KR20190033103A - Novel oxime derivative compounds and antibody-drug conjugate comprising thereof - Google Patents

Abstract

The present invention relates to a novel oxime derivative compound and an antibody-drug complex comprising the same. An oxime linker according to the present invention is excellent in stability compared to an existing oxime linker, has a high synthesis yield in the process of forming the antibody-drug complex, increases anticoagulant properties and drug delivery efficiency of the antibody-drug complex due to a hydrophobicity reducing effect of a drug, by the linker of the present invention, and has high structural stability, thereby having excellent utility as a therapeutic agent.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel oxime derivative compound and an antibody-drug complex comprising the same. BACKGROUND ART < RTI ID = 0.0 >

The present invention relates to novel oxime derivative compounds and antibody-drug complexes comprising them.

The antibody-drug complex (Andibody-Drug Conjugate, ADC) is a drug that has been developed to replace existing antibody drugs. Antibodies play a role in reaching specific cancer cells, drugs kill cancer cells, and antibody-drug complexes have the advantage of selectively killing specific cancer cells. Linkers that bind the antibody and the drug play an important role in effectively causing the antibody-drug complex to be transported into cancer cells.

However, since the linker of the existing antibody-drug complex has a low structural stability, the drug is rapidly degraded while circulating in the blood and affects the normal cells, or most of the drug is highly hydrophobic, so that several linker-drugs are bound to the antibody There is a problem that they are aggregated by hydrophobicity and rapidly removed in the blood.

For example, the SMCC linker is a representative hydrophobic linker used in trastuzumab emtansine (Kadcyla ^® ), in which the linker is composed of a single moiety and has two different functional groups in one molecule , When the antibody is encapsulated in cancer cells, the linker-drug complex is separated and the cancer cells are killed by the first degradation of the antibody without breaking the linker. However, the SMCC linker has a succinimidyl group and a maleimide group at the ends thereof, so that the hydrophobicity of the linker itself is very high. Therefore, when an antibody-drug complex is formed, aggregation due to an increase in hydrophobicity occurs, Can not bind several drugs to the drug, and there is an unstable problem.

An example of a linker composed of at least one moiety in which a different functional group is introduced into a molecule through a peptide bond or the like is a hydrazone linker. The hydrazone linker is a compound having two different functional groups bonded to each other through a peptide bond and has a hydrazide group at one end and is bonded to a substance having an aldehyde group to form -C = N-NH- The hydrazine bond of CO- is generated. Although the C = N bond is designed to decompose under acidic pH conditions in the cell, there is a side effect that the hydrazone bond is degraded even at pH conditions that are not acidic. In addition, since the hydrolysis occurs, the linker is degraded during the circulation in the blood, and there is a risk that the drug is toxic to not only cancer cells but also normal cells due to the disappearance of antibody that plays a specific targeting role.

Finally, there is a peptide linker that attaches amino acid valine, valine, and citrulline through peptide bonds. Although it is designed to be degraded by proteolytic enzymes in the cell, there is a concern that the linker may be cleaved by proteolytic enzymes present in cells as well as in cells.

Therefore, it is still necessary to develop a linker capable of overcoming such a problem for developing an antibody-drug complex having excellent efficacy. In particular, development of a linker capable of enhancing the drug delivery efficiency and stability of the antibody- do.

In order to solve the problems of the conventional linker and to develop an excellent linker, the inventors of the present invention have conducted extensive research and have found that an oxime derivative compound having a hydrophobic moiety on one side of the oxime bond and a hydrophilic moiety on the other side thereof And it was confirmed that it can be used as a structurally stable linker having both hydrophobicity and hydrophilicity. In addition, it has been confirmed that the hydrophobic value of the antibody-drug complex can be lowered so that the drug rod efficiency and drug delivery efficiency can be increased in the antibody, and the stability can be improved through the degradation property only in the intracellular acidic pH condition. .

Accordingly, the present invention relates to an oxime derivative compound that can be utilized as an amphipathic linker capable of enhancing drug delivery efficiency while having structural stability, and it is possible to bind a plurality of drugs to an antibody by the anti- The present invention is characterized in that it provides a linker and an antibody-drug complex having excellent stability in the body since it has a linkage which is degraded only under specific acidic environmental conditions of the lysosome of a cell.

In order to solve the above problems of the conventional linkers and to provide a linker and an antibody-drug complex (ADC) excellent in the stability in the body and the drug delivery efficiency, the present invention provides an oxime derivative compound represented by the formula (1) Salt.

The present invention also provides an antibody-linker-drug complex represented by the general formula (2) or a pharmaceutically acceptable salt thereof.

The oxime linker according to the present invention is superior in stability to a conventional oxime linker, has a high synthesis yield when forming an antibody-drug complex, and exhibits anticoagulant properties of the antibody-drug complex by the effect of reducing the hydrophobicity of the drug by the linker of the present invention And has high structural stability, and thus has excellent utility as a therapeutic agent.

Figures 1a and 1b show profiles of HPLC traces over time in the oxime ligation reaction according to one embodiment of the present invention, wherein 1a represents the RP over the entire residence time range during the 12 hour reaction, -HPLC chromatogram, and 1b represents an expanded RP-HPLC chromatogram for a retention time of 35-50 minutes in an 8 hour reaction.
Figures 2a and 2b show HPLC chromatograms of crude oxime linker 2a and purified oxime linker 2b according to one embodiment of the present invention.
Figure 3 is a positive mode ESI-MS analysis for an oxime linker showing a deconvoluted mass spectrum according to an embodiment of the present invention, wherein the 393 Da peak is [M + Na ⁺ -H ₂ O] ⁺ The 411 Da peak represents [M + Na] ⁺ .
Figure 4 shows the degradation profiles of oxime linkers in PBS at pH 7.0 and pH 4.0 at 37 ° C according to one embodiment of the present invention.
FIGS. 5A, 5B and 5C show RP-HPLC chromatogram results of the oxime linker-DM1 complex according to an embodiment of the present invention, wherein DM1, DM5, and DM5 complexes are purified, Linker-DM1 complex.
Figure 6 is a positive mode ESI-MS analysis of the oxime linker-DM1 complex (50 min and 51 min) showing a deconvoluted mass spectrum according to one embodiment of the present invention, wherein the 1,130 Da peak is [M + Na ⁺ -H ₂ O] ⁺ and the 1,148 Da peak represents [M + Na] ⁺ .
7 is a SPR sensorgram analysis of the EDC / NHS activated D- glucuronic acid bound to the immobilized Herceptin ^® in accordance with one embodiment of the present invention; (A) an activated CM5 chip surface using the EDC / NHS, (B) Herceptin (Herceptin ^®) immobilized, (C) washing non-binding to the glycine buffer Herceptin, (D) disabling D- glucuronic acid, (E) active D-glucuronic acid.
Figure 8 is an SPR sensorgram analysis of the EDC / NHS activated the immobilized Herceptin ^® DM1- oxime linker conjugate according to one embodiment of the present invention; (A) EDC / activated CM5 chip surface using NHS, (B) Herceptin ^® immobilized, (C) removing the unbound glycine, (D) a DM1- oxime linker conjugated to the activation of immobilized Herceptin ^®.
9 is Herceptin ^® (A) in accordance with one embodiment of the present invention, (B) ADC-AL and (C) the standard protein; The size of thyroglobulin (670 kDa), r-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B (Vitamin B, 1.35 kDa) (D) protein standard curve (AD in order from top).
10a and 10b show HIC (butyl) -HPLC chromatogram results of Herceptin-amphipathic oxycin linker-DM1 complex (ADC-AL, 10a) and cassia (Kadcyla ^® , 10b) according to an embodiment of the present invention .
Figure 11 shows a MS spectrum result of Herceptin (Herceptin ^®) and ADC-AL fragment (Fc, LC, Fd regions) in accordance with one embodiment of the present invention: (a) Herceptin (Herceptin ^®), Fc region, (b) Fc region of an ADC-AL, (c) Herceptin (Herceptin ^®) LC area, (d) LC area of ADC-AL, (e) Herceptin (Herceptin ^®) and Fd region (f) Fd region of ADC-AL.
12a and 12b are MDA-MB-231, according to one embodiment of the present invention (Her2 negative cell lines, 12a), SK-BR- 3 Herceptin (Herceptin ^®) for (Her2-positive cell lines, 12b), caviar ssail La ( Kadcyla ^® ) or ADC-AL, respectively, and the cell viability was confirmed.

The present invention provides an oxime derivative compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof.

Further, there is provided an antibody-drug complex comprising the compound represented by the formula (1) of the present invention as a linker.

In the present invention, "substituted" means that at least one hydrogen atom is replaced with a halogen atom, a hydroxy group, a nitro group, a cyano group, an amino group, an amidino group, a hydrazine, a hydrazone, a carboxyl group or a salt thereof, a sulfonic acid group or a salt thereof, An alkyl group having 1 to 10 carbon atoms, an alkenyl group having 1 to 10 carbon atoms, an alkynyl group having 1 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an arylalkyl group having 7 to 20 carbon atoms, a heteroaryl group having 4 to 20 carbon atoms, ≪ / RTI > to 20 heteroarylalkyl groups.

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

The present invention relates to an oxime derivative compound represented by the formula (1) or a pharmaceutically acceptable salt thereof. Specifically, the present invention relates to an amphipathic oxime linker having a structure represented by the following formula (1).

[Chemical Formula 1]

The oxime derivative compound of the present invention or a pharmaceutically acceptable salt thereof is a parent compound having a hydrophilic moiety in -R ₁ -X on one side of the oxime bond and a hydrophobic moiety in -R ₂ -Y on the other side May be a compound having a mating property. The oxime derivative compound may specifically be included as a linker in the formation of an antibody-drug complex. When the oxime derivative compound of the present invention is used as a linker, since the amphiphilic property including the hydrophilic moiety and the hydrophobic moiety is exhibited, the hydrophobic value of the drug-antibody complex is low even when the number of the drug bound to the antibody is increased, The oxime bond is decomposed only in the condition that the structure stability is excellent. Therefore, when the oxime derivative compound of the present invention is used as a linker, it has an excellent effect of improving the stability of the body and the delivery efficiency of the drug due to the improvement of the aggregation phenomenon of the antibody-drug complex and reducing the side effects of decomposition of the drug in normal cells .

The oxime derivative compound of Formula 1 of the present invention is characterized in that it has an oxime bond. The oxime bond has a property that the bond is decomposed only at an acidic pH and has high stability at a neutral pH.

In one embodiment of the present invention, the pH stability of the oxime linker of Formula 1 and the conventional hydrazone linker was confirmed. As a result, the hydrazone bond was decomposed by about 30% or more in the conventional hydrazone linker at pH 7.0, It was confirmed that the oxime linker has an effect of separating the oxime bond at less than 10%. This is because when the antibody-drug complex is formed using the oxime linker of the present invention and injected into the body, the linker is hardly decomposed at the normal cell environment (pH 7.0), and the side effects of the drug on the normal cells do not appear , And it can be said that it is possible to lower the side effect problem of hydrazon linker to 1/3 level in the past.

In the above formula (1), R ₁ is C n (H ₂ O) n, and n is an integer of 1 to 10. Specifically, R ₁ may be a monosus, diose, triose, tetrose, pentose, hexose, heptose, octose, nonose or decose monosaccharide. , Alpha-form or beta-form monosaccharides are all included in the present invention. Preferably, R ₁ may be tetrose, pentose, hexose, heptose, octose, nonose or decose, wherein n is an integer from 4 to 10. More preferably, R ₁ may be pentose or hexose, wherein n is 5 or 6, and examples of the pentose include xylose arabinose, ribulose, ricinose, ribose, deoxyribose and derivatives thereof But is not limited thereto. Examples of the hexose include, but are not limited to, glucose, fructose, galactose, tagatose, rosuvose, fucose, quinobose, rhamnose, altrose or allose. And R < ₁ > includes all of the monosaccharides in the open or non-open form.

The oxime derivative compound of the present invention can contain a carbohydrate moiety on one side of the oxime bond, thereby reducing the hydrophobic value upon formation of the antibody-drug complex. If the hydrophobic value is decreased in the complex, the number of drugs to be bound to the antibody can be increased, so that even a small amount of the complex can have an excellent therapeutic effect, and the aggregation phenomenon of the complex can be reduced and the stability in the body can be enhanced.

In Formula 1, R ₂ may be a substituted or unsubstituted C ₁ -C ₂₀ alkyl group. Preferably an unsubstituted C ₁ -C ₂₀ alkyl group. When the number of carbon atoms of the alkyl group in R ₂ is out of the above range, the hydrophobic property of the linker becomes stronger, which may affect the amphipathic nature.

In Formula 1, R ₃ is hydrogen (-H) or a hydroxyl group (-OH), preferably hydrogen.

In Formula 1, X may be a functional group capable of binding to the lysine of the antibody, and specifically, a functional group capable of binding with lysine of an antibody having hydrophilicity. The X may be any one selected from the group consisting of a carboxyl group (-COOH), an aldehyde group (-CHO), and a ketone group (-CO-R ₄ ). When the oxime derivative of the present invention is used as a linker in an antibody-drug complex, the hydrophilic functional group can bind to the lysine of the antibody. When the compound of the present invention is used as a linker, the antibody-drug complex does not aggregate even when a complex of a plurality of linker-hydrophobic drugs binds to the antibody due to the hydrophilic property of the antibody binding site. This is particularly important in that the problems of conventional linkers and antibody-drug complexes that can be rapidly removed in the blood by aggregation of antibody-drug complexes, lowering drug delivery efficiency and causing drug toxicity to normal cells are important.

The R ₄ may be a substituted or unsubstituted C ₁ -C ₂₀ alkyl group.

) 및 싸이올기(-SH)로 이루어진 군에서 선택된 어느 하나 일 수 있다. 본 발명의 화학식 1의 옥심 유도체를 링커로 사용하여 항체-약물 복합체를 형성하는 경우, 결합된 약물의 소수성을 낮춰, 항체-약물 복합체가 응집되는 문제를 개선할 수 있다. 이는 특히, 항체-약물 복합체의 응집에 의해 혈액 내에서 빠르게 제거되어, 약물 전달 효율을 낮추고 정상 세포에 약물 독성을 일으킬 수 있는 종래 링커 및 항체-약물 복합체의 문제점을 해결할 수 있다는 점에서 중요하다. In the above formula (1), Y may be a functional group binding to a drug and specifically a hydrophobic functional group capable of binding to a hydrophobic drug. Y is a maleimide group (

) And a thiol group (-SH). When the oxime derivative of formula (I) of the present invention is used as a linker to form an antibody-drug complex, the hydrophobicity of the bound drug can be lowered and the problem of aggregation of the antibody-drug complex can be improved. This is particularly important in that the problems of conventional linkers and antibody-drug complexes that can be rapidly removed in the blood by aggregation of antibody-drug complexes, lowering drug delivery efficiency and causing drug toxicity to normal cells are important.

According to one embodiment of the present invention, the oxime derivative may be a compound of formula 1-1.

[Formula 1-1]

In Formula 1-1, a is an integer of 1 to 20, and X and Y are as defined in Formula 1.

The oxime derivative of the present invention may be a compound represented by the following general formula (1-2).

[Formula 1-2]

In Formula 1-2, X and Y are as defined in Formula 1 above.

The oxime derivative compound having the structure of Formula 1-1 or 1-2 includes a hydrophilic functional group, a hydrophobic functional group, and an oxime bond in each of X and X on one side. Therefore, when the compound of the general formula (1-2) is used as a linker, the hydrophobic property of the complex formed can be lowered, and the stability of the complex and the drug delivery efficiency can be set.

More specifically, the oxime derivative of the present invention may be any one selected from the group consisting of formulas 1-3, 1-4, 1-5, 1-6, 1-7 and 1-8.

[Formula 1-3]

[Formula 1-4]

[Formula 1-5]

[Chemical Formula 1-6]

[Chemical Formula 1-7]

[Chemical Formula 1-8]

In the above formulas 1-7 or 1-8, R ₄ is as defined in formula (1).

According to one embodiment of the present invention, an oxime derivative of the formula 1-3 containing a carboxyl group in X and a maleimide group in Y was synthesized and its characteristics were confirmed.

The oxime derivative compound of Formula 1-3 according to one embodiment of the present invention is an amphiphilic compound having a hydrophilic moiety including a carbohydrate and a carboxyl group on one side and a hydrophobic moiety including a C ₆ alkyl group and a maleimide group on another side . When the oxime derivative compound of Formula 1-1 having the above-mentioned amphipathic property is included as a linker in the antibody-drug complex, hydrophobicity of the bound drug may be lowered and the problem of aggregation of the antibody-drug complex may be improved. This is particularly important in that the problems of conventional linkers and antibody-drug complexes that can be rapidly removed in the blood by aggregation of antibody-drug complexes, lowering drug delivery efficiency and causing drug toxicity to normal cells are important. In addition, by lowering the hydrophobic property of the drug, the number of drugs (DAR) to be bound per antibody can be increased, and the therapeutic efficiency by the antibody-drug complex can be enhanced.

According to one embodiment of the present invention, when the oxime linker-DM1 conjugate is formed by incorporating the compound of the general formula (1-3) as a linker and then binding it to Herceptin, four oxime linker-DM1 conjugates Antibody-drug complex was formed. The linker of the present invention has an excellent effect in that it has a form in which a larger number of drugs are bound than the conventional antibody-drug complex cacillas, which can increase the drug load ratio of the antibody-drug complex. In addition, the oxime linker-DM1 conjugate contained in the complex had a significantly lower hydrophobic value (Clog P) than that of the SMCC-DM1 conjugate of cassia, and the aggregation rate of the antibody-drug complex comprising the oxime linker of the present invention Is lower than the syllabary, which means that it has excellent stability and transmission efficiency in the body and has an excellent effect as a therapeutic agent.

In this aspect, the invention may be an antibody-drug complex or a pharmaceutically acceptable salt thereof.

In the present invention, an antibody-drug conjugate (ADC) is used in the sense that it includes all of complexes in which an antibody and a drug are bound by a linker compound, and an antibody comprising a linker according to an embodiment of the present invention - Drug complexes may be referred to as ADC-AL.

The antibody-drug complex (ADC) of the present invention may contain the oxime derivative of the above formula (1) or a pharmaceutically acceptable salt thereof as a linker, and specifically may be represented by the following formula (2).

(2)

), 싸이올기(-SH)로 이루어진 군에서 선택된 어느 하나이며, Drug은 약물이고, 그리고 m은 1 내지 10의 정수이다.In Formula 2, Ab is the antibody, R ₁ is a _{Cn (H 2 O) n,} n is an integer from 1 to 10, R ₂ is an alkyl group a substituted or unsubstituted C ₁ -C ₂₀ ring, R ₃ X is any one selected from the group consisting of a carboxyl group (-COOH), an aldehyde group (-CHO) and a ketone group (-CO-R ₄ ), Y is a maleimide group

), Thiol group (-SH), Drug is a drug, and m is an integer of 1 to 10.

R ₁ , R ₂ , R ₃ , R ₄ , X and Y in the general formula (2) are the same as those in the general formula (1), and overlapping descriptions are omitted.

As an example, the antibody-drug complex of the present invention may have the structure of Formula (2-1).

[Formula 2-1]

Wherein a is an integer of 1 to 20, and m, X and Y are as defined in the above formula (2).

Ab in the above formula (2) may be an antibody.

In the present invention, an antibody is used in its broadest sense and specifically includes a monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from two or more antibodies (for example, a bispecific antibody), and a target biological activity ≪ / RTI > Antibodies are proteins produced by the immune system that can recognize and bind specific antigens. In its structural aspect, the antibody typically has a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains). Each antibody has two regions, mainly a variable region and a constant region. The variable region located at the distal end of the arm of Y binds and interacts with the target antigen. The variable region comprises a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a specific antigen. The constant region in the tail of Y is recognized and interacted by the immune system. Target antigens typically have multiple binding sites, also known as epitopes, that are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen can have one or more corresponding antibodies.

In one embodiment of the present invention, the antibody may be an antibody that immunospecifically binds to an antigen. Antibodies specific for an antigen may be obtained commercially or may be prepared by methods known in the art, such as recombinant expression techniques. Nucleotide sequences encoding antibodies specific for an antigen can be obtained, for example, from the Gene Bank database or literature, or by conventional cloning and sequencing. The antibody of the present invention may be an antibody for treating a disease such as cancer or an immunological disease, and includes all commercially available antibody therapeutic agents.

Chimeric anti -CD20 antibody rituximab for the treatment of Hodgkin's lymphoma Examples of antibodies available for the treatment of a humanized antibody, wherein -HER2 Trast jumap (Herceptin ^®, Genentech, Inc.), for the treatment of non-metastatic breast cancer (Rituxan ^®, Genentech, Inc.), Thank setuk when the chimeric anti--EGFR antibody for the treatment of epidermal growth factor positive cancers such as cancer of the head and neck (eobi Tuxtla ^®, imkeulron Systems, Inc.), a humanized for the treatment of breeding the anti-integrin αvβ3 antibody ratio Thaksin ^® (Med immune Co.), Valley mumap (Benlysta ^®, Belimumab), the Cooley jumap (Soliris ^®, Eculizumab) ^®, Paris jumap to (Raptiva ^®, Efalizumab), Alessio wave septeu ( Alefacept ^®, Amevive), Ivry Thank Tomorrow (Ibritumomab), Toshio Thank Tomorrow (Bexxar ^®, Tositumomab), O Pato mumap (Arzerra ^®, Ofatumumab), Mu our lead ^{-CD3 (Orthoclone-OKT3 ®, Muromonab} -CD3), Brent Took Thank bay even when tin (Adcetris ^®, Brentuximab vedotin), jemtu jumap (Gemtuzumab), Valley-to-jumap ^{(Campath-1H ®, Alemtuzumab)} , ahbatasepteu (Orencia ^®, Abatacept), Bella other septeu (Nulojix ^®, Belatacept), Ibis removable Thank (Yervoy ^®, Ipilimumab), Trapani-to mumap (Vectibix ^®, Panitumumab), katu film somap (Removab ^®, Catumaxomab), apsik when Thank (ReoPro ^®, Abciximab), pertussis jumap (Perjeta ^®, Pertuzumab), five jumap (Xolair ^®, Omalizumab), Kana Kinugawa Thank (Ilaris ^®, Canakinumab), reel or septeu (Arcalyst ^®, Rilonacept), right stereo Kinugawa Thank (Stelara ^®, Ustekinumab), the Cleveland jumap (Zenapax ^®, Daclizumab), Thank when Basil rigs (Simulect ^®, Basiliximab), sat silica jumap (Actemra ^®, Tocilizumab), Natalie jumap (Tysabri ^®, Natalizumab), to nosu Thank (Prolia ^®, denosumab), Pavilion jumap (Synagis ^®, Pavilizumab), when inflation Rick Thank (Remicade ^®, Infliximab), ¥ Tane septeu (Enbrel ^®, etanercept), Oh, unlike mumap (Humira ^®, adalimumab), Sertoli jumap (Cimzia ^®, Certolizumab), goaltender mumap (Simponi ^®, golimumab), Romi platforms Steam (Nplate ^®, Romiplostim), bevacizumab (Avastin ^®, Bevacizumab), Raney non - mainstream Thank (Lucentis ^®, Ranibizumab) or sick River septeu including, (Eylea ^®, Aflibercept), but is not limited thereto.

The antibody-drug complex of the present invention may be linked directly to the antibody by a linker or linker-drug complex. Specifically bind to the lysine residue of the antibody.

In the formula (2), Drug means a drug, and the drug may be a compound separated or purified from a naturally occurring substance or an artificially synthesized compound. Specifically, the antibody-drug complex of the present invention is not limited as long as it has biological activity in the body, and more specifically, the antibody-drug complex of the present invention contains a linker of the oxime derivative compound of the formula (1) Is a moiety having a functional group or a bond capable of binding to Y, which is a hydrophobic moiety, is included in the present invention. The functional group or bond capable of binding to the hydrophobic moiety Y of formula (1) may be a thiol group, a maleimide group or a disulfide bond, but is not proposed here. If it is possible to bond with the oxime derivative compound of the present invention, It is not subject to restrictions. Therefore, the antibody-drug complex of the present invention is included in the present invention as long as it has a thiol group or a maleimide group and has a therapeutic activity. For example, the drug includes all compounds that have killing activity against cells or have therapeutic or improving activity against damaged cells or tissues. The drug may be, for example, an immunotherapeutic agent or an anti-cancer agent that is commercially produced and sold, but is not limited thereto.

As a specific example, a drug that forms a complex with an antibody specific for a tumor cell may be included in the present invention without being limited thereto, as long as it is a compound known to have killing activity against tumor cells. The anticancer agent may be, for example, DM1 (N2'-deacetyl-N2 '- (3-mercapto-1-oxopropyl ) -maytansine), DM2, DM3 (N2'-deacetyl-N2'- (4-methyl-4-mercapto-1-oxopentyl) -1-oxopentyl) -maytansine, but is not limited thereto.

The technical feature of the present invention is that the antibody or drug is in the form of a "linker" capable of forming an antibody-drug complex by linking the antibody and drug to the antibody, , The antibody-drug complex can be formed using the linker of the present invention, so that the scope of the present invention is not limited to the specific kind of antibody and drug.

In Formula 2, m means the number of linker-drug conjugates that can be bound per antibody, and the number thereof may be different depending on the type of the antibody and the drug.

Specifically, when the antibody is trastumum and the inclusion of the oxime linker of formula (1-1), m may be an integer of 1 to 10. According to one embodiment of the present invention, It was experimentally confirmed that four DM1 drugs were bound. This is because the drug-drug complex comprising the linker of the present invention has a higher drug loading efficiency and delivery efficiency than the conventional antibody-drug complex, have.

According to one embodiment of the present invention, when the antibody-drug complex according to Formula 2 comprises an oxime derivative compound represented by Formula 1-1 and DM1 is included as a drug, the structure of Formula 2-2 Lt; / RTI >

[Formula 2-2]

In addition, the present invention may be a drug delivery composition comprising the oxime derivative according to Formula 1 or a pharmaceutically acceptable salt thereof.

The composition for drug delivery of the present invention may be a complex of a drug having biological activity in an invivo and an oxime derivative compound according to the formula (1), or a complex of the drug-oxime derivative compound and an antibody. In this case, the drug can be efficiently delivered to the target site by the target specificity of the antibody.

In this aspect, the present invention may be a pharmaceutical composition for improving or treating disease comprising the antibody-drug complex.

The pharmaceutical composition is not limited in the type of disease in that the disease can be different depending on the type of the antibody and the drug constituting the complex.

According to one embodiment of the present invention, the composition may be an anti-cancer composition, and more specifically, a composition for treating breast cancer.

The pharmaceutical compositions according to the invention may be administered orally (e.g., by ingestion or inhalation) or parenterally (e.g., by injection, sedation, implantation, suppository) and the injection may be, for example, , Subcutaneous injection, intramuscular injection or intraperitoneal injection. The anticancer agent according to the present invention may be formulated into tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, . The various forms of the pharmaceutical composition according to the present invention can be prepared by a known technique using a pharmaceutically acceptable carrier commonly used in each formulation. Examples of pharmaceutically acceptable carriers are excipients such as excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffers, encapsulating agents, sweeteners, solubilizers, bases, , Suspending agents, stabilizers, coloring agents and the like.

The pharmaceutical composition according to the present invention may contain about 0.01 to 95% by weight of the compound of the present invention or a pharmaceutically acceptable salt thereof, though it varies depending on the form of the medicine.

The specific dose of the anticancer composition of the present invention may vary depending on the kind of the mammal including the treated person, the weight, the sex, the degree of the disease, the judgment of the doctor, and the like. Preferably, for oral administration, 0.01 to 50 mg of active ingredient per kilogram of body weight per day is administered, and in the case of parenteral administration, 0.01 to 10 mg of active ingredient per kilogram of body weight per day is administered. The total daily dose may be administered once or several times depending on the severity of the disease, the judgment of the physician, and the like.

The compound of formula (I) according to the present invention can be used as a linker in an antibody-drug complex (ADC), specifically, as a linker having an amphipathic characteristic, a problem of high hydrophobicity of conventional linkers when forming an antibody- drug complex, And can have excellent drug delivery efficiency and structural stability. Specifically, when the oxime derivative compound of the present invention having the above-mentioned amphiphilic property is included in the antibody-drug complex as a linker, the hydrophobicity of the bound drug can be lowered and the problem of aggregation of the antibody-drug complex can be improved. This is particularly important in that the problems of conventional linkers and antibody-drug complexes that can be rapidly removed in the blood by aggregation of antibody-drug complexes, lowering drug delivery efficiency and causing drug toxicity to normal cells are important. In addition, by lowering the hydrophobic property of the drug, the number of drugs (DAR) to be bound per antibody can be increased, and the therapeutic efficiency by the antibody-drug complex can be enhanced.

[Example]

Example 1. Preparation and Materials of Experiment

D-glucuronic acid, ammonium acetate and acetic acid were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Amino-oxyhexyl maleimide hydrochloride was purchased from Pe Bioscience (Hong Kong, China). For ADC-AL synthesis, Y biologics, Inc. (Herceptin) was purchased from Daejeon, Korea and the cytotoxic drug DM1 (N2'-deacetyl-N2'- (3-mercapto-1-oxopropyl) -maytansine) was obtained from AbCam (Cambridge, UK) . For RP-HPLC analysis, an Agilent 1260 system equipped with a C18 column (Shim-pack GIS-ODS) was connected to a UV-Vis detector which was detected at 214 nm. Acetonitrile (ACN) and trifluoroacetic acid (TFA) (both HPLC grade) were purchased from Sigma-Aldrich (St. Louis, Mo., USA). ESI-MS was carried out at Korea Advanced Institute of Science and Technology (Ochang, Korea). The Chem-droTM program was used for Clog P calculation. For SPR experiments, a 4-channel BIAcore 3000 optical sensor instrument with CM5 chip was used (GE Healthcare, USA). (NHS), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), potassium phosphate dibasic and monobasic, citric acid Sodium citrate, ethylene diamine tetra acetic acid (EDTA) and dimethyl sulfoxide (DMSO) were purchased from Sigma (USA). All chemicals except ACN and TFA used analytical grade. The Amicon Ultra-15 centrifugal filter unit (100 kD molecular weight cut-off) was obtained from Millipore (Germany). SEC, HIC-HPLC and LC-QTOF MS were carried out by the Osong Medical Innovation Foundation (Osong, Korea). Tumor cell lines (MDA-MB-231 and SK-BR-3) were obtained from Korean Cell Line Bank. Cell culture media and components containing RPMI 1640, fetal bovine serum (FBS), penicillin and dulbeco phosphate-buffered saline (DPBS) were purchased from Welgene, Inc. (Gyeongsan, Korea). ^{EZ-Cytox ® kit (EZ-} 3000) for cell toxicity are determined (state) and services purchased from Lab-one, which was used for the plate reader (Synergy mX) purchased from Biotek (USA). The thermal behavior of the composites was investigated using differential scanning calorimetry DSC (DSC-60, Shimadzu, Japan) and UV-1800 spectroscopy (Shimadzu Inc., Kyoto, Japan).

Example 2. Experimental Method

2-1. Synthesis of oxime linker

D-glucuronic acid and amino-oxyhexyl maleimide were bound together to form an oxime linker. Aminomaleimide has an amino-oxy functional group, and D-glucuronic acid in the open chain form contains an aldehyde group. These two functional groups are ligated through a condensation reaction to form an oxime functional group (see Scheme 1 below).

[Reaction Scheme 1]

D-glucuronic acid solution (40 mM) was prepared in 100 mM ammonium acetate buffer (pH 4.5). Amino-oxyhexyl maleimide hydrochloride (4 mM) was dissolved in methanol. Oxime ligation was performed by mixing 1 mM amino-oxyhexyl maleimide and 10 mM D-glucuronic acid in a molar ratio of 1:10. The reaction was carried out at ambient temperature for 24 hours and observed with RP-HPLC equipped with a C18 column.

2-2. HPLC analysis and oxime linker purification

The formation and quantification of the oxime linkers was monitored by RP-HPLC system with gradient elution mode. (Acetonitrile-water (90:10, v / v) and 0.1% TFA) in 100% Buffer A (acetonitrile-water (5:95, v / v) and 0.1% Respectively. After each analysis was complete, the column was equilibrated with Buffer A for 30 minutes to restore to initial conditions. Oxime linkers appearing as a single peak at 43 minutes of HPLC trace (78% ACN elution) were separated and combined for lyophilization. The resulting powder was dissolved in methanol for the next step. The oxime synthesis yield was calculated from the peak area of the HPLC chromatogram.

2-3. Oxime  Electrospray ionization mass spectrometry of linker ( Electrospray Ionization  Mass Spectrometry, ESI-MS) analysis

The molecular weight of the oxime linker was analyzed by Synapt G2 High Definition Mass Spectrometry (HDMS) -Quadrupole time-of-flight (QTOF) mass spectrometer equipped with an electrospray ion source (Waters, Milford, MA, USA). The instrument was calibrated using NaF solution. Samples were dissolved in 100% MeOH and introduced by direct injection at a flow rate of 20 l / min into an ion source operating in a positive mode. All spectra were obtained in the range of 50 to 1500 m / z. Leucine encephalin was used as a lock mass for accurate mass measurement correction.

2-4. PH stability of oxime linker

In order to confirm the pH dependency stability of the oxime linker, experiments were conducted in comparison with the hydrazone linker decomposed under acidic conditions. A crude oxime linker solution was prepared as in Example 2-2 using 50% methanol in 50% 100 mM ammonium acetate buffer (pH 4.5) at about 11 mM. The oxime linker solution was diluted with 1 X PBS (1: 9, v / v) at pH 7.0 and pH 4.0 by adjusting the pH with 1N NaOH and 6N HCl. The diluted oxime linker solution was continuously stirred in an incubator at 37 DEG C for 2 days. At different time intervals (starting at t = 0), an aliquot of solution (50 μl) was collected and analyzed by RP-HPLC. The oxime linker peak area for estimating the concentration of the truncated oxime linker was calculated using the following [Formula 1] using the initial HPLC area (at t = 0) as 100%.

[Formula 1]

% Cleaved Oxime = [1- ( Area of Oxime (t) / Area of Oxime (t ₀ ) )] x 100%

2-5. Oxime linker-DM1 conjugation

In the case of the DM1-oxime complex, the maleimide group of the oxime linker and the thiol group of the DM1 drug were conjugated (see Scheme 2 below).

[Reaction Scheme 2]

A DM1 stock solution (about 6.7 mM; free thiol form) was prepared in DMSO. The DM1 stock solution was diluted in sodium citrate buffer (35 mM at pH 6.0 with 150 mM NaCl and 2 mM EDTA) to reach a DM1 concentration of 1.3 mM. The purified oxime linker was mixed with DM1 solution for complex formation. Complex formation efficiency was compared between purified oxime linker and crude oxime linker (without RP-HPLC fraction). In both cases, the molar ratio of DM1 to oxime linker was 1: 1 and the complexation reaction was carried out at room temperature with stirring for 4 to 20 hours. The complexation product was analyzed by RP-HPLC and ESI-MS in the same manner as used for oxime linker analysis.

2-6. Calculation of hydrophobic value (Clog P)

When hydrophobic DMl is complexed to the amphipathic oxime linker, its hydrophobicity should be substantially reduced due to the carbohydrate moiety of the synthesized oxime linker. This phenomenon can be estimated by calculation of the hydrophobic value (Clog P) which enables the estimation of the hydrophobicity of the compound. This is defined as the ratio of the concentration of compound distributed in the aqueous phase to the organic phase. Clog P estimation is typically performed using a program such as DaylightTM or ChemDroM software. The Clog P calculation program divides the molecule into fragments and adds the fragment values and the structure-dependent correction values in the Hansch and Leo database to predict the log P of the various organic molecules. Typically, when Clog P > 0, it is characterized by a more hydrophobic material partitioned in the organic phase, whereas Clog P < 0 represents a hydrophilic molecule.

2-7. Bio conjugation of antibody drug conjugates

21 μM of purified Herceptin ^® was buffer exchanged with 50 mM potassium phosphate and 2 mM EDTA (pH 7.0). The carboxylic acid of the oxime linker was activated with EDC and NHS solution to conjugate the carboxylic acid group of the oxime linker and the amine group of the antibody through amine coupling. The mole ratio of oxime, EDC, and NHS was 1: 7: 10.

The activated crude oxime linker solution in 100 mM ammonium acetate buffer (pH 4.5) was added to the antibody solution so that the final oxime linker to Ab mole ratio was 10: 1. The reaction proceeded with mixing in phosphate buffer for 4 hours at room temperature. The oxime linker-modified antibody was then purified via dialysis filtration through a 100 kD MWCO Amicon Ultra-15 centrifugal filter unit for sodium citrate buffer (35 mM at pH 6.0) containing 150 mM NaCl and 2 mM EDTA .

The antibody-oxime linker intermediate was then reacted with the DMl drug. DM1 dissolved in DMSO was added to the oxime linker-antibody so that the molar ratio of DM1 to antibody was 10: 1. The reaction was carried out in citrate buffer for 6 to 12 hours with stirring at room temperature. The conjugation mixture was purified through the same 100 kD Amicon filter unit with 1 x PBS (pH 7.4) to remove excess DM1. The final ADC-AL product was stored at -20 ° C and analyzed by surface plasmon resonance (SPR), size exclusion-HPLC, SEC-HPLC, HIC (butyl) HPLC and LC-QTOF MS.

2-8. Bond analysis using surface plasmon resonance

Using a CM5 sensor chip mounting surface plasmon resonance (SPR) biosensor (Biacore 3000, GE Healthcare, USA ) (1) to Herceptin ^® and (2) DM1- oxime complex of Herceptin ^® to the D- glucuronic acid The molecular-to-molecule binding was analyzed.

Herceptin (Herceptin ^® ) in potassium phosphate buffer (34 μM at pH 7.0) containing 2 mM EDTA was immobilized on CM5 chip surface activated with 70 mM EDC and 50 mM NHS in the same phosphate buffer (pH 7.0). Unbound Herceptin was washed with glycine (pH 1.5). 5 mM of inactivated D-glucuronic acid was supplied to confirm that D-glucuronic acid did not bind to Herceptin. Thereafter activate D- glucuronic acid (5 mM) was provided for the combined analysis of the Herceptin (Herceptin ^®). To - - the active D- glucuronic acid about one mole of Herceptin (Herceptin ^®) molar binding ratio was calculated in terms of the increase in resonance units (resonance unit, RU) as a molar concentration. [Reaction Scheme 3] below shows the SPR reaction formula for binding of activated D-glucuronic acid to Herceptin.

[Reaction Scheme 3]

To convert each resonance unit (RU) to molar concentration, 1 RU corresponded to approximately 1 pg / mm ^{2 of} sample. DM1- SPR binding analysis of Herceptin ^® to the oxime linker complex was similar to the previous method of D- glucuronic acid (Scheme 4). However, since the inactivated D-glucuronic acid was found not to bind Herceptin, the activated oxime linker-DM1 (1 mM) was immobilized on the CM5 chip surface and loaded directly onto Herceptin ^® .

[Reaction Scheme 4]

2-9. Size exclusion of ADC-AL and HIC (butyl) HPLC analysis

The characteristics of ADC-AL were confirmed by SEC and HIC-HPLC. SEC was eluted with 1xPBS (pH 7.0) at 25 占 폚 at a flow rate of 1 ml / min using a Sepax SEC300 column. The chromatogram was recorded at 220 nm. Herceptin approximate molecular weight of the (Herceptin ^®) and ADC-AL was determined using a calibration curve based on the retention time (retention time) of the protein standards and the molecular weight.

HIC (butyl) -HPLC was loaded with Propac butyl column. The method was performed in 100% mobile phase B (75% v / v 50 mM sodium phosphate, pH 7.0) in 100% mobile phase A (95% v / v 50 mM sodium phosphate, 5% v / v isopropanol, 1.5 M 25% v / v isopropanol, pH 7.0) for 15 min. The flow rate was 1 ml / min, the temperature was 25 ° C, and the UV detection was performed at 214 nm. HIC chromatogram of ADC-AL was compared to the HIC chromatogram of Herceptin (Herceptin ^®) and cache ssail La (Kadcyla ^®, reference ADC).

2-10. ADC -AL liquid Chromatography / QTOF  ( Quadrapole time of flight ) analysis

The antibody and ADC-AL samples were dissolved in 1x PBS (pH 7.4). Tris (2-carboxyethyl) phosphine (TCEP) was added to the sample at 10 mM and the mixture was incubated at 60 ° C for 1 hour to reduce disulfide bonds. After cooling the sample to 37 ℃, Streptococcus payioh presence (Streptococcus pyogenes- derived immunoglobulin-degrading enzyme (IdeS) and then incubated at 37 ° C for 2 hours to digest the antibody and ADC-AL in the hinge region of the heavy chain to obtain light chain, Fd region, and Fc Area.

LC / MS / MS was performed using a Waters Acquity UPLC (ethylene bridge hybrid (BEH) C4) system coupled with a Synapt G2-Si HDMS (High Definition Mass Spectrometry) system with electrospray ion positive mode. The antibody fragment was separated by gradient elution using water as mobile phase A and acetonitrile as mobile phase B (both containing 0.1% formic acid).

The gradient elution was used as follows; 0 to 10% B for 0 to 2 minutes, 90% B for 10 to 90% B for 15 to 17 minutes, 90% to 10% B for 17.0 to 17.1 minutes, 10% B for 17.1 to 20 minutes .

MS scans were performed on Synapt G2-Si HDMS. The electrospray ionization voltage was 3.0kV and the sampling cone voltage was 60V. The source temperature was 120 占 폚 and the MS scan speed was 1 second at a mass range of 700-4000 m / z.

2-11. Cell death (cytotoxicity) analysis

Herceptin and ADC-AL assays for tumor cell viability were performed using WST-1 assay. MDA-MB-231 and SK-BR-3 cell lines were treated with about 10,000 cells per well in a 96-well plate and adhered overnight in a humidified atmosphere of 5% CO ₂ at 37 ° C. The next day, cells of only PBS buffer or Herceptin (Herceptin ^®), a cache ssail La (Kadcyla ^®) and ADC-AL was treated with drugs including from 0.001 to 5㎍ / ml concentration.

Repeat for 3 wells for each cell treatment for accurate experiments. Four days after treatment, WST-1 proliferation assay was performed [Jung, S. et al. Oleic acid-embedded nanoliposome as a selective tumoricidal agent. Colloids and Surfaces B: Biointerfaces 2016, 146, 585-589., 40]. The results are expressed as relative values (%) to the control (PBS treated cells only).

[Example 3] Results and discussion

3-1. Reverse phase RP for oxime linker-HPLC analysis

RP-HPLC was performed to observe the residence time and synthesis yield of the oxime linker of the present invention. Retention times of D-glucuronic acid and amino-oxyhexyl maleimide in the HPLC trace were 16 and 40 minutes respectively, but the oxime product eluted at 43 minutes (number 3 in Figure 1)

As shown in Figures 1a and 1b, it is an interesting result that the residence time (43 min) of oxime is longer than the amino-oxymaleimide (40 min, number 2 in Figure 1) despite the presence of hydrophilic carbohydrate moieties. During the 12 hour reaction, the peaks of D-glucuronic acid and amino-oxymaleimide drastically decreased while the peaks corresponding to the oxime product increased. After about 4 hours, almost all of the amino-oxymaleimide was consumed and the oxime synthesis appeared to be complete. The yield of oxime synthesis calculated from HPLC chromatogram area was over 90%.

The carbohydrate oxime linker was then purified by fractionation of the 43 minute peak of RP-HPLC and lyophilized. The purified oxime linker was re-introduced into RP-HPLC to confirm the purity, and some cohesive peaks were observed after 43 minutes; 46, 47 minutes (see FIG. 2B). It was shown that the oxime product could be damaged during the freeze drying process. To solve this problem, the present invention used a carbohydrate oxime ligation mixture as a crude product without RP-HPLC purification.

As shown in Figures 2a and 2b, the crude oxime linker showed a relatively clearer peak than the purified (Figure 2a): main peak of 43 min (ACN elution 78%) corresponding to the oxime product, Very small peaks at 16 and 40 minutes corresponding to D-glucuronic acid and amino-oxymaleimide, respectively.

3-2. ESI-MS analysis of oxime linkers

The positive mode ESI-MS analysis spectrum showed that the molecular weight of the oxime linker was 388 Da. The ESI-MS results showed two main peaks; 393 Da for [M + Na ⁺ -H ₂ O] ⁺ and 411 Da for [M + Na] ⁺ , indicating that the formed oxime product is the same as the molecular weight and possibly the theoretical value of the structural formula (FIG.

3-3. Stability of oxime linker to pH

In order to confirm the pH stability of the oxime linker according to the present invention, the pH stability of an oxime linker and a hydrazone linker, each having an oxime and hydrazone bond decomposed under acidic conditions, were compared.

Both linkers were incubated in 1xPBS (37 ° C) for 50 hours at pH 7.0 and pH 4.0 and analyzed by RP (C18) -HPLC. At pH 7.0, about 30% of the hydrazone linker was degraded, but the oxime linker synthesized according to the present invention showed excellent stability, degrading to less than 10% in the same buffer. Also, when incubated in PBS at pH 4.0, both linkers were rapidly degraded (Figure 4).

Therefore, it was confirmed that the carbohydrate oxime linker of the present invention is an acid-decomposable linker that is very stable at neutral pH and cleaved at an acidic pH such as lysosome pH. However, conventional hydrazone linkers used as comparative examples are partially degraded at neutral pH, which adversely affects normal cells during blood circulation. As a result, it can be seen that the carbohydrate oxime linker according to the present invention is more efficient and stable than conventional hydrazone linkers.

In addition, the half-life of the hydrazone and oxime linkers was calculated using a formula assuming an exponential decay in PBS of pH 7.0.

N (t) = N ₀ exp (-λ t)

Where N (t) is the concentration of the linker at time t, N ₀ is the initial concentration at t = 0, and 1 / λ is the exponential decay constant.

The half-life was calculated as t1 / ₂ = ln2 / lambda. The oxazole linker exhibited a slow degradation with a half-life of about 99 h, while the hydrazone linker exhibited a relatively rapid degradation, an average half-life of about 43 h. Therefore, it can be seen that the oxime linker according to the present invention is more stable than the conventional hydrazone linker under neutral pH conditions, and has an effect of being cleaved at an acidic pH condition close to the lysosome environment, thereby being more suitable for systemic administration.

3-4. RP-HPLC and ESI-MS analysis of oxime linker-DM1 complex

Due to the isomer, DM1 produced two RP-HPLC peaks at residence times of 62.0 and 66.0 min (Fig. 5A). The purified oxime linker complexed with DM1 and the reaction mixture was analyzed by RP-HPLC to reveal six peaks; All peaks appeared between 50.0 minutes and 55.0 minutes; 50.6, 51.2, 52.6, 53.2, 53.7, 54.3 minutes (Fig. 5B). Each peak was collected for identification using ESI-MS. The peak eluted at 50.6 and 51.2 min was found to be the oxime linker-DM1 complex. However, besides the oxime linker-DM1 complex, other substances eluted in ESI-MS and the product state was not clear. This result appears to be related to previous RP-HPLC results, in which the lyophilized oxime linker exhibits some cohesive peaks.

Thus, a crude oxime linker solution was used to obtain a more specific oxime linker-DM1 complex. When conjugated using crude oxime linker solution, two peaks appeared at 50.4 and 51.0 minutes (Fig. 5C). Since the linker-drug complex exhibits a shorter residence time than DM1 (51.0 and 62.0 min), it can be seen that the hydrophobicity of DM1 is substantially reduced by complex formation.

In addition, crude oxime linker-DM1 complex (50.0 and 51.0 min) was purified by RP-HPLC, then evaporated and analyzed by positive mode ESI-MS. The eluted peaks at 50.4 and 51.0 min showed the same deconvoluted ESI-MS spectrum, indicating that the two peaks are the same material (isomer) [42]. The theoretical molecular weight of the oxime linker-DM1 complex is 1,126 Da. The ESI-MS results for the two complexes showed two major peaks (Figure 6). [M + Na ⁺ -H ₂ O] ⁺ is 1,130 Da, and [M + Na] ⁺ is 1,148 Da, which represents the expected complex formation. These results are consistent with the ESI-MS results of the DM1 peak.

3-5. Comparison of hydrophobic values (Clog P) between oxime-DM1 complex and SMCC-DM1

Table 1 shows ClogP values for DM1, SMCC linkers, amphiphilic oxime linkers and DM1-linker complexes.

Oxime linker -DM1 SMCC linker -DM1
( reference - cadillac ) Clog  P Oxime linker - -2.41 - SMCC linker 0.41 DM1 drug DM1 drug 4.18 Oximelinker-DM1 - 2.17 - SMCC-DM1 4.99

The ClogP value of the oxime linker is -2.41, the ClogP value of the SMCC linker (used in the reference ADC, Kadcyla ^® ) is 0.41, and the oxime linker according to the present invention shows a lower hydrophobicity. When DM1 (Clog P = 4.18) complexed with the oxime linker, the hydrophobicity of the complex was significantly reduced to 2.17, but increased to 4.99, the hydrophobic value of the complex when combined with the SMCC linker. Thus, through ClogP comparison, it can be expected that the antibody-drug conjugate (ADC-AL) using the amphiphilic oxycin linker of the present invention can improve the aggregation problem of the complex due to hydrophobic interactions.

3-6. Oxime  Linker-DM1 complex and Herceptin ^®  Between On conjugation  About SPR  Bonding study

The SPR sensorgram is shown in Fig. Herceptin (Herceptin ^®) to CM5 chips (the cost that is, the carboxyl group is esterified with EDC / NHS) were fixed on (step B 7), activated D- glucuronic shown that the conjugate on the surface of the antibody (Step E of FIG. 7), whereas inactive D-glucuronic acid was not reactive (step D of FIG. 7).

The change in the resonance unit (RU) at each step is also shown in Table 2 by changing the molar concentration.

RU _b RU _a △ RU Pmol E / N activation 18564 19385 821 7.1 Ab conjugation 19385 23279 3894 0.026 D-glu conjugation 23279 23280 One 0.005 Activated D-glu conjugation 23280 23409 129 0.66

As shown in Table 2, about 0.66 pmol of activated D-glucuronic acid was conjugated with 0.026 pmol of Herceptin, which results in a binding molar ratio of about 1:25 between fixed Herceptin and activated D-glucuronic acid Is high.

To Make sure there are not modified D- glucuronic acid does not bind to Herceptin, fixed to the activated oxime linker -DM1 conjugate Herceptin (Herceptin ^®) was loaded directly. Figure 8 shows the results of SPR analysis of Herceptin for the sensorgram and the oxime-DM1 complex activated in Table 3.

RU _b RU _a △ RU Pmol E / N activation 18942 19227 285 2.4 Ab conjugation 19227 23485 4258 0.028 Activated Oxime-DM1 conjugation 23485 23614 129 0.11

For the oxime linker-DM1 complex, the molar-to-mole conjugation ratio was about 1: 4 lower than D-glucuronic acid. Because DM1 is hydrophobic and can have steric hindrance to hydrophilic antibodies, the available space for structurally accessible structures is reduced, suggesting that the coupled oxime linker-DM1 complex is smaller than D-glucuronic acid [44]. However, the drug-antibody complex (ADC-AL) according to the present invention exhibited a better drug-to-antibody ratio than the cassia.

3-7. Size Exclusion Chromatography Analysis for ADC-AL

FIG. 9 shows the results of the SEC chromatogram for the ADC-AL of the present invention.

Herceptin (about 145 kD) eluted at 7.8 min and showed agreement with the standard curve. The ADC-AL containing the amphipathic oxime linker of the present invention was eluted at about the same time (7.8 minutes) and its molecular weight appeared similar to Herceptin. Both chromatograms showed no aggregation or fragmentation peaks, indicating that both samples had high purity.

3-8. HIC (butyl) HPLC analysis for ADC-AL

The ADC-AL of the present invention was prepared by random conjugation between the amine group (lysine residue) of Herceptin and the NHS functionalized ester group of the carbohydrate oxime linker-DM1 complex. HIC-HPLC was performed to determine the effect of the linker-drug complex on the hydrophobicity of the ADC.

Figure 10a shows that Herceptin eluted at 10.8 min and ADC-AL eluted at 11.8 min. The peak of ADC-AL was broader and shifted to the right compared to the sharp peak of Herceptin. Through this, ADC-AL can be conjugated to DM1 and expected to be more hydrophobic than Herceptin. In addition, the synthesized ADC-AL appeared homogeneous in its molecular entity and drug to antibody ratio.

As shown in Fig. 10b, in the case of cadillac (Kadcyla ^® ), unconjugated Herceptin was eluted at 8.4 min and the conjugated cassia (Kadcyla ^® ) was moved to the right. Several linker-DMl conjugates were conjugated and several peaks appeared. Hence, it has been shown that the cassette has heterogeneous and various drug to antibody ratios.

3-9. LC-QTOF analysis results for ADC-AL

Herceptin and the ADC-AL according to the present invention were cleaved with the IdeS enzyme and reduced to light chain, Fd region and Fc region. Fig. 11 shows QTOF-LC / MS data.

The Fc region of Herceptin has glycans and the exact molecular weights including these glycans are 25,237 Da (G0F glycans and Fc) and 25,399 Da (G1F glycans and Fc). The molecular weights of antibody light chain and Fd region were 23,442 Da and 25,382 Da, respectively. Compared with Herceptin (Herceptin ^®), ADC-AL of this invention showed an increase in molecular weight from 1,937 Da light chain. This means that two molecules of the carbohydrate oxime linker-DM1 complex are bound to each light chain of Herceptin [46]. These results are also consistent with HIC (butyl) -HPLC results that the synthesized ADC-AL is homogeneous. Since the antibody molecule has two light chains, the fact that two linker-drug complex molecules are bound to each light chain of Herceptin means that four linker-drug complexes are conjugated to the whole antibody molecule. This result is consistent with the result of combining with the 1: 4 DAR of the previous SPR data.

3-10. Cytotoxicity assay (WST-1 assay).

In vitro Herceptin on cellular potency (Herceptin ^®), caviar ssail La (Kadcyla ^®) and the ADC-AL effects of the present invention was confirmed by the WST-1 analyzes for the two cell lines; MDA-MB-231 (HER2 negative cell line) and SK-BR-3 (HER2 positive cell line). These cell lines have been established by human epidermal growth factor receptor 2 (HER2) expression levels which are important biomarkers of breast cancer.

When processing the Herceptin (Herceptin ^®), caviar ssail La (Kadcyla ^®) and ADC-AL according to the invention in MDA-MB-231 cells, respectively, and incubated 4 days, there was no sensitivity due to the lack of HER2 expression (FIG. 12a) .

However, when SK-BR-3 cells overexpressing HER2 were treated with Herceptin, cell proliferation was reduced by 40% in a dose-dependent manner. In the case of the ADC-AL of the present invention, cell growth was suppressed to 52%, and Kadcyla ( ^R ) showed about 90% maximal apoptosis (Fig. 12B).

In conclusion, Herceptin (Herceptin ^®), caviar ssail La (Kadcyla ^®) and ADC-AL according to the invention exhibit high selectivity for the HER2-positive breast cancer cell line, free DM1 is the equivalent efficacy in all cell lines irrespective of HER2 over-expressing . ADC-AL showed a 12% increased activity in SK-BR-3 cells compared to Herceptin ^®.

Claims (8)

An oxime derivative of the formula (1): &lt; EMI ID =
[Chemical Formula 1]

(In the formula 1,
R ₁ is C n (H ₂ O) n, n is an integer of 1 to 10,
R ₂ is a substituted or unsubstituted C ₁ -C ₂₀ alkyl group,
R ₃ is hydrogen or a hydroxy group,
X is a carboxyl group, an aldehyde group or a ketone group, and
Y is a maleimide group or a thiol group) The method according to claim 1,
Wherein R ₁ is tetraose, pentose, hexose, heptose, octose, nonose or decose, wherein n is an integer of 4 to 10, or an oxime derivative or a pharmaceutically acceptable salt thereof. The method according to claim 1,
Wherein the oxime derivative is a compound represented by the following formula (1-1) or a pharmaceutically acceptable salt thereof.
[Formula 1-1]

(In the above formula 1-1,
a is an integer of 1 to 20,
X is a carboxyl group, an aldehyde group or a ketone group, and
Y is a maleimide group or a thiol group) An antibody-drug conjugate of the formula (2): &lt; EMI ID =
(2)

(In the formula (2)
Ab is an antibody,
R ₁ is C n (H ₂ O) n, n is an integer of 1 to 10,
R ₂ is a substituted or unsubstituted C ₁ -C ₂₀ alkyl group,
R ₃ is hydrogen or a hydroxy group,
X is a carboxyl group, an aldehyde group or a ketone group,
Y is a maleimide group or a thiol group,
Drug is a drug, and m is an integer from 1 to 10) 5. The method of claim 4,
Wherein R ₁ is an antibody-drug complex in which n is an integer of 4 to 10, such as tetrose, pentose, hexose, heptose, octose, 5. The method of claim 4,
Wherein the antibody-drug complex is a complex of formula (2-1).
[Formula 2-1]

(In the above formula (2-1)
Ab is an antibody,
X is a carboxyl group, an aldehyde group or a ketone group,
Y is a maleimide group or a thiol group,
A is an integer from 1 to 20, and m is an integer from 1 to 10, 5. The method of claim 4,
Said antibody is selected from the group consisting of trastuzumab, rituximab, cetuximab, vitaxin, velimovat, eculizumab, epiparum, alepepept, ibritumom, tositumom, opatumat, , Rhamnosep, rhamnose, rhamnose, rhamnose, rhamnose, rhamnose, rhamnose, rhamnose, rhamnose, rhamnose, rhamnose, But are not limited to, but are not limited to, streptomycin, streptomycin, stevanumab, daclizumab, basilicicum, tociliumum, natalizumab, denosumab, pavilionumip, infliximab, entanesecept, Wherein the antibody-drug complex is selected from the group consisting of raloxifene, xanthan gum, steam, bevacizumab, ranibizumab, and influenzacept. A pharmaceutical delivery composition comprising the oxime derivative according to claim 1 or a pharmaceutically acceptable salt thereof.

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