: 30), AHGSAAHGCA (SEQ ID NO: 31), or AHGSAASGHA (SEQ ID NO: 32).

[M _motif1 ] _n1 -X _b- [M _motif2 ] The _n2 structure is ACGHAAAGHA (SEQ ID NO: 33), ACGHAAHGAA (SEQ ID NO: 34), AHGCAAAGHA (if X is alanine (A) when there is no linker of X _b ) SEQ ID NO: 35), AHGCAAHGAA (SEQ ID NO: 36), AAGHAAAGHA (SEQ ID NO: 37), AAGHAACGHA (SEQ ID NO: 38), AAGHAAHGCA (SEQ ID NO: 39), AAGHAAHGAA (SEQ ID NO: 40), AHGAAAHGAA (SEQ ID NO: 41), AHGAAACGHA (SEQ ID NO: 41) Number 42), AHGAAAHGCA (SEQ ID NO: 43), or AHGAAAAGHA (SEQ ID NO: 44).

[M _motif1 ] _n1 -X _b- [M _motif2 ] The _n2 structure is ACGHAATGHA (SEQ ID NO: 45), ACGHAAHGTA (SEQ ID NO: 46), AHGCAATGHA (if X is a threonine (T) when there is no linker of X _b ) SEQ ID NO: 47), AHGCAAHGTA (SEQ ID NO: 48), ATGHAATGHA (SEQ ID NO: 49), ATGHAACGHA (SEQ ID NO: 50), ATGHAAHGCA (SEQ ID NO: 51), ATGHAAHGTA (SEQ ID NO: 52), AHGTAAHGTA (SEQ ID NO: 53), AHGTAACGHA (SEQ ID NO: 53) Number 54), AHGTAAHGCA (SEQ ID NO: 55), or AHGTAATGHA (SEQ ID NO: 56).

[M _motif1 ] _n1 -X _b- [M _motif2 ] The _n2 structure is the absence of a linker of X _b , for example, if X is tyrosine (Y), ACGHAAYGHA (SEQ ID NO: 57), ACGHAAHGYA (SEQ ID NO: 58), AHGCAAYGHA (SEQ ID NO: Number 59), AHGCAAHGYA (SEQ ID NO: 60), AYGHAAYGHA (SEQ ID NO: 61), AYGHAACGHA (SEQ ID NO: 62), AYGHAAHGCA (SEQ ID NO: 63), AYGHAAHGYA (SEQ ID NO: 64), AHGYAAHGYA (SEQ ID NO: 65), AHGYAACGHA (SEQ ID NO: 65) 66), AHGYAAHGCA (SEQ ID NO: 67), or AHGYAAYGHA (SEQ ID NO: 68).

[M _motif1 ] _n1 -X _b- [M _motif2 ] The _n2 structure is the absence of a linker of X _b , for example, if X is aspartic acid (D), ACGHAADGHA (SEQ ID NO: 69), ACGHAAHGDA (SEQ ID NO: 70), AHGCAADGHA ( SEQ ID NO: 71), AHGCAAHGDA (SEQ ID NO: 72), ADGHAADGHA (SEQ ID NO: 73), ADGHAACGHA (SEQ ID NO: 74), ADGHAAHGCA (SEQ ID NO: 75), ADGHAAHGDA (SEQ ID NO: 76), AHGDAAHGDA (SEQ ID NO: 77), AHGDAACGHA (SEQ ID NO: 77) Number 78), AHGDAAHGCA (SEQ ID NO: 79), or AHGDAADGHA (SEQ ID NO: 80).

_{[M motif1] n1 -X b -} [M motif2] n2 structure is the absence of a linker of X _b, for example when X is lysine (K) ACGHAAKGHA (SEQ ID NO: 81), ACGHAAHGKA (SEQ ID NO: 82), AHGCAAKGHA (SEQ ID NO: Number 83), AHGCAAHGKA (SEQ ID NO: 84), AKGHAAKGHA (SEQ ID NO: 85), AKGHAACGHA (SEQ ID NO: 86), AKGHAAHGCA (SEQ ID NO: 87), AKGHAAHGKA (SEQ ID NO: 88), AHGKAAHGKA (SEQ ID NO: 89), AHGKAACGHA (SEQ ID NO: 90), AHGKAAHGKA (SEQ ID NO: 91), or AHGKAAKGHA (SEQ ID NO: 92).

_{[M motif1] n1 -X b -} [M motif2] n2 structure is the absence of a linker X _b For example, if X is phenylalanine (F) ACGHAAFGHA (SEQ ID NO: 93), ACGHAAHGFA (SEQ ID NO: 94), AHGCAAFGHA (SEQ ID NO: Number 95), AHGCAAHGFA (SEQ ID NO: 96), AFGHAAFGHA (SEQ ID NO: 97), AFGHAACGHA (SEQ ID NO: 98), AFGHAAHGCA (SEQ ID NO: 99), AFGHAAHGFA (SEQ ID NO: 100), AHGFAAHGFA (SEQ ID NO: 101), AHGFAACGHA (SEQ ID NO: 102), AHGFAAHGFA (SEQ ID NO: 103), or AHGFAAFGHA (SEQ ID NO: 104).

When X _b is present, a peptide consisting of 1 to 20 amino acid residues may be further included at the 6th position at the 5 'end in the amino acid sequences of SEQ ID NOs: 5 to 104. In this case, X may be selected from the group consisting of serine (S), alanine (A), threonine (T), tyrosine (Y), aspartic acid (D), lysine (K) and phenylalanine (F).

N1 and n2 may each be two or more, for example, when n1 and n2 are 2, _AC MHA (SEQ ID NO: 1), AHGCA (SEQ ID NO: 2), and _AXGHA if M _{motif 1} and M _{motif 2} each include the same amino acid sequence Either one consisting of (SEQ ID NO: 3) and AHGXA (SEQ ID NO: 4) includes a sequence in which each is repeated twice, or a different sequence such as ACGHA and AHGCA, ACGHA and AXGHA, ACGHA and AHGXA, AHGCA and ACGHA , AHGCA and AXGHA, AHGCA and AHGXA, AXGHA and ACGHA, AXGHA and AHGCA, AXGHA and AHGXA, AHGXA and ACGHA, AHGXA and AHGCA, or AHGXA and AXGHA, respectively. When n1 and n2 are 3-10, M _motif1 and M _motif2 are each any one of a sequence consisting of ACGHA (SEQ ID NO: 1), AHGCA (SEQ ID NO: 2), AXGHA (SEQ ID NO: 3), and AHGXA (SEQ ID NO: 4). It may include the same sequence corresponding to one, or may be repeated 3-10 times each including a different sequence.

Preferably, n1 and n2 may each be 1, and there may not be a linker of X _b , wherein the motif according to the present invention is one or more sequences selected from the group consisting of SEQ ID NOs: 5 to 104, for example. It may include.

The motif may bind to the heavy or light chain C-terminus of the antibody, specifically the heavy chain C-terminus, thereby providing a modified antibody and a drug conjugate comprising the same, which significantly increase the drug conjugation yield. The yield of the antibody-drug conjugate can be increased by increasing the yield of the drug. Drugs conjugated in high yield can be specifically delivered to target cancer cells by modified antibodies, thereby increasing the therapeutic effect, and lowering the production cost of the antibody-drug conjugate therapeutics due to the high yield of conjugated antibody-drug conjugates.

The motif may be directly bonded in the form of a fusion through an amino bond with the parent antibody, or in a form in which the terminal functional group of the parent antibody and the terminal functional group in the motif are chemically bonded, or mediated to a linker connecting the terminal functional group and the drug in the motif. Linker-mediated forms of coupling are also possible.

The linker may be in the form of linking a drug with a specific residue in a motif, and may have a reactive site having an electrophilic group that reacts to a nucleophilic residue (eg, cysteine) present on a motif among modified antibodies. The linker may comprise, for example, reactive functional groups, amino acids and self-cutting spacers that bind to the motif.

The functional groups are i) maleimide groups, acetamide groups, or derivatives thereof, ii) aziridine groups, arylhalides, acryloyl groups, or derivatives thereof, iii) alkylation reactors, arylation reactors, pyridyl disulfides, thionitrobenzo Ikic acid, or a derivative thereof. Specifically, the linker may specifically include, for example, i) a maleimide group or a derivative thereof-valine-citurulline-para-aniline benzoic acid (PABA); Or ii) an acetamide group or derivative thereof-valine-citurulline-paraaniline benzoic acid (PABA), but is not limited thereto.

The binding of the moiety to the drug via the linker may be carried out using known methods such as alkylation, disulfide interchange and transthioesterification. This allows the antibody and the drug to be conjugated via the thiol group of the cysteine residue in the motif.

In one embodiment, for maleimide groups used for thiol-linker linkages, the nucleophilic reactivity of the thiol of the cysteine residue to the maleimide group is present in the protein, such as other amino acid functional groups, such as amino groups of lysine residues or Because it is about 1,000 times higher than the N-terminal amino group, it can be used to specifically bind to cysteine. Thus, the modified antibody-drug conjugates via maleimide groups, derivatives thereof or acetamide groups or derivatives thereof, such as bromo acetamide groups, iodo acetamide groups, can be seen that cysteine is bound to the drug by thioether linkage. .

The antibody may be one or more selected from the group consisting of monoclonal antibodies, bispecific antibodies, chimeric antibodies, human antibodies and humanized antibodies. Moreover, both modified antibodies, such as a bispecific antibody, antibody fragments, etc. can be used. 'Antibody fragments' refer to fragments that have at least the binding function to the antigen, and are single-chain antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F (ab') 2 fragments, Fd, scFv, domain antibodies, mini Body, single chain antibody (scAb), derivatives of antibody constant regions, phosphorus based on protein scaffolds and the like.

In some cases, the antibody may be selected from the group consisting of IgA, IgD, IgE, IgG and IgM.

The antibody specifically includes cancer specific antigens, cell surface receptor proteins, cell surface proteins, transmembrane proteins, signaling proteins, cell survival regulators, cell proliferation regulators, molecules associated with tissue development or differentiation, lymphokines, cytokines. May have binding capacity and specificity for Cain, a molecule involved in cell cycle regulation, an angiogenesis related molecule, or an angiogenesis related molecule, for example, (1) BMPR1B (bone morphogenetic protein receptor-IB type, gene Bank grant number NM_001203);

(2) E16 (LAT1, SLC7A5, GenBank Accession No. NM_003486);

(3) STEAP1 (six transmembrane epithelial antigens of the prostate, Genbank Accession No. NM_012449);

(4) 0772P (CA125, MUC16, GenBank Accession No. AF361486);

(5) MPF (MPF, MSLN, SMR, megakaryocyte enhancing factor, mesothelin, Genbank Accession No. NM — 005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, Solute Carrier Family 34 (Sodium Phosphate), Member 2, Type II Sodium-Dependent Phosphate Transporter 3b, GenBank Accession No. NM_006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, Sema Domain, 7 Thrombospondin Repeats (Type 1 and Similar Type 1), Transmembrane Domain (TM) And short cytoplasmic domain, (semaphorin) 5B, Genbank Accession No. AB040878);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genebank Accession No. AY358628);

(9) ETBR (endothelin type B receptor, Genbank Accession No. AY275463);

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genebank Accession No. NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, Prostate Cancer Related Gene 1, Prostate Cancer Related Protein 1, Prostate 6 Transmembrane Epithelial Antigen 2, 6 Transmembrane Prostate Protein, Genebank Authorization number AF455138);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subgroup M, member 4, Genbank Accession No. NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP — 003203 or NM — 003212);

(14) CD21 (CR2 (complementary receptor 2) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792 Genbank Accession No. M26004);

(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29, Genbank Accession No. NM — 000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchoring protein 1a), SPAP1B, SPAP1C, GenBank Accession No. NM_030764);

17 HER2 (Genbank approval number M11730)

(18) ErbB receptors selected from EGFR, HER3 and HER4

(19) NCA (Genbank Accession No. M18728);

(20) MDP (GenBank Accession No. BC017023);

(21) IL20Rα (GenBank Accession No. AF184971);

(22) Brevican (Genbank Accession No. AF229053);

(23) EphB2R (GenBank Accession No. NM_004442);

(24) ASLG659 (Genbank Accession No. AX092328);

(25) PSCA (Genbank Accession No. AJ297436);

(26) GEDA (Genbank Accession No. AY260763);

(27) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, NP_443177.1);

(28) CD22 (B-cell receptor CD22-B isotype, NP-001762.1);

(29) CD79a, CD79A, CD79α, and immunoglobulin-associated alpha, which are covalently interacting with CD79a (Ig beta (CD79B) and forming complexes on the surface with IgM molecules, are signals involved in B cell differentiation Forwarded, Genbank approval number NP_001774.1);

(30) CXCR5 (Bucket Lymphoma Receptor 1, a G protein coupled receptor activated by CXCL13 chemokines, acts on lymphocyte migration and humoral defense, participates in HIV-2 infection, and develops AIDS, lymphoma, myeloma and leukemia Considered to be related to, Genbank approval number NP_001707.1);

(31) HLA-DOB (beta subunit of MHC class II molecules (Ia antigen), binding to peptides and presenting in CD4 + T lymphocytes, Genbank Accession No. NP — 002111.1);

(32) P2X5 (purine receptor P2X ligand-gate ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, the lack of which may contribute to the pathophysiology of idiopathic detrusor instability Yes, Genbank approval number NP_002552.2);

(33) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank Accession No. NP — 001773.1);

(34) Lymphocyte antigen 64 (RP105), a type I membrane protein of the LY64 (leucine rich repeat (LRR) family), modulates B cell activation and apoptosis, and its loss of function is attributed to systemic lupus erythematosus patients. Associated with increased disease activity, GenBank Accession No. NP_005573.1);

(35) FcRH1 (Fc receptor-like protein 1, a putative receptor for immunoglobulin Fc domains containing C2 Ig-like and ITAM domains, may be involved in B lymphocyte differentiation, Genbank accession number NP_443170.1) ;

(36) IRTA2 (associated gene deregulation by immunoglobulin macrophage receptor translocation, a putative immunoreceptor capable of acting on B cell development and lymphoma development, occurs in some B cell malignancies, Genbank approval number NP_112571.1);

(37) TENB2 (estimated transmembrane proteoglycan associated with EGF / Heregulin family of growth factors and follistatin, Genbank Accession No. AF179274);

(38) MAGE-C1 / CT7 (testicular cancer overexpressed protein);

(39) androgen receptor, PTEN, human kallikrein-related peptidase 3 (protein overexpressed in prostate cancer);

(40) CD20;

(41) CD30;

(42) CD33;

(43) CD52;

(44) EpCam;

(45) CEA;

(46) gpA33;

(47) Mucins;

(48) TAG-72;

(49) Carbonic anhydrase IX;

(50) PSMA;

(51) folate receptor (a family of proteins expressed by the FoLR gene, which has high binding capacity with Folic acid and carries 5-methyltetrahydrofolate into cells);

(52) gangliosides (GD2, GD3, GM2);

(53) carbohydrate Lewis-Y;

(54) VEGF;

(55) VEGFR;

(56) aVb3;

(57) a5b1;

(58) ERB3;

(59) c-MET;

(60) EphA3;

(61) TRAIL-R1, TRAIL-R2;

(62) RANKL;

(63) FAP; And

(64) may have binding capacity to one or more targets selected from the group consisting of tenascin, but is not limited thereto.

In one embodiment according to the present invention, an antibody (Farletuzumab) that binds to a folate receptor comprising a heavy chain of SEQ ID NO: 115 and a light chain of SEQ ID NO: 116 was used as a parent antibody. By introducing a metal ion binding motif to the heavy chain terminal of the parent antibody to prepare a variety of modified antibodies according to the sequence and placement of the peptide motif, the difference in the conjugate ratio of the drug according to the modified antibody was measured. The modified antibody may include one or more heavy chains selected from the group consisting of SEQ ID NOs: 117 to 121.

In another embodiment according to the present invention, an antibody (Trastuzumab) that specifically binds to Her2 was used as the parent antibody. By introducing a metal ion binding motif to the heavy chain terminal of the parent antibody to prepare a variety of modified antibodies according to the sequence and placement of the peptide motif, the difference in the conjugate ratio of the drug according to the modified antibody was measured.

As a result, it was confirmed that the antibody-drug conjugate according to the present invention had a similarly high drug conjugation yield when the motifs were introduced into Farletuzumab and Trastuzumab. Therefore, the motif according to the present invention can be utilized as a platform technology for binding a drug to an antibody in antibody-drug conjugate preparation regardless of the type of antibody.

In one embodiment, the antibody may comprise the variable region of the parent or modified antibody and CH1, CH2 and CH3 of IgG2 or IgG4. For example, the VH and VL of Farletuzumab, Trastuzumab or its modified antibodies can be used and include CH1, CH2, CH3 of IgG2 or IgG4. For example, the variable region of the Farletuzumab antibody may include a heavy chain variable region of SEQ ID NO: 122 and / or a light chain variable region of SEQ ID NO: 123.

In another embodiment, the antibody may comprise a Fab of the parent antibody or a modified antibody and an Fc of IgG2 or IgG4. Specifically, it may include a form in which the Fab portion of Farletuzumab, Trastuzumab or its modified antibody and the Fc portion of IgG2 or IgG4 are fused. For example, the Fab of the Farletuzumab antibody may comprise a heavy chain variable region of SEQ ID NO: 122 and a CH1 containing sequence (SEQ ID NO: 124) and / or a light chain variable region of SEQ ID NO: 123. The Trastuzumab antibody may comprise a heavy chain of SEQ ID NO: 127 and / or a light chain of SEQ ID NO: 128.

The drug combined with the modified antibody in the present invention can be used without limitation any drug that has a therapeutic effect of the disease, and particularly preferred is a drug for treating cancer having a proliferation inhibitory effect of tumor cells.

The drug may be conjugated to the cysteine group or serine group of the motif introduced at the terminal of the modified antibody.

Drugs that may be used in the modified antibody-drug conjugates of the present invention specifically include any compound, moiety or group that has a cytotoxic or cytostatic effect, and (i) a microtubulin inhibitor, mitosis inhibitor, topoiso Chemotherapeutic agents that can function as merase inhibitors, or DNA intercalators; (ii) protein toxins that can function enzymatically; (iii) micro RNA (miRNA), siRNA, shRNA capable of inhibiting the expression of certain oncogenes; Or (iv) radioisotopes.

Such drugs include, for example, maytansinoids, orstatin, aminopterin, actinomycin, bleomycin, thalisomycin, camptocecin, N8-acetyl spermidine, 1- (2 chloroethyl) -1, 2-dimethyl sulfonyl hydrazide, esperamycin, etoposide, 6-mercaptopurine, dolastatin, tricortesene, calicheamicin, taxane, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mito Mycin A, mitomycin C, chlorambucil, duocarmycin, nucleolytic enzymes, toxins derived from bacteria or plants, cisplatin, irinotecan, paclitaxel and docetaxel, but is not limited thereto.

In some cases, the drug may react with an amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, which can react to form covalent bonds with electrophilic groups on the linker and linker reagent, And one or more nucleophilic groups selected from the group consisting of arylhydrazide groups.

In another aspect, the present invention provides a therapeutic composition comprising the antibody-drug conjugate as an active ingredient. The drug of the modified antibody-drug conjugate in the composition is characterized by the use of an antibody conjugate for topical delivery of a drug that kills or inhibits tumor cells in the treatment of cancer, with the drug moiety being targeted to the antibody-antigen into the tumor and into the cell. Enable accumulation

The present invention also provides a method of inhibiting proliferation of target cells by contacting the target cells with cancer, autoimmune, inflammatory or infectious diseases or diseases using the modified antibody-drug conjugate as an active ingredient.

Treatable cancers in the present invention are liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, One or more selected from cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor It doesn't work. As a specific example, a modified antibody-drug conjugate may be contacted in KB cells, which are cancer cells amplified with in vitro folate receptors, to induce cell proliferation inhibition. Therefore, the inhibitory method using the modified antibody-drug conjugate of the present invention as an active ingredient has an effect of killing or reducing the rate of proliferation and inhibiting cells associated with the disease.

Unless otherwise defined in the technical and scientific terms used in the present invention, those having ordinary skill in the art to which the present invention pertains generally have meaning. In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by the following examples, and it will be apparent to those skilled in the art that various modifications or changes can be made within the idea and scope of the present invention.

Example 1 Preparation of Expression Vector pAV4

Expression vector cloning was performed using the pAV4 vector, which was developed and modified for the purpose of using the parent vector pSGHV0 (GenBank Accession No. AF285183) to manufacture antibodies in the industry. The parent vector is overexpressed into cells when a human-derived protein is expressed using bacteria such as Escherichia coli, but in the case of a protein that is difficult to obtain as an active substance, a high concentration of the protein of interest having a physiological activity outside the cell is obtained using animal cells. It is a research vector produced for the purpose of easy purification by expression. However, since there are various limitations to use for production in industry, the vector has been improved to be used in industry in order to use the high expression amount, which is the biggest advantage of this vector for production. In addition, in the case of an antibody, a heavy chain and a light chain must simultaneously express two proteins, so a vector suitable for this purpose has been developed.

Example 2 Vector Preparation of Modified Antibodies Incorporating ACGHA, a Parent Antibody Having a Binding Capability to the Folate Receptor, and a Metal Ion Binding Motif

To prepare a parental antibody (Fwt) vector capable of binding to a folate receptor, the heavy chain of SEQ ID NO: 125 and the light chain coding cDNA of SEQ ID NO: 126 were respectively synthesized into codon optimized sequences to maximize expression in CHO cells. This gene was cloned into XhoI / NotI and ApaI / SmaI of the pAV4 vector, respectively, to prepare a parental antibody vector (pFwt).

Fwt Antibody Amino Acid Sequence

division	order	number
Heavy chain	EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	SEQ ID NO: 115
Light chain	DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVKLSKLSKLSKVKKKVKVKDR	SEQ ID NO: 116

2-1. Preparation of Modified Antibody FM2 from Fwt, a Folate Receptor Binding Parent Antibody:

XhoI-Q5-F was used as a template to prepare FM2 (Fwt-ACGHAACGHA (SEQ ID NO: 5), FM2), a modified antibody of Fwt having two metal ion binding motifs (ACGHA), as a template. PCR was performed using forward primers (5'-GCTCCTCGAGGCCACCATGGGATGGAGCTGT ATCATCC-3 ': SEQ ID NO: 105) and M2 reverse primer (5'-CCATGCGGCCGCTCATTTAGGCATGGCCA CAAGCAGCATGGCCACAGGCACCCGGAGACAGGGAGAGGC-3': SEQ ID NO: 106). The amplified nucleotide was cleaved with two restriction enzymes, XhoI and NotI, present at the ends, and conjugated with an expression vector pFwt having an XhoI / NotI cleavage to prepare a modified antibody vector (pFM2).

2-2. Preparation of Folate receptor-binding modified antibody FM1:

In order to prepare FM1 (Fwt-GGGACGHA, pFM1), a trastuzimab modified antibody having only one metal ion binding motif (ACGHA), a forward primer (5'-GCTCCTCGAGGCCACCATGGGATGGAGCTGT ATCATCC-3 ') was used as a template. ) And reverse primers (5'-CCATGCGGCCGCTCATTTAGGCATGGCC ACAAGCA CCTC CACCACCCGGAGACAGGGAGA-3 ': SEQ ID NO: 108) were used to amplify by PCR using the site-directed mutagenesis (Enzymex EzChange Site-directed mutagenesis kit, Ez004S) method. The amplified nucleotide was cleaved with two restriction enzymes, XhoI and NotI, present at the ends, and conjugated with an expression vector pFwt having an XhoI / NotI cleavage to prepare a modified antibody vector (pFM1).

2-3. Preparation of Folate receptor-binding modified antibody FM2L:

In order to prepare a modified antibody FM2L (Fwt-ACGHAGGGACGHA, pFM2L), in which a metal ion binding motif (ACGHA) is linked with a three amino acid linker, a forward primer (5'-GCTCCTCGAGGCCACCATGGGATGGAGCTGTATCATCC-3 ': Site-directed mutagenesis (SEQ ID NO: 109) and reverse primers (5'-CCATGCGGCCGCTCATTTAGGCATGGCCACAAGCACCTCCACCAGCATGGCCACAGGCACCCGGAGACAGGGAGAGGC-3 ': SEQ ID NO: 110) using two methods: site-directed mutagenesis (enzymeics EzChange site-directed mutagenesis kit, PCR using two metal ion methods) Glycine linkers were added between the motifs. The amplified nucleotide was cleaved with two restriction enzymes, XhoI and NotI, present at the ends, and conjugated with an expression vector pFwt having an XhoI / NotI cleavage to prepare a modified antibody vector (pFM2L).

2-4. Preparation of Folate receptor-binding modified antibody FM2a:

In the ACGHAACGHA (SEQ ID NO: 5), two metal ion binding motifs present in the FM2 modified antibody, a modified antibody FM2a (Fwt-ASGHAACGHA ( SEQ ID NO: 26), a forward primer (5'-GCTCCTCGAGGCCACCATGGGATGGAGCTGT ATCATCC-3 ') and a reverse primer (5'-CAGATTGCGGCCGCTCATTAGGCATGGCCACAAGCAGCATGGCCGA AGGCACCCG: SEQ ID NO: 112) was used to replace inner cysteine with serine using PCR. The amplified nucleotide was cleaved with two restriction enzymes, XhoI and NotI, present at the ends, and conjugated with the expression vector pFwt having the XhoI / NotI cleavage to prepare a modified antibody vector (pFM2a).

2-5. Preparation of Folate receptor-binding modified antibody FM2b:

In the ACGHAACGHA, the two metal ion binding motifs present in the FM2 modified antibody, FM2b (Fwt-ACGHAASGHA (SEQ ID NO: 21)), in which only one metal ion binding motif exists, by replacing the outer cysteine with serine To prepare pFM2b), a forward primer (5'-GCTCCTCGAGGCCACCATGGGATGGAGCTGTATCATCC-3 ': SEQ ID NO: 113) and a reverse primer (5'-CAGATTGCGGCCGCTCATTAGGCATGGCCTGAAGCAGCATGGCCACA GGCACCC-3): Internal cysteine was replaced with serine by PCR using site-directed mutagenesis (EzChange Site-directed mutagenesis kit, Ez004S). The amplified nucleotide was cleaved with two restriction enzymes, XhoI and NotI, present at the ends, and conjugated with an expression vector pFwt having an XhoI / NotI cleavage to prepare a modified antibody vector (pFM2b).

FM antibody

division	order	number
FM2 heavy chain	EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ACGHAACGHA	SEQ ID NO: 117
FM2a heavy chain	EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ASGHAACGHA	SEQ ID NO: 118
FM2b heavy chain	EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ACGHAASGHA	SEQ ID NO: 119
FM1 heavy chain	EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGACGHA	SEQ ID NO: 120
FM2L heavy chain	EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ACGHAGGGACGHA	SEQ ID NO: 121

Example 3 Expression and Purification of Fwt and Folate Receptor Binding Antibody

Chinese hamster ovary cells (CHO-K1) was used to confirm the protein expression of the Fwt and its metal ion binding motif modified antibodies (FM1, FM2, FM2L, FM2a, FM2b) prepared in Example 2. CHO-K1 was incubated in Dulbecco's Modified Eagle Media (DMEM) containing 10% FBS (Fetal Bovine Serum) and antibiotics at 37 ° C, 5% CO ₂ , incubator. One day before the introduction of Fwt and its modified antibody expression vector, cells were inoculated at a concentration of 5 × 10 ⁶ / ml in a 100 mm culture dish, followed by incubation with 800 μl of DMEM without FBS and antibiotics, and 10 μg of Fwt or modified. The antibody expression vector was mixed and maintained at room temperature for 1 minute, and then mixed with 20 μg of PEI (Polyethylenimine, linear, Polysciences Inc (Cat. No: 23966, MW ~ 25,000)) and left at room temperature for 10 to 15 minutes. . At this time, the cells cultured one day ago were washed with PBS and 6 ml of DMEM was added. Expression vectors of Fwt or its modified antibodies left at room temperature for 10-15 minutes were added to this culture dish. The next day, the protein expression was confirmed by washing with PBS and adding IMDM (Cat. No 12200-028, Gibco, Iscove's Modified Dulbecco's Medium) medium without FBS.

Thus expressed Fwt and its metal ion binding motif modified antibody was purified as follows. Specifically, in order to purify the Fwt and its metal ion binding motif-modified antibody secreted into the cell culture, the culture medium was centrifuged to remove the cells, and then only the supernatant was taken and the supernatant was equilibrated with the equilibration buffer HiTrap Protein A HP. (GE Healthcare, USA) The protein was eluted by injection into the column, washing well with equilibration buffer, and changing the pH with Glycine buffer (100 mM Glycine, pH 2.8). The solution was dialyzed with phosphate buffer, concentrated using Vivaspin20 (Sartorius, USA), and finally purified protein was obtained with high purity.

Example 4 Preparation of Antibody-Drug Conjugate by Reaction of Fwt Modified Antibody with Maleimide Group and Cysteine

In the present invention, a modified antibody of MMAE and Fwt was conjugated to prepare an FMx (metal ion-binding motif variant of Fwt) -MMAE conjugate. As a single methyl auristatin E (see Formula 2), a conjugation derivative of Auristatin known as MMAE,

Maleimide groups that selectively bind to thiol groups through valine-citurulline, which is degraded by protease in cells, and para-aniline benzoic acid (PABA), a self-decomposing spacer group It has a structure to be connected. Collectively, it is called MC (maleimido caproic acid) -VC (valine-citurulline) -PAB-MMAE. Auristatin is a substance with strong intracellular toxicity and IC ₅₀ value in the cell proliferation inhibition test is known to be 200 ~ 300 pM. .

In the present invention, after adding 3 equivalents of TCEP, a reducing agent per equivalent of purified antibody, and reacting at 4 ° C. for 30 minutes to reduce thiol groups, 3 equivalents of MC-vc-PAB-MMAE are added and reacted at room temperature for 2 hours. The reaction was terminated by the addition of excess cysteine, and excess MC-vc-PAB-MMAE and TCEP were removed through dialysis in a centrifugal filtration filter and phosphate buffer to remove the final purified FMx-MC-vc-PAB-MMAE. Prepared.

The yield of conjugation to the heavy chain of each modified antibody is shown in Table 3 below.

Conjugation yield of each modified antibody through maleimide group

Modified antibody	FM1	FM2	FM2a	FM2b	FM2L
Junction yield	62.7%	64.1%	64.5%	97.5%	66.3%

As can be seen in Table 3 above, the yield of drug conjugation to the heavy chain between the modified antibodies shows a very large difference. Modified antibodies such as FM1, FM2, FM2a, and FM2L show conjugation yields of about 63-66% under the same drug conjugation conditions, while FM2b shows very high drug conjugation yields of 97.5%. In the case of FM2b, nearly identical conjugation yields were shown in samples from two different transfection batches.

Example 5 Preparation of Antibody-Drug Conjugate by Reaction of Fwt Modified Antibody with Bromoacetamide Group

In this example, an antibody-drug conjugate that binds to a thiol group of cysteine through a bromoacetamide group was prepared. Bromoacetamide is bound to MMAE via valine-citurulline, which is degraded by protease in cells, and para-aniline benzoic acid (PABA), a self-decomposing spacer group. With the structure, MMAE is bound to the modified antibody through the coupling of bromoacetamide and thiol group. This is called br (bromo acetamide) -VC (valine-citurulline) -PAB-MMAE.

In the present invention, after adding 3 equivalents of TCEP, a reducing agent per equivalent of purified antibody, and reacting at 4 ° C. for 30 minutes to reduce thiol groups, 3 equivalents of br-vc-PAB-MMAE are added and reacted at 37 ° C. for 2 hours. The reaction was terminated by the addition of excess cysteine, and excess br-vc-PAB-MMAE and TCEP were removed via dialysis in a centrifugal filtration filter and phosphate buffer to remove the final purified FMx-acetamide-vc-PAB-MMAE. Prepared.

The yield of conjugation to the heavy chain of each modified antibody is shown in Table 4 below.

Conjugation yield of each modified antibody through bromoacetamide group

Modified antibody	FM1	FM2	FM2a	FM2b	FM2L
Junction yield	42%	44%	38%	73%	49%

As can be seen in Table 4, the yield of drug conjugated to the heavy chain between each of the modified antibodies shows that the conjugate yield of FM2b is superior to other modified antibodies. In the conjugation reaction via iodoacetamide, FM2b showed superior conjugation yield compared to other modified antibodies.

Conjugation yield of each modified antibody through iodoacetamide group

Modified antibody	FM1	FM2	FM2a	FM2b	FM2L
Junction yield	51%	53%	44%	80%	55%

Example 6 Production of Trastuzumab Modified Antibodies

As in the method used in Example 2, a modified antibody was prepared by introducing a metal ion motif containing cysteine at the C-terminus of trastuzumab.

6-1. Preparation of Modified Antibody HM2 from Trastuzumab:

To prepare HM2 (HR-ACGHAACGHA (SEQ ID NO: 5), HM2), a modified antibody of trastuzumab having two metal ion binding motifs (ACGHA), as a template, a vector (pHR) of the parent antibody trastuzumab was used. PCR was performed using forward primers (5'-GCTCCTCGAGGCCACCATGGGATGGAGCTGT ATCATCC-3 ': SEQ ID NO: 111) and M2 reverse primers (5'-CCATGCGGCCGCTCATTTAGGCATGGCCA CAAGCAGCATGGCCACAGGCACCCGGAGACAGGGAGAGGC-3': SEQ ID NO: 112). The amplified nucleotides were cleaved with two restriction enzymes, XhoI and NotI, present at the ends, and conjugated with an expression vector pHR having an XhoI / NotI cleavage to prepare a modified antibody vector (pHM2).

6-2. Preparation of the modified antibody HM2a of Trastuzumab:

In the ACGHAACGHA (SEQ ID NO: 5), two metal ion-binding motifs present in the HM2 modified antibody, a substitution antibody HM2a (HR-ASGHAACGHA ( SEQ ID NO: 26), a forward primer (5'-GCTCCTCGAGGCCACCATGGGATGGAGCTGT ATCATCC-3 ': SEQ ID NO: 111) and a reverse primer (5'-CAGATTGCGGCCGCTCATTAGGCATGGCCACAAGCAGCATGGCCGA AGGCAG-3CC): SEQ ID NO: 112) was used to replace inner cysteine with serine using PCR. The amplified nucleotide was cleaved with two restriction enzymes, XhoI and NotI, present at the ends, and conjugated with an expression vector pHR having an XhoI / NotI cleavage to prepare a modified antibody vector (pHM2a).

6-3. Preparation of the modified antibody HM2b of Trastuzumab:

In the ACGHAACGHA, two metal ion binding motifs present in the HM2 modified antibody, HM2b (HR-ACGHAASGHA (SEQ ID NO: 21)), a variant antibody in which only one metal ion binding motif exists by replacing the outer cysteine with serine To prepare the pHM2b), the forward antibody (5'-GCTCCTCGAGGCCACCATGGGATGGAGCTGTATCATCC-3 ': SEQ ID NO: 109) and the reverse primer (5'-CAGATTGCGGCCGCTCATTAGGCATGGCCTGAAGCAGCATGGCCACA GGCACGA) Internal cysteine was replaced with serine by PCR using site-directed mutagenesis (EzChange Site-directed mutagenesis kit, Ez004S). The amplified nucleotide was cleaved with two restriction enzymes, XhoI and NotI, present at the ends, and conjugated with an expression vector pHR having an XhoI / NotI cleavage to prepare a modified antibody vector (pHM2b).

Example 7 Expression and Purification of Trastuzumab Modified Antibodies

Chinese hamster ovary cells (CHO-K1) was used to confirm the protein expression of the trastuzumab modified antibodies (HM2, HM2a, HM2b) prepared in Example 6. CHO-K1 was incubated in Dulbecco's Modified Eagle Media (DMEM) containing 10% FBS (Fetal Bovine Serum) and antibiotics at 37 ° C, 5% CO ₂ , incubator. One day before the introduction of the modified antibody expression vector, cells were inoculated at a concentration of 5 × 10 ⁶ / ml in a 100 mm culture dish, followed by incubation with 800 μl of DMEM without antibiotics and 10 μg of the modified antibody expression vector. After maintaining for 1 minute at room temperature, and mixed with 20 ㎍ PEI (Polyethylenimine, linear, Polysciences Inc (Cat. No: 23966, MW ~ 25,000)) and left at room temperature for about 10-15 minutes. At this time, the cells cultured one day ago were washed with PBS and 6 ml of DMEM was added. Expression vectors of the modified antibodies left at room temperature for 10-15 minutes were added to the culture dish. The next day, the protein expression was confirmed by washing with PBS and adding IMDM (Cat. No 12200-028, Gibco, Iscove's Modified Dulbecco's Medium) medium without FBS.

The modified antibody expressed as above was purified as follows. Specifically, in order to purify the modified antibody secreted into the cell culture, the culture medium was centrifuged to remove the cells, and then only the supernatant was taken and the supernatant was equilibrated with the equilibration buffer HiTrap Protein A HP (GE Healthcare, USA) column. The protein was eluted by changing the pH with Glycine buffer (100mM Glycine, pH 2.8) after washing with equilibration buffer. The solution was dialyzed with phosphate buffer, concentrated using Vivaspin20 (Sartorius, USA), and finally purified protein was obtained with high purity.

Example 8 Preparation of Antibody-Drug Conjugate by Conjugation of Modified Antibody of Trastuzumab with MC-vc-PAB-MMAE

In the present invention, the modified antibody of trastuzumab produced in Example 6, 7 was conjugated with MMAE to prepare a HMx (metal ion binding motif variant of Trastuzumab) -MMAE conjugate. In the present invention, after adding 3 equivalents of TCEP, a reducing agent per equivalent of purified antibody, and reacting at 4 ° C. for 30 minutes to reduce thiol groups, 2.5 equivalents of MC-vc-PAB-MMAE are added and reacted at room temperature for 2 hours. The reaction was terminated by the addition of excess cysteine, and excess MC-vc-PAB-MMAE and TCEP were removed through dialysis in a centrifugal filtration filter and phosphate buffer to remove the final purified FMx-MC-vc-PAB-MMAE. Prepared.

The yield of conjugation to the heavy chain of each modified antibody is shown in Table 6 below.

Conjugation yield of each modified antibody through maleimide group

Modified antibody	HM2	HM2a	HM2b
Junction yield	55.5%	62.4%	85.5%

As can be seen in Table 6 above, the yield of drug conjugation to the heavy chain between the modified antibodies shows a very large difference. The modified antibodies of HM2 and HM2a show about 55-62% conjugation yield under the same drug conjugation conditions, while HM2b shows very high drug conjugation yield of 85.5%. These results show that the sequence of M2b has the same high conjugation yield when introduced into other antibodies as well as Farletuzumab.

Example 9 Production of Modified Antibody Substituted with Serine in M2b (ACGHAASGHA: SEQ ID NO: 21) Sequence

In the previous example, it was confirmed that M2b (ACGHAASGHA) in which the rear cysteine was substituted with serine in M2 (ACGHAACGHA), which is a metal ion binding motif, showed much higher drug binding ability than the M2 sequence. In order to confirm that the serine substitution sites showed the same effect in the substitution of other amino acids, a modified antibody was substituted with various amino acids at this position.

Modified antibody which substituted another amino acid at serine site in M2b sequence

Modified antibody	C-terminal metal ion binding motif sequence
M2b or M2b-S	ACGHAASGHA (SEQ ID NO: 21)
M2b-A	ACGHAAAGHA (SEQ ID NO: 33)
M2b-T	ACGHAATGHA (SEQ ID NO 45)
M2b-Y	ACGHAAYGHA (SEQ ID NO 57)
M2b-D	ACGHAADGHA (SEQ ID NO: 69)
M2b-K	ACGHAAKGHA (SEQ ID NO: 81)
M2b-F	ACGHAAFGHA (SEQ ID NO: 93)

The modified antibodies having the C-terminal sequence as shown in Table 7 above were introduced into FM2b or FM2b-S, respectively, of FM2b-A, FM2b-T, FM2b-Y, FM2b-D, FM2b-K, and FM2b-F. Modified antibodies were produced.

Example 10 Antibody-Drug Conjugate Production by Conjugation of FM2b Modified Antibody to MC-vc-PAB-MMAE

In the present invention, FM2b-X-MMAE conjugate was prepared by conjugating MMAE with FM2b-X (X = A, T, Y, D, K, or F) modified antibodies produced as in Example 9. In the present invention, after adding 3 equivalents of TCEP, a reducing agent per equivalent of purified antibody, and reacting at 4 ° C. for 30 minutes to reduce thiol groups, 2.5 equivalents of MC-vc-PAB-MMAE are added and reacted at room temperature for 2 hours. The reaction was terminated by the addition of excess cysteine and excess MC-vc-PAB-MMAE and TCEP were removed via dialysis in a centrifugal filtration filter and phosphate buffer solution to obtain the final purified FMb-X-MC-vc-PAB- MMAE was prepared. The yield of conjugation to the heavy chain of each modified antibody is shown in Table 8 below.

DAR2 yield when conjugated with FM2b-X modified antibody and MC-vc-PAB-MMAE

Modified antibody	FM2b	FM2b-A	FM2b-T	FM2b-Y	FM2b-D	FM2b-K	FM2b-F
Junction yield	72.8%	62.5%	69%	74.5%	58.9%	75.3%	72.9%

As can be seen in Table 8 above, substitution of other amino acids in the rear serine site of the ACGHAASGHA, which is the M2b sequence, shows a similar result to that of FM2b.

Example 11. Purification of Antibody-Drug Conjugates That Are DAR2

Each modified antibody prepared as in the above embodiment four-drug conjugate is modified number of the bonding substance (DAR: drug-to-antibody ratio) per antibody because it is different, to compare the cytotoxicity of the drug from in vitro Is not easy. Therefore, in order to purify the modified antibody-drug conjugate with the same DAR, the modified antibody-drug conjugate was purified using hydrophobic chromatography. Phenyl column chromatography was used to purify the modified antibody-drug conjugate having a DAR of 2. The column was equilibrated with 10 mM sodium succinate, 0.5 M NaCl, pH 5.0 buffer, and then the modified antibody-drug conjugate was injected into the column. After washing the column with the same buffer, the buffer containing 30% acetonitrile was added to elute the modified antibody-drug conjugate according to DAR. The eluted modified antibody-drug conjugates were exchanged for buffer using dialysis in a buffer solution of 10 mM sodium succinate, 30 mM sucrose, pH 6.0.

Example 12. Stability Testing of In Vitro Antibody-Drug Conjugates

FM2b-S-D2, FM2b-F-D2, FM2b-K-D2, FM2b-Y, which are antibody-drug conjugates having two conjugated MC-vc-PAB-MMAE drugs as in Examples 9, 10, and 11 above Produced -D2. The produced antibody-drug conjugates were incubated at 25 ° C. and 50 ° C., respectively, to measure the change in the number of drug conjugates and the change in aggregation.

Each test sample was prepared at a concentration of 110 uL at a concentration of 1 mg / mL, each of 12. Six samples of each antibody-drug conjugate were stored at 25 ° C. and the remaining six samples were kept at 0 ° C. for 1, 3, 5, 7, and 14 days to measure changes in DAR and monomer. At 25 ° C, the content of DAR2 was decreased by 1.5 ~ 2% for each sample, and the purity of monomer was observed about 0.3 ~ 4%, but the difference was not large. Changes in DAR2 content and monomer purity at 50 ° C were larger than those observed at 25 ° C, but the differences between samples were not significant. Therefore, it was confirmed that the difference between the antibody variants is not very large.

Contents of monomers on Days 0, 1, 3, 5, 7, and 15 at 25 ° C and 50 ° C storage conditions of FM2b-S-D2, FM2b-F-D2, FM2b-K-D2, and FM2b-Y-D2

Storage temperature	sample		Day 0	Day 1	Day 3	Day 5	Day 7	Day 15
25 ℃	FM2b-S-D2	97.6	97.6	97.2	96.4	95.6	95.1
	FM2b-F-D2	97	97.8	97.2	96.6	96.1	95
	FM2b-K-D2	97.6	97.6	97.3	96.6	96.1	96.3
	FM2b-Y-D2	96.7	96.6	97	95.8	95.4	94.9
50 ℃	FM2b-S-D2	97.6	93.9	88.8	82.8	79.1	68.9
	FM2b-F-D2	97	93.7	86.9	80.9	76.9	63.5
	FM2b-K-D2	97.6	93.9	87.7	81.8	77.8	66.8
	FM2b-Y-D2	96.7	93.9	88.4	82.7	77.9	66.6

Contents of DAR 2 on Days 0, 1, 3, 5, 7, and 15 at 25 ° C and 50 ° C storage conditions of FM2b-S-D2, FM2b-F-D2, FM2b-K-D2, and FM2b-Y-D2

Storage temperature	DAY		Day 0	Day 1	Day 3	Day 5	Day 7	Day 15
25 ℃	FM2b-S-D2	98.9	98.6	98.6	98.2	98.4	98.1
	FM2b-F-D2	98.2	98.4	98.4	98.2	98.3	97.9
	FM2b-K-D2	97.9	97.4	97.5	97.3	97.3	97
	FM2b-Y-D2	97.6	97.6	97.4	96.6	95.5	93.2
50 ℃	FM2b-S-D2	98.9	97.1	93.6	91.8	90.7	87.9
	FM2b-F-D2	98.2	96.5	93.2	89.4	88.3	85.1
	FM2b-K-D2	97.9	96.2	94.6	91.8	90.8	88.9
	FM2b-Y-D2	97.6	95.8	91.3	89.4	87.6	84.4

Example 13. In Vitro Cell Proliferation Inhibition Test

In order to compare the in vitro cell proliferation inhibitory activity of the modified antibody-drug conjugates, cell growth inhibitory activity was performed using KB-cells overexpressed with the folate receptor. KB-cells were diluted in DMEM / F12 medium with 10% FBS adjusted to 1 × 10 ⁴ / well and 100 μl cell culture was added to each well of a 96-well plate. It was. The well plates were then incubated for 24 hours in an incubator set at 5% carbon dioxide and 37 ° C. to attach the cells to the plates. Each test sample was diluted in medium and added to final concentrations of 6.45 nM, 3.23 nM, 1.61 nM, 0.806 nM, 0.403 nM, 0.202 nM, 0.101 nM, 0.0504 nM, 0.0252 nM and 0.0126 nM, together with the medium in the control wells. (No drug) was added. After incubation for 5 days, CellTiter 96- AQueous One Solution reagent [MTS-based assay; MTS forms purple formazan by dehydrogenase of living cells, and proliferation is measured by the amount of purple formazan produced], followed by incubation for 2 hours in an incubator set at 37 ° C. It was. Cell lysis was measured at 490 nm using an absorbance spectrometer to determine viability (%).

13-1. Comparison of cell growth inhibitory activity between FM2-D2 and FM2b-D2 (or FM2b-S-D2):

FM2-D2 (modified antibody-drug conjugate having DAR 2 as MMAE drug conjugate of modified antibody FM2) and FM2b purified after conjugation of the parent antibody Fwt with MMAE in the above example After treating -D2 (modified antibody-drug conjugate having DAR 2 as MMAE drug conjugate of modified antibody FM2b) to KB cells as described above, the cell growth inhibition of the drug was compared.

As can be seen in Figure 3, the antibody-drug conjugates FM2-D2 and FM2b-D2 show superior anticancer efficacy compared to the parent antibody. FM2-D2 and FM2b-D2 show nearly identical cancer cell growth inhibition. These results show that there is no difference in activity of inhibiting cancer cell growth when the same antibody DAR is used as a modified antibody-drug conjugate between the modified antibodies FM2 and FM2b.

13-2. Comparison of cell growth inhibitory activity between FM2-D2 and FM2b-D2 (or FM2b-S-D2):

To compare cell growth inhibition between FM2b-S and other variants-based antibody-drug conjugates, MC-vc-PAB-MMAE was conjugated to FM2b-S, -F, and -Y variants and purified to have the same DAR. Then, after treating the KB cells as described above, the cell growth inhibition of the drug was compared.

As can be seen in Figure 4, FM2b-S-D2 and FM2b-F-D2, FM2b-Y-D2 shows almost the same cell growth inhibition. The respective IC ₅₀ values were 0.25 nM for FM2b-S-D2, 0.26 nM for FM2b-F-D2, and 0.24 nM for FM2b-Y-D2. These results show that there is no difference in the activity of inhibiting cancer cell growth between antibody-drug conjugates based on antibody variants substituted with phenylalanine and tyrosine instead of the serine of FM2b-S.

From these results, when the antibody-drug conjugate was prepared by using the antibody to which the M2b sequence was introduced, there was a difference in the anti-cancer activity as the antibody-drug conjugate while having a superior conjugated yield of drugs compared to other modified antibodies. Shows that there is no. In addition, even if the amino acid is substituted in the serine site in the ACGHA-ASGHA M2b sequence, it was confirmed that there is no difference in the drug yield, stability, and anticancer activity. Therefore, when introducing ACGHA-AXGHA (X is an amino acid other than cysteine), a motif as used in FM2b or HM2b, the conjugate yield is superior to other modified antibodies, thereby providing a more efficient conjugation yield. And economic antibody-drug conjugates.

The antibody-drug conjugate produced by the antibody variant according to the present invention can greatly improve the productivity of the antibody-drug conjugate by increasing the conjugation yield of the drug. In addition, the drug conjugated to the terminal portion of the antibody through position-specific conjugation does not inhibit the structural stability of the parent antibody, thereby maintaining the antigen specificity and structural stability of the parent antibody originally, and the high antigen possessed by the parent antibody. Specificity allows the drug conjugated to the antibody to be specifically delivered to cancer cells.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (14)

1. An antibody-drug conjugate wherein a modified antibody comprising a motif represented by the following structural formula (1) at the terminal of the antibody is bound to a drug via a linker:

   Structural Formula (1)

   _{X a - [M motif1] n1} -X b - [M motif2] n2

   _Wherein M _motif1 or M _motif2 each independently include any one of a sequence consisting of ACGHA (SEQ ID NO: 1), AHGCA (SEQ ID NO: 2), AXGHA (SEQ ID NO: 3), and AHGXA (SEQ ID NO: 4), wherein X in SEQ ID NO: 3 or 4 includes amino acid residues other than cysteine,

   X _a And X _b is a peptide consisting of 0 to 20 amino acid residues each independently selected from the group consisting of A (alanine), S (serine), G (glycine),

   n1 and n2 are integers of 1-10, respectively.
2. The antibody-drug conjugate of claim 1 wherein the M _motif2 comprises AXGHA (SEQ ID NO: 3) and X is an amino acid residue other than cysteine.
3. The method of claim 1, wherein X of M _{motif 1} or M _{motif 2} is serine (S), glycine (G), alanine (A), threonine (T), tyrosine (Y), aspartic acid (D), lysine (K) And phenylalanine (F). An antibody-drug conjugate, characterized in that the amino acid residue is selected from the group consisting of.
4. The antibody-drug conjugate of claim 1, wherein the motif represented by the structural formula (1) comprises at least one sequence selected from the group consisting of SEQ ID NOs: 5 to 104.
5. The antibody-drug conjugate of claim 1, wherein the motif is introduced at the heavy chain C-terminus of the antibody.
6. The antibody-drug conjugate of claim 1 wherein the linker comprises a reactive functional group that binds to a motif, an amino acid, and a self cleaving spacer.
7. The method of claim 1, wherein the drug is maytansinoid, oristatin, aminopterin, actinomycin, bleomycin, thalisomycin, camptothecin, N8-acetyl spermidine, 1- (2 chloroethyl)- 1,2-dimethyl sulfonyl hydrazide, esperamycin, etoposide, 6-mercaptopurine, dolastatin, tricortesene, calicheamicin, taxane, methotrexate, vincristine, vinblastine, doxorubicin, melphalan Antibody-drug conjugate, characterized in that at least one selected from, mitomycin A, mitomycin C, chlorambucil, duocarmycin, nucleases, toxins derived from bacteria or plants, cisplatin, irinotecan, paclitaxel and docetaxel.
8. The antibody-drug conjugate of claim 1, wherein the antibody is at least one selected from the group consisting of monoclonal antibodies, bispecific antibodies, chimeric antibodies, human antibodies and humanized antibodies.
9. The antibody-drug conjugate of claim 1 wherein the antibody is selected from the group consisting of IgA, IgD, IgE, IgG and IgM.
10. The method of claim 1, wherein the antibody is a cancer specific antigen, cell surface receptor protein, cell surface protein, transmembrane protein, signaling protein, cell survival regulator, cell proliferation regulator, molecule associated with tissue development or differentiation, lymphokah An antibody-drug conjugate, having binding capacity and specificity for phosphorus, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, or molecules involved in angiogenesis.
11. The method of claim 1, wherein the antibody,

    (1) BMPR1B (bone morphogenic protein receptor-IB type, Genbank Accession No. NM_001203);

    (2) E16 (LAT1, SLC7A5, GenBank Accession No. NM_003486);

    (3) STEAP1 (six transmembrane epithelial antigens of the prostate, Genbank Accession No. NM_012449);

    (4) 0772P (CA125, MUC16, GenBank Accession No. AF361486);

    (5) MPF (MPF, MSLN, SMR, megakaryocyte enhancing factor, mesothelin, Genbank Accession No. NM — 005823);

    (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, Solute Carrier Family 34 (Sodium Phosphate), Member 2, Type II Sodium-Dependent Phosphate Transporter 3b, GenBank Accession No. NM_006424);

    (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, Sema Domain, 7 Thrombospondin Repeats (Type 1 and Similar Type 1), Transmembrane Domain (TM) And short cytoplasmic domain, (semaphorin) 5B, Genbank Accession No. AB040878);

    (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genebank Accession No. AY358628);

    (9) ETBR (endothelin type B receptor, Genbank Accession No. AY275463);

    (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genebank Accession No. NM_017763);

    (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, Prostate Cancer Related Gene 1, Prostate Cancer Related Protein 1, Prostate 6 Transmembrane Epithelial Antigen 2, 6 Transmembrane Prostate Protein, Genebank Authorization number AF455138);

    (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subgroup M, member 4, Genbank Accession No. NM_017636);

    (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP — 003203 or NM — 003212);

    (14) CD21 (CR2 (complementary receptor 2) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792 Genbank Accession No. M26004);

    (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29, Genbank Accession No. NM — 000626);

    (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchoring protein 1a), SPAP1B, SPAP1C, GenBank Accession No. NM_030764);

    17 HER2 (Genbank approval number M11730)

    (18) ErbB receptors selected from EGFR, HER3 and HER4

    (19) NCA (Genbank Accession No. M18728);

    (20) MDP (GenBank Accession No. BC017023);

    (21) IL20Rα (GenBank Accession No. AF184971);

    (22) Brevican (Genbank Accession No. AF229053);

    (23) EphB2R (GenBank Accession No. NM_004442);

    (24) ASLG659 (Genbank Accession No. AX092328);

    (25) PSCA (Genbank Accession No. AJ297436);

    (26) GEDA (Genbank Accession No. AY260763);

    (27) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, NP_443177.1);

    (28) CD22 (B-cell receptor CD22-B isotype, NP-001762.1);

    (29) CD79a, CD79A, CD79α, and immunoglobulin-associated alpha, which are covalently interacting with CD79a (Ig beta (CD79B) and forming complexes on the surface with IgM molecules, are signals involved in B cell differentiation Forwarded, Genbank approval number NP_001774.1);

    (30) CXCR5 (Bucket Lymphoma Receptor 1, a G protein coupled receptor activated by CXCL13 chemokines, acts on lymphocyte migration and humoral defense, participates in HIV-2 infection, and develops AIDS, lymphoma, myeloma and leukemia Considered to be related to, Genbank approval number NP_001707.1);

    (31) HLA-DOB (beta subunit of MHC class II molecules (Ia antigen), binding to peptides and presenting in CD4 + T lymphocytes, Genbank Accession No. NP — 002111.1);

    (32) P2X5 (purine receptor P2X ligand-gate ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, the lack of which may contribute to the pathophysiology of idiopathic detrusor instability Yes, Genbank approval number NP_002552.2);

    (33) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank Accession No. NP — 001773.1);

    (34) Lymphocyte antigen 64 (RP105), a type I membrane protein of the LY64 (leucine rich repeat (LRR) family), modulates B cell activation and apoptosis, and its loss of function is attributed to systemic lupus erythematosus patients. Associated with increased disease activity, GenBank Accession No. NP_005573.1);

    (35) FcRH1 (Fc receptor-like protein 1, a putative receptor for immunoglobulin Fc domains containing C2 Ig-like and ITAM domains, may be involved in B lymphocyte differentiation, Genbank accession number NP_443170.1) ;

    (36) IRTA2 (associated gene deregulation by immunoglobulin macrophage receptor translocation, a putative immunoreceptor capable of acting on B cell development and lymphoma development, occurs in some B cell malignancies, Genbank approval number NP_112571.1);

    (37) TENB2 (estimated transmembrane proteoglycan associated with EGF / Heregulin family of growth factors and follistatin, Genbank Accession No. AF179274);

    (38) MAGE-C1 / CT7 (testicular cancer overexpressed protein);

    (39) androgen receptor, PTEN, human kallikrein-related peptidase 3 (protein overexpressed in prostate cancer);

    (40) CD20;

    (41) CD30;

    (42) CD33;

    (43) CD52;

    (44) EpCam;

    (45) CEA;

    (46) gpA33;

    (47) Mucins;

    (48) TAG-72;

    (49) Carbonic anhydrase IX;

    (50) PSMA;

    (51) folate receptor (a family of proteins expressed by the FoLR gene, which has high binding capacity with Folic acid and carries 5-methyltetrahydrofolate into cells);

    (52) gangliosides (GD2, GD3, GM2);

    (53) carbohydrate Lewis-Y;

    (54) VEGF;

    (55) VEGFR;

    (56) aVb3;

    (57) a5b1;

    (58) ERB3;

    (59) c-MET;

    (60) EphA3;

    (61) TRAIL-R1, TRAIL-R2;

    (62) RANKL;

    (63) FAP; And

    (64) An antibody-drug conjugate, having an ability to bind to one or more targets selected from the group consisting of Tenascin.
12. The antibody-drug conjugate of claim 1 wherein the antibody comprises a variable region and CH1, CH2 and CH3 of IgG2 or IgG4, or Fab and Fc of IgG2 or IgG4.
13. The antibody-drug conjugate of claim 1 wherein the drug is conjugated to the cysteine or X of the motif.
14. 14. A composition for preventing or treating cancer, comprising the antibody-drug conjugate according to any one of claims 1 to 13.

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