WO2024225837A1 - Maleimide-containing linker-payload conjugate and novel antibody-drug conjugate including same - Google Patents

Abstract

The present invention provides a maleimide unit-containing linker-payload conjugate and a novel antibody-drug conjugate including same.

Description

Maleimide-containing linker-payload conjugates and novel antibody-drug polymers comprising the same

The present invention relates to a linker-payload conjugate comprising a maleimide unit and a novel antibody-drug polymer comprising the same.

Antibody-Drug Conjugate (ADC) is a new drug that combines the specificity of antibodies and the cytotoxicity of drugs to increase TI. About 10 types of ADC have been approved by the FDA so far, but the number is not large. Early ADCs used a method of binding to the amine group of lysine exposed on the surface of antibodies or a bond using cysteine obtained by reducing the inter-chain disulfide bond of native antibodies.

Since the introduction of Paul Ehrlich’s “magic bullet,” a variety of monoclonal antibody (mAb) therapeutics have been developed. These mAbs have effectively replaced small-molecule compounds for cancer therapy, offering the remarkable advantage of precise targeting. The advent of antibody-drug conjugates (ADCs) was a notable milestone in the market in the early 2000s. According to the global sales forecast of ADCs from 2020 to 2026, the market shares of T-DM1 (Kadcyla®) and T-Dxd (Enhertu®) are projected to exceed $2.4 billion and $6.2 billion, respectively. This clearly shows that ADCs are now gaining ground in a space previously dominated by mAbs and small-molecule drugs.

Meanwhile, metastatic breast cancer accounts for 15-20% of breast cancers, occurs frequently in young people under 50 years of age, and is a disease with a poor prognosis that exhibits rapid cancer cell growth, high recurrence rate, and metastasis.

Breast cancer, which has risen to the top of the list of cancers for women in Korea, recorded approximately 222,000 patients in 2019, an increase of approximately 41.8% compared to 2015, showing a steep increase in incidence. According to the 2019 cause of death statistics, the breast cancer mortality rate in Korea was 5.1 per 100,000 people, an increase of 6.8% compared to the previous year, and it was the number one cause of death for people in their 30s among cancer-related deaths.

The most basic treatment for breast cancer is surgical resection of the lesion, and for metastatic breast cancer treatment, endocrine therapy, chemotherapy, and targeted therapy are mainly used. Many breast cancer patients receive endocrine therapy as a first-line treatment, but they experience disease progression due to resistance during treatment, and in the case of patients who must receive chemotherapy, there is a problem that their quality of life inevitably worsens due to side effects such as vomiting, general weakness, and hair loss.

In the case of existing anticancer drugs, since they target cancer cells that are constantly dividing and cause serious side effects in other organs of the body where proliferation is active, the development of targeted anticancer drugs that target only specific target factors of cancer cells is emerging, and representative breast cancer targeted anticancer drugs include Herceptin (trastuzumab), Perjeta (pertuzumab), and Avastin (bevacizumab).

Antibody-Drug Conjugate (ADC) applied to anticancer drugs is a technology that conjugates chemicals, toxins, radioactive substances, enzymes, etc. that are effective in treating diseases to antibodies as a way to maximize the resistance and efficacy of existing antibody-targeted anticancer drugs.

Current antibody-drug conjugates indicated for the treatment of metastatic breast cancer include Kadcyla (trastuzumab emtansine), which was approved by the FDA in 2013, Enhertu (lastuzumab deruxtecan), and Trodelvy (sasituzumab govitecan).

Most of the drugs used in existing antibody-drug polymers are highly toxic substances that are involved in DNA cleavage or microtubule inhibition, such as DM1 of the maytansinoid series or MMAE of the auristatin series. However, the linker connecting the antibody and the drug is unstable and the drug-to-antibody ratio (DAR) is not constant, so the absorption rate in the blood is low and the exposure time in the blood is long, which may cause problems with cytotoxicity.

Although the recently approved Enhertu and Trodelvy use relatively less toxic drugs, there is still a need for the development of new antibody-drug conjugate anticancer agents that are more stable and have fewer toxic side effects.

In order to exhibit accurate efficacy as a pharmaceutical, it is important that the antibody and drug are always combined at a constant ratio. In addition, the number of drugs combined also affects the in vivo pharmacokinetic properties of the ADC.

Currently, among the FDA-approved ADCs, two drugs are known to target HER2-positive BC: Kadcyla (T-DM1) and Enhertu. Both ADCs use trastuzumab, an anti-HER2-positive BC IgG1, and use maleimide as a linker terminal functional group that binds to the inter-chain disulfide bond of the antibody.

Enhertu uses deruxtecan as the drug and has a DAR of approximately 7.7, which is relatively homogeneous, but is still heterogeneous.

Kadcyla is an early-stage ADC in which the tubulin inhibitor drug DM1 is conjugated to the SMCC linker, a non-cleavable linker, via the lysine residue of trastuzumab. It is a heterogeneous ADC with a DAR of approximately 3.5. Kadcyla®, the first ADC approved for solid tumors (breast cancer), has grown into a blockbuster drug in the past few years. The payload of Kadcyla®, DM1, has a narrow therapeutic index (TI) range, making it difficult to use it alone in patients. However, DM1, a drug with relatively good solubility, high stability, and excellent efficacy, is also a drug very suitable for ADC development.

Trastuzumab, the antibody of Kadcyla®, is a recombinant monoclonal antibody targeting HER2. The target of trastuzumab, HER2 protein, is overexpressed in various cancer types, including breast cancer, stomach cancer, colon cancer, ovarian cancer, lung cancer, and head and neck cancer. Since Kadcyla®, many HER2-targeting ADCs have been developed, and HER2 currently accounts for a significant portion of ADC targets.

Previously, ADC synthesized by reducing inter-chain disulfide bonds was known to have an optimal DAR value of 2 to 4. This is because a higher DAR may show weakness in PK properties even though the efficacy is better. However, if the high DAR of the ADC can be maintained while controlling toxicity and stability, the higher and more uniform the DAR, the better the ADC can be evaluated.

Meanwhile, linkers that can be used to manufacture ADCs include non-cleavable linkers and cleavable linkers. ADCs containing non-cleavable linkers undergo catabolism by cytoplasmic and lysosomal proteases after internalization, and cytotoxic drugs are released in a form bound to the linker. Therefore, ADCs with non-cleavable linkers have the advantage of high stability during systemic blood circulation, but have the disadvantage that the efficacy of the original cytotoxic drug may be reduced because the cytotoxic drug exists in a form bound to the linker rather than as a single cytotoxic drug.

There are many different types of cleavable linkers. Chemically cleavable linkers are acid-labile linkers that are stable at neutral pH in blood but are cleaved by acid in the acidic environment of endosomes or lysosomes (pH 5-6) within cells, and reducible linkers that are reduced and cleaved by reducing substances that are relatively abundant in cancer cells. pH-dependent linkers are unstable in blood circulation, and there is a concern about systemic toxicity. Enzymatically cleavable linkers are designed to be cleaved by enzymes that are relatively abundant in target cells, and are mainly designed to cleave peptide bonds or be cleaved by specific enzymes such as β-glucuronidase.

In addition to whether the linker is cleavable, the site at which the linker is attached to the antibody is also an important factor in determining the efficacy and side effects of ADC drugs. The linker attachment site can be classified into lysine, interchain cysteine residue amino acid, and site-specific.

Looking at ADC drugs currently in preclinical and clinical trials with payloads of DM1 and DM4, SMCC, SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate), SPDB (N-succinimidyl-4-(2-pyridyldithio)butanoate) etc. are used, and the structure is to connect the SH at the end of the payload with a linker and then connect it to the lysine residue of the antibody.

As mentioned above, the conjugation of lysine residues causes various problems such as non-specific and diverse DAR generation-induced side effects and regulatory issues, so the strategy of linking DM1 to cysteine residues of antibodies was chosen. However, this also had limitations in producing ADCs with uniform DARs. Maintaining a constant DAR has become a key competitive edge in ADC development, and although various methods are currently being attempted, satisfactory results have not been reported.

The present invention relates to a novel linker-payload that can be used to manufacture ADCs. It has been confirmed that when the linker-payload including the novel maleimide group of the present invention is used, an ADC having a highly uniform DAR (e.g., DAR = 8.0) can be manufactured through a very simple synthetic process with a high conjugation yield.

Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is a linker with considerable efficiency and simplicity, characterized by the presence of maleimide and succinimide ester functionalities on both sides. Upon attachment to the free thiol of DM1, SMCC forms a linkage with the maleimide functionality via 1, 4-addition. Consequently, SMCC-DM1 is expected to bind to antibodies by attacking the succinimide ester with the free amine of antibody surface lysines. IgG1 displays a surface distributed with approximately 70–80 lysine residues, thereby exhibiting inherent heterogeneity even without antibody engineering. Heterologous ADCs are known to have several vulnerabilities during clinical pharmacokinetic evaluation. Therefore, SMCC-DM1 offers a simple and convenient synthetic approach, but has clear limitations.

The inventors of the present invention have endeavored to generate a linker similar to SMCC through simple synthesis, and have developed a homogeneous ADC utilizing an improved linker compared to SMCC linker using the antibody-drug (trastuzumab-DM1) combination validated in Kadcyla®. Specifically, when using DM1 as a payload, the facile combination of free thiol and maleimide functionalities of DM1 was considered. Taking advantage of the presence of free thiols in both trastuzumab and DM1, a dimaleimide linker featuring maleimide functionalities at both ends was designed. Subsequently, experiments were conducted to determine whether the stability and efficacy of the ADC differ depending on the length of the dimaleimide linker.

Furthermore, to synthesize a more homogeneous ADC than Kadcyla®, all four interchain disulfide bonds were reduced before conjugating the linker payload to most of the obtained free thiol functionalities. Subsequently, all synthesized ADCs were analyzed and showed a DAR close to 8, reminiscent of T-Dxd (Enhertu®), confirming that this novel ADC has a higher DAR (DAR = 7~8) than the conventional one (DAR = 2~4) while carrying DM1 as a payload, can be prepared through a very simple synthetic process, and can provide an ADC with a high DAR and a high conjugation yield.

In addition, for the purpose of providing a treatment for metastatic breast cancer, a novel single ADC compound with improved efficacy and low side effects was developed, and the novel ADC according to the present invention exhibited the desired level of efficacy in an in vivo mouse model, and it was confirmed that it can be used as a pharmaceutical composition for preventing or treating metastatic breast cancer, thereby completing the present invention.

Prior art literature

(Patent Document 0001) Republic of Korea Registered Patent No. 10-2442906

(Patent Document 0002) Republic of Korea Publication Patent No. 10-2023-0008723

(Patent Document 0003) Republic of Korea Publication Patent No. 10-2019-0038579

(Non-patent literature 0001) Chemical Science (2022), 13(10), 2909-2918

(Non-patent literature 0002) Bioconjugate Chem. 2021, 32, 8, 1525-1534

(Non-patent literature 0003) Clin Cancer Res (2004) 10 (20): 7063-7070.

(Non-patent literature 0004) J. Med. Chem. 2014, 57, 16, 6949-6964

(Non-patent literature 0005) Mol. Pharmaceutics 12, 3986-3998.

The present invention seeks to provide a linker-payload conjugate comprising a maleimide unit and a novel antibody-drug polymer comprising the same.

One aspect of the present invention provides a linker-payload conjugate represented by the following general formula I:

[General Formula Ⅰ]

M ₁ -SU-M ₂ -XP

In the above formula,

M ₁ and M ₂ are maleimide moieties,

SU is a first spacer unit represented by (SU ₁ ) _a -(SU ₂ ) _b -(SU ₃ ) _c , and SU ₁ , SU ₂ , and SU ₃ may be the same or different, and a, b, and c are each independently integers from 0 to 3,

X is a second spacer unit expressed as (X ₁ ) _x -(X ₂ ) _y -(X ₃ ) _z , wherein X ₁ , X ₂ , and X ₃ may be the same or different, and x, y, and z are each independently integers from 0 to 3,

P is the payload.

Another aspect of the present invention provides a ligand-drug conjugate represented by the following general formula II:

[General Formula II]

Ab-(M ₁ -SU-M ₂ -XP) _n

In the above formula,

Ab is a ligand,

M ₁ and M ₂ are maleimide moieties,

SU is a first spacer unit represented by (SU ₁ ) _a -(SU ₂ ) _b -(SU ₃ ) _c , and SU ₁ , SU ₂ , and SU ₃ may be the same or different, and a, b, and c are each independently integers from 0 to 3,

X is a second spacer unit expressed as (X ₁ ) _x -(X ₂ ) _y -(X ₃ ) _z , wherein X ₁ , X ₂ , and X ₃ may be the same or different, and x, y, and z are each independently integers from 0 to 3,

P is the payload,

n is an integer from 1 to 20.

Another aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising the ligand-drug conjugate.

Another aspect of the present invention provides a method comprising the step of administering the pharmaceutical composition to a subject in need thereof.

The novel maleimide-containing linker-payload conjugate according to the present invention can be used to prepare an antibody-drug conjugate (ADC), and when the linker of the present invention is used, there is an advantage in that an ADC having a highly uniform DAR (e.g., DAR = 8.0) can be provided.

Figure 1 shows a connection method of ADC 1-5 according to one embodiment (Embodiment 3) of the present invention.

FIG. 2 shows the structure of an antibody-drug polymer manufactured according to one embodiment of the present invention (Comparative Example 1 and Comparative Example 2), where (a) is Comparative Example 1 and (b) is Comparative Example 2.

Figure 3 shows the results of DAR analysis according to the conjugation method according to an experimental example of the present invention.

Figure 4 shows the results of DAR analysis of an antibody-drug polymer (ABC-002) according to an experimental example of the present invention.

Figure 5 shows the results of measuring the yield of ADC according to an experimental example of the present invention.

Figure 6 shows hydrophobic interaction chromatography (HIC) data of (a) trastuzumab; (b)-(f) ADC 1-5, respectively, according to an experimental example of the present invention.

Figure 7 shows the MS analysis results according to an experimental example of the present invention, showing one linker-payload detected in the light chain (left) and three linker-payloads detected in the heavy chain (right), namely (a) linker-payload 2 (MW ~972); (b) linker-payload 3 (MW ~986); and (c) linker-payload 4 (MW ~1,000).

Figure 8 shows the mass analysis results of ADC 2-4 according to an experimental example of the present invention; TIC spectra and DAR calculation table. (a) ADC 2; (b) ADC 3; and (c) ADC 4.

Figure 9 is a size exclusion chromatography (SEC) spectrum for aggregates of ADC 2-4 according to an experimental example of the present invention, namely (a) ADC 2; (b) ADC 3; and (c) ADC 4.

Figure 10 shows the results of an in vitro test comparing the cytotoxicity against tumor cells SK-BR-3 according to an experimental example of the present invention: (a) DM1 and linker-payload 2; (b) trastuzumab and ADC 2.

Figure 11 shows a graph comparing the binding affinity of trastuzumab, T-Dxd, and ADC 2 for one week as a result of ELISA analysis for binding affinity according to an experimental example of the present invention.

Figure 12 shows the results of SDS-PAGE analysis according to an experimental example of the present invention.

FIG. 13 shows ¹ H- and ¹³ C-NMR spectra of linkers and linker payloads of the present invention, respectively: (a) linker 1; (b) linker-payload 1; (c) linker 2; (d) linker-payload 2; (e) linker 3; (f) linker-payload 3; (g) linker 4; (h) linker-payload 4; (i) linker 5; and (j) linker-payload 5.

Figure 14 shows the results of a xenograft anticancer activity efficacy test using the HER2 positive cell line SK-BR3 according to one embodiment of the present invention. (a) is a measurement of the degree of inhibition of transplanted tumor growth, and (b) is a result of a toxicity test according to weight change.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used with a meaning that can be commonly understood by a person of ordinary skill in the art to which the present invention belongs. In addition, terms defined in commonly used dictionaries shall not be ideally or excessively interpreted unless explicitly specifically defined.

As used herein, the singular form may include the plural form unless the context clearly indicates otherwise.

When it is said in this specification that a part “includes” a certain component, this does not exclude other components, but rather may include other components, unless otherwise specifically stated.

In addition, all numbers and expressions indicating the amounts of components, reaction conditions, etc. described in this specification should be understood as being modified by the term “about” in all cases unless otherwise specified.

As used herein, “pharmaceutically acceptable” means that which is approved by a governmental or equivalent regulatory body for use in animals, and more specifically in humans, by avoiding significant toxic effects when used at usual pharmaceutical doses, is listed in a pharmacopoeia, or is otherwise generally recognized by the pharmacopoeia.

Additionally, the experimental procedures specified in this specification are identical to the experimental procedures commonly performed in the art unless specifically described.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a linker-payload conjugate represented by the following general formula I:

[General Formula Ⅰ]

M ₁ -SU-M ₂ -XP

In the above formula,

M ₁ and M ₂ are maleimide moieties,

SU is a first spacer unit represented by (SU ₁ ) _a -(SU ₂ ) _b -(SU ₃ ) _c , and SU ₁ , SU ₂ , and SU ₃ may be the same or different, and a, b, and c are each independently integers from 0 to 3,

X is a second spacer unit expressed as (X ₁ ) _x -(X ₂ ) _y -(X ₃ ) _z , wherein X ₁ , X ₂ , and X ₃ may be the same or different, and x, y, and z are each independently integers from 0 to 3,

P is the payload.

In one specific embodiment of the present invention, SU ₁ , SU ₂ , and SU ₃ may each independently be selected from the group consisting of a substituted or unsubstituted C _1-30 alkylene, a substituted or unsubstituted C _1-30 heteroalkylene, a substituted or unsubstituted C _2-30 alkenylene, a substituted or unsubstituted C _3-30 cycloalkylene, a substituted or unsubstituted C _6-30 arylene, and a substituted or unsubstituted C _6-30 heteroarylene.

In another specific embodiment of the present invention, SU ₁ , SU ₂ , and SU ₃ may each independently be selected from the group consisting of a substituted or unsubstituted C _1-20 alkylene, a substituted or unsubstituted C _1-20 heteroalkylene, a substituted or unsubstituted C _2-20 alkenylene, a substituted or unsubstituted C _3-20 cycloalkylene, a substituted or unsubstituted C _6-20 arylene, and a substituted or unsubstituted C _6-20 heteroarylene.

In another specific embodiment of the present invention, SU ₁ , SU ₂ , and SU ₃ may each independently be selected from the group consisting of a substituted or unsubstituted C _1-15 alkylene, a substituted or unsubstituted C _1-15 heteroalkylene, a substituted or unsubstituted C _2-15 alkenylene, a substituted or unsubstituted C _3-15 cycloalkylene, a substituted or unsubstituted C _6-15 arylene, and a substituted or unsubstituted C _6-15 heteroarylene.

In another specific embodiment of the present invention, SU ₁ , SU ₂ , and SU ₃ may each independently be selected from the group consisting of a substituted or unsubstituted C _1-20 alkylene, a substituted or unsubstituted C _1-10 heteroalkylene, a substituted or unsubstituted C _2-10 alkenylene, a substituted or unsubstituted C _3-10 cycloalkylene, a substituted or unsubstituted C _6-10 arylene, and a substituted or unsubstituted C _6-10 heteroarylene.

In another specific embodiment of the present invention, the SU ₁ , SU ₂ , or SU ₃ When substituted, SU ₁ , SU ₂ , or SU ₃ may not contain thiopropionic acid, disulfide, or oligonucleotide.

In another specific embodiment of the present invention, SU ₁ , SU ₂ , and SU ₃ may each independently be selected from the group consisting of -(CH ₂ ) n -, -C _3-6 cycloalkyl-, -C _3-6 cycloaryl-, -C _3-6 heteroaryl-, and -(CH ₂ CH ₂ O) m -, wherein n is each independently an integer from 1 to 10, and wherein m may be an integer from 1 to 10.

In another specific embodiment of the present invention, the SU is -(CH ₂ ) _n -, wherein n can be an integer of 2 or more, and specifically, n can be an integer of 2 or more and 30 or less, an integer of 2 or more and 25 or less, an integer of 2 or more and 20 or less, an integer of 2 or more and 19 or less, an integer of 2 or more and 18 or less, an integer of 2 or more and 17 or less, an integer of 2 or more and 16 or less, an integer of 2 or more and 15 or less, an integer of 2 or more and 14 or less, an integer of 2 or more and 13 or less, an integer of 2 or more and 12 or less, an integer of 2 or more and 11 or less, an integer of 2 or more and 10 or less, an integer of 2 or more and 9 or less, an integer of 2 or more and 8 or less, an integer of 2 or more and 7 or less, an integer of 2 or more and 6 or less, an integer of 2 or more and 5 or less, an integer of 3 or more and 5 or less, an integer of 4 or more and 5 or less, but is not limited thereto.

In another specific embodiment of the present invention, X ₁ , X ₂ , and X ₃ may each independently be selected from the group consisting of a substituted or unsubstituted C _1-30 alkylene, a substituted or unsubstituted C _1-30 heteroalkylene, a substituted or unsubstituted C _2-30 alkenylene, a substituted or unsubstituted C _3-30 cycloalkylene, a substituted or unsubstituted C _6-30 arylene, and a substituted or unsubstituted C _6-30 heteroarylene.

In another specific embodiment of the present invention, X ₁ , X ₂ , and X ₃ may each independently be selected from the group consisting of a substituted or unsubstituted C _1-20 alkylene, a substituted or unsubstituted C _1-20 heteroalkylene, a substituted or unsubstituted C _2-20 alkenylene, a substituted or unsubstituted C _3-20 cycloalkylene, a substituted or unsubstituted C _6-20 arylene, and a substituted or unsubstituted C _6-20 heteroarylene.

In another specific embodiment of the present invention, X ₁ , X ₂ , and X ₃ may each independently be selected from the group consisting of a substituted or unsubstituted C _1-15 alkylene, a substituted or unsubstituted C _1-15 heteroalkylene, a substituted or unsubstituted C _2-15 alkenylene, a substituted or unsubstituted C _3-15 cycloalkylene, a substituted or unsubstituted C _6-15 arylene, and a substituted or unsubstituted C _6-15 heteroarylene.

In another specific embodiment of the present invention, X ₁ , X ₂ , and X ₃ may each independently be selected from the group consisting of a substituted or unsubstituted C _1-10 alkylene, a substituted or unsubstituted C _1-10 heteroalkylene, a substituted or unsubstituted C _2-10 alkenylene, a substituted or unsubstituted C _3-10 cycloalkylene, a substituted or unsubstituted C _6-10 arylene, and a substituted or unsubstituted C _6-10 heteroarylene.

In another specific embodiment of the present invention, at least one of X ₁ , X ₂ , and X ₃ is a substituted C _1-10 heteroalkylene, and when substituted, is substituted with a group consisting of halo, =O, OH, NH ₂ , SH, NO ₂ , N ₃ , CN, OR ₁ , SR ₁ , OC(O)R ₁ , OC(O)NHR ₁ , OC(O)OR ₁ , CONHR ₁ , CON(R ₁ ) ₂ , NHC(O)R ₁ , C(O)R ₁ , NHR ₁ , N(R ₁ ) ₂ , C(O)R ₁ , OS(O) ₂ R ₁ , -OP(O)(OR ₁ )(OR ₁ ), OP(O)(NHR ₁ )(NHR ₁ ), and C _1-3 alkyl, Here, R ₁ can be selected from the group consisting of H, OH, C _1-5 alkyl, C _1-5 heteroalkyl, C _3-8 aryl and C _3-8 heteroaryl.

In another specific embodiment of the present invention, when any one of X ₁ , X ₂ , and X ₃ is absent and the other one is C _2-10 alkyl, the remaining one may not be an ester.

In another specific embodiment of the present invention, X is selected from the group consisting of -S-(CH ₂ ) _a1 -NHC(O)-(CH ₂ ) _b1 -S-, -S-(CH ₂ ) _a1 OC(O)-(CH ₂ ) _b1 -S-, -S-(CH ₂ ) _a1 C(O)-(CH ₂ ) _b1 -S-, -S-(CH ₂ ) _a1 NH-(CH ₂ ) _b1 -S-, -S-(CH ₂ ) _a1 C(O)NH-(CH ₂ ) _b1 -S- and -S-(CH ₂ ) _a1 OC(O)NH-(CH ₂ ) _b1 -S-, wherein a1 and b1 can each independently be an integer from 1 to 5.

In another specific embodiment of the present invention, X is -S-(CH ₂ ) _a1 -NHC(O)-(CH ₂ ) _b1 -S-, wherein a1 and b1 can each independently be an integer from 1 to 3.

The term "payload" of the present invention means a molecule to be linked to a target molecule. By way of example, the payload may be, but is not limited to, a compound, a peptide, a polypeptide, a protein, and/or a drug molecule.

In another embodiment of the present invention, the payload can be at least one active agent selected from a chemotherapeutic agent and a toxin.

In another embodiment of the present invention, the active agent can be an immunomodulating compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent or a combination thereof.

In another embodiment of the present invention, the active agent may be independently selected from:

(a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, Carboquone, meturedopa, uredopa, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, calystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, Duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, Lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophores, aclacinomycin, actinomycin, antrmycin, azaserine, bleomycin, cactinomycin, carabicin, carninomycin, carzinophilin, Chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, Aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocin, mitoguazone, Mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, and anguidine, urethane, Vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate or acid of any of the foregoing;

(b) monokines, lymphokines, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factor, interferon, interferon-α, interferon-β, interferon-γ, colony stimulating factor (“CSF”), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (“IL”), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF, kit ligand, or a combination of any of the foregoing;

(c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, amanitin derivatives, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, a dolastatin analogue, cryptophycin, camptothecin, camptothecin derivatives and metabolites, rhizoxin, a rhizoxin derivative, CC-1065, a CC-1065 analogue or derivative, a duocarmycin, an enediyne antibiotic, esperamicin, an epothilone, azonafide, apridine, a toxoid, or a combination of any of the foregoing;

(d) an affinity ligand (wherein the affinity ligand is a substrate), an inhibitor, a stimulant, a neurotransmitter, a radioisotope, or a combination of any of the foregoing;

(e) a radiolabel, ³² P, ³⁵ S, a fluorescent dye, an electron dense reagent, an enzyme, biotin, streptavidin, dioxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to the target, or a combination of any of the foregoing;

(f) immunomodulating compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or a combination of any of the foregoing;

(g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene;

(h) 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole;

(i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine;

(j) aromatase inhibitors;

(k) protein kinase inhibitors;

(l) lipid kinase inhibitors;

(m) antisense oligonucleotides;

(n) ribozyme;

(o) vaccines; and

(p) Anti-angiogenic agent.

In another specific embodiment of the present invention, the activator may be, but is not limited to, DM1.

The linker-payload conjugate of the present invention can be used to prepare antibody-drug conjugates (ADCs).

In the above linker-payload conjugate, the linker may additionally comprise a cleavable linker. For example, the linker may be in a form that is cleavable under intracellular conditions, i.e., such that the drug can be released from the antibody through cleavage of the linker in the intracellular environment.

Another aspect of the present invention provides a ligand-drug conjugate represented by the following general formula II:

[General Formula II]

Ab-(M ₁ -SU-M ₂ -XP) _n

In the above formula,

Ab is a ligand,

M ₁ and M ₂ are maleimide moieties,

SU is a first spacer unit represented by (SU ₁ ) _a -(SU ₂ ) _b -(SU ₃ ) _c , and SU ₁ , SU ₂ , and SU ₃ may be the same or different, and a, b, and c are each independently integers from 0 to 3,

X is a second spacer unit expressed as (X ₁ ) _x -(X ₂ ) _y -(X ₃ ) _z , wherein X ₁ , X ₂ , and X ₃ may be the same or different, and x, y, and z are each independently integers from 0 to 3,

P is the payload,

n is an integer from 1 to 20.

In one specific embodiment of the present invention, the ligand is covalently bonded to the maleimide moiety by a thioether bond, wherein the thioether bond includes a sulfur atom of a cysteine of the ligand.

In another embodiment of the present invention, the ligand comprises a cysteine-terminal amino acid motif; and the thioether linkage comprises a sulfur atom of a cysteine of the amino acid motif.

In another embodiment of the present invention, the ligand can be a monoclonal antibody, a polyclonal antibody, an antibody fragment, a Fab, a Fab', a Fab-SH, a F(ab')2, a Fv, a single-chain Fv ("scFv"), a diabody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody or a fusion protein comprising an antigen-binding portion of an antibody.

In another specific embodiment of the present invention, the ligand is muromonab-CD3 abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab, etanercept, basiliximab, gemtuzumab, alemtuzumab, ibritumomab, adalimumab, alefacept, omalizumab, efalizumab, tositumomab, cetuximab, ABT-806, Bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab, romiplostim, AMG-531, golimumab, ustekinumab, ABT-874, belatacept, belimumab, atacicept, anti-CD20 antibodies, canakinumab, tocilizumab, atlizumab, mepolizumab, pertuzumab, HuMax CD20, tremelimumab, Consisting of ticilimumab, ipilimumab, IDEC-114, inotuzumab, HuMax EGFR, aflibercept, HuMax-CD4, teplizumab, otelixizumab, catumaxomab, anti-EpCAM antibody IGN101, adecatumomab, oregovomab, dinutuximab, girentuximab, denosumab, bapineuzumab, motavizumab, efumgumab, raxibacumab, LY2469298, and veltuzumab. Can be selected from a group.

In another specific embodiment of the present invention, the ligand may be, but is not limited to, trastuzumab.

In another embodiment of the present invention, the payload can be at least one active agent selected from a chemotherapeutic agent and a toxin.

In another embodiment of the present invention, the active agent can be an immunomodulating compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent or a combination thereof.

In another embodiment of the present invention, the active agent may be independently selected from:

(a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, Carboquone, meturedopa, uredopa, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, calystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, Duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, Lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophores, aclacinomycin, actinomycin, antrmycin, azaserine, bleomycin, cactinomycin, carabicin, carninomycin, carzinophilin, Chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, Aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocin, mitoguazone, Mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, and anguidine, urethane, Vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate or acid of any of the foregoing;

(b) monokines, lymphokines, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factor, interferon, interferon-α, interferon-β, interferon-γ, Colony stimulating factor (“CSF”), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (“IL”), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF, kit ligand, or a combination of any of the foregoing;

(c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, amanitin derivatives, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, a dolastatin analogue, cryptophycin, camptothecin, camptothecin derivatives and metabolites, rhizoxin, a rhizoxin derivative, CC-1065, a CC-1065 analogue or derivative, a duocarmycin, an enediyne antibiotic, esperamicin, an epothilone, azonafide, apridine, a toxoid, or a combination of any of the foregoing;

(d) an affinity ligand (wherein the affinity ligand is a substrate), an inhibitor, a stimulant, a neurotransmitter, a radioisotope, or a combination of any of the foregoing;

(e) a radiolabel, ³² P, ³⁵ S, a fluorescent dye, an electron dense reagent, an enzyme, biotin, streptavidin, dioxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to the target, or a combination of any of the foregoing;

(f) immunomodulating compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or a combination of any of the foregoing;

(g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene;

(h) 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole;

(i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine;

(j) aromatase inhibitors;

(k) protein kinase inhibitors;

(l) lipid kinase inhibitors;

(m) antisense oligonucleotides;

(n) ribozyme;

(o) vaccines; and

(p) Anti-angiogenic agent.

In another specific embodiment of the present invention, the activator may be, but is not limited to, DM1.

The ligand-drug polymer of the present invention can be used to prepare an antibody-drug polymer (ADC).

The ligand-drug polymer of the present invention is characterized by having a Drug-to-Antibody Ratio (DAR) of 3 to 8, specifically a DAR of 4 to 8, 5 to 8, 6 to 8, or 7 to 8.

In another specific embodiment of the present invention, the linkage between Ab and M ₁ and the linkage between X and P can each be independently cleavable or non-cleavable.

Another aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising the ligand-drug conjugate.

The term "treatment" of the present invention means any action taken to statistically significantly cure, heal, alleviate, alleviate, alter, relieve, improve, enhance or otherwise affect an illness (e.g., a disease) and symptoms of a disease, or to prevent or delay the onset of symptoms, complications and biochemical markers, or otherwise prevent or inhibit further progression of the disease, illness or disorder.

In one specific embodiment of the present invention, the cancer can be selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, uterine corpus cancer or melanoma.

In another specific embodiment of the present invention, the cancer may be specifically a cancer associated with Her2 expression or underexpression, and specifically may be at least one selected from the group consisting of biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, malignant melanoma, laryngeal cancer, oral cancer, and skin cancer, and specifically may be breast cancer, and more specifically, metastatic breast cancer, but is not limited thereto.

The term "HER2" of the present invention refers to the second member of the EGFR family, which has tyrosine kinase activity.

The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, at least one selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

The carrier, excipient and diluent that may be included in the pharmaceutical composition according to the present invention may be at least one selected from lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

Additives for tablets, powders, granules, capsules, pills and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinyl pyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ethers, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

Additives that can be used in the liquid formulation according to the present invention include water, diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc.

The syrup according to the present invention may use a solution of white sugar, other sugars or sweeteners, and may also use a flavoring agent, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity increasing agent, etc., as needed.

Purified water may be used in the emulsion according to the present invention, and an emulsifier, preservative, stabilizer, fragrance, etc. may be used as needed.

The suspension according to the present invention may use suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, and the like. Surfactants, preservatives, stabilizers, colorants, and fragrances may also be used as needed.

The injection according to the present invention includes: solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, nonvolatile oils such as sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nitrile acid amide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO ₃ ), carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid disodium, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cisplatinum, sodium alginate, Tween 80, and aluminum monostearate.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

In addition, the present invention provides a kit for preventing or treating cancer comprising the pharmaceutical composition.

The kit according to the present invention may, in addition to the composition described above, include, without limitation, other components, compositions, solutions, devices, etc. that are usually necessary for the prevention or treatment of cancer, and in particular, may include instructions for proper use and storage of the composition according to the present invention.

Another aspect of the present invention provides a method comprising the step of administering the pharmaceutical composition to a subject in need thereof.

In the present invention, the term "subject" means a subject requiring treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

In the present invention, “administration” means providing a composition of the present invention to a subject by any appropriate method.

The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically) depending on the intended method, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the time, but can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount, for example, 0.1 to 10 μmol. In the present invention, the "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered simultaneously, separately, or sequentially, and can be administered singly or in multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitantly administered drugs, and may increase or decrease depending on the route of administration, severity of obesity, sex, weight, age, etc.

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are only intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

<Example 1> Preparation of di-maleimide linker

<1-1> 1,1'-(ethane-1,2-diyl)bis(1H-pyrrole-2,5-dione): linker 1

Maleic anhydride (0.980 mg, 10 mmol) dissolved in DMF (3 ml) was added to a flame-dried 250 ml 3-necked round-bottom flask equipped with nitrogen gas. 1,2-Ethylenediamine (0.300 mg, 5 mmol) dissolved in DMF (0.6 ml) was slowly added using an addition funnel. The reaction mixture was stirred at 60 °C for 1 h. When mixed, amic acid was formed and a white strand-like substance was generated, which was redissolved with continuous stirring. Then, acetic anhydride (1.23 g, 12 mmol) was added, followed by sodium carbonate (0.2 g, anhydrous). The mixture was stirred at 60 °C overnight, cooled to room temperature, precipitated, washed with ice water, and dried in vacuo. The product was obtained by purification by silica column chromatography (hexane: EtOAc = 2:1).

Obtained form: White solid; ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.66 (4H, s), 3.71 (4H, s); ¹³ C NMR (101 MHz, CDCl ₃ ) δ 170.64, 134.31, 36.64.

<1-2> 1,1'-(propane-1,3-diyl)bis(1H-pyrrole-2,5-dione): linker 2

A flame-dried 250-mL 3-necked round-bottom flask equipped with nitrogen gas was added maleic anhydride (0.980 mg, 10 mmol) dissolved in DMF (3 ml). 1,3-Diaminopropane (0.371 mg, 5 mmol) was dissolved in DMF (0.6 ml) and slowly added using an addition funnel. The reaction mixture was stirred at 60 °C for 1 h. Upon mixing, amic acid was formed and a white strand-like substance was generated, which was redissolved with continuous stirring. Then, acetic anhydride (1.23 g, 12 mmol) was added, followed by sodium carbonate (0.2 g, anhydrous). The mixture was stirred at 60 °C overnight, cooled to room temperature, precipitated, washed with ice water, and dried in vacuo. The product was obtained by purification by silica column chromatography (hexane: EtOAc = 2:1).

Obtained form: white solid; ^1H NMR (400 MHz, CDCl ₃ ) δ 6.70 (4H, s), 3.53 (4H, t, J = 7.2 Hz); 13C NMR (101 MHz, CDCl ₃ ) δ 170.74, 134.30, 35.46, 27.55.

<1-3> 1,1'-(butane-1,4-diyl)bis(1H-pyrrole-2,5-dione): linker 3

A flame-dried 250-mL 3-necked round-bottom flask equipped with nitrogen gas was added maleic anhydride (0.980 mg, 10 mmol) dissolved in DMF (3 ml). 1,4-Diaminobutane (0.441 mg, 5 mmol) dissolved in DMF (0.6 ml) was slowly added using an addition funnel. The reaction mixture was stirred at 60 °C for 1 hour. Upon mixing, amic acid was formed and a white strand-like substance was generated, which was redissolved with continuous stirring. Then, acetic anhydride (1.23 g, 12 mmol) was added, followed by sodium carbonate (0.2 g, anhydrous). The mixture was stirred at 60 °C overnight, cooled to room temperature, precipitated, washed with ice water, and dried in vacuo. The product was obtained by purification by silica column chromatography (hexane: EtOAc = 2:1).

Obtained form: White solid; ^1H NMR (400 MHz, CDCl ₃ ) δ 6.8 (4H, s), 3.53 (4H, t, J = 6.0 Hz), 1.58 (4H, m); 13C NMR (100 MHz, CDCl ₃ ) δ 170.90, 134.23, 37.28, 25.90.

<1-4> 1,1'-(pentane-1,5-diyl)bis(1H-pyrrole-2,5-dione): Linker 4

Maleic anhydride (0.980 mg, 10 mmol) dissolved in DMF (3 ml) was added to a flame-dried 250 ml 3-necked round bottom flask equipped with nitrogen gas. 1,5-Diaminopentane (0.511 mg, 5 mmol) dissolved in DMF (0.6 ml) was slowly added using an addition funnel. The reaction mixture was stirred at 60 °C for 1 hour. When mixed, amic acid was formed and a white strand-like substance was generated, which was redissolved with continuous stirring. Then, acetic anhydride (1.23 g, 12 mmol) was added, followed by sodium carbonate (0.2 g, anhydrous). The mixture was stirred at 60 °C overnight, cooled to room temperature, precipitated, washed with ice water, and dried in vacuo. The product was obtained by purification by silica column chromatography (hexane: EtOAc = 2:1).

Obtained form: White solid; ^1H NMR (400 MHz, CDCl ₃ ) δ 6.68 (4H, s), 3.49 (4H, t, J = 7.2 Hz), 1.60 (4H, m), 1.26 (2H, m); 13C NMR (100 MHz, CDCl ₃ ) δ 170.95, 134.19, 37.66, 28.15, 23.99.

<1-5> 1,1'-(hexane-1,6-diyl)bis(1H-pyrrole-2,5-dione): Linker 5

Maleic anhydride (0.980 mg, 10 mmol) dissolved in DMF (3 ml) was added to a flame-dried 250 ml 3-necked round-bottom flask equipped with nitrogen gas. 1,6-Diaminohexane (0.581 mg, 5 mmol) dissolved in DMF (0.6 ml) was slowly added using an addition funnel. The reaction mixture was stirred at 60 °C for 1 h. Upon mixing, amic acid was formed and a white strand-like substance was generated, which was redissolved with continuous stirring. Then, acetic anhydride (1.23 g, 12 mmol) was added, followed by sodium carbonate (0.2 g, anhydrous). The mixture was stirred at 60 °C overnight, cooled to room temperature, precipitated, washed with ice water, and dried in vacuo. The product was obtained by purification by silica column chromatography (hexane: EtOAc = 2:1).

Obtained form: White solid; ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.68 (4H, s), 3.49 (4H, t, J = 7.2 Hz), 1.56 (4H, m), 1.29 (4H, m); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 170.99, 134.16, 37.79, 28.46, 26.28.

<1-6> 1-(3-(3-((2-Aminoethyl)thio)-2,5-dioxopyrrolidin-1-yl)propyl)-1H-pyrrole-2,5-dione trifluoroacetic acid salt

Dimaleimide was added to the vial, followed by boc-2-aminoethanethiol and triethylamine in 5 ml of dichloromethane. The mixture was stirred overnight at room temperature, and the solvent was evaporated. The mixture was purified by silica column chromatography (hexane: EtOAc = 5:1) to obtain the boc-protected dimaleimide product. More than 20 equivalents of trifluoroacetic acid were added to the obtained boc-dimaleimide, dissolved in 2 ml of dichloromethane, and stirred at room temperature. After confirming that boc-dimaleimide had completely disappeared by TLC, all the solvent was removed to obtain dimaleimide trifluoroacetic acid salt with the boc-protecting group removed.

<Example 2> Preparation of a conjugate of maleimide linker and DM1

DM1 and the maleimide linker prepared in Example 1 were added in a ratio of 1:1 to 1:2, 1 equivalent of triethylamine was added, dissolved in dichloromethane, and stirred at room temperature. After the reaction was completed, the solvent was removed, and the resultant was purified by column chromatography and analyzed.

<2-1> 1,1'-(ethane-1,2-diyl)bis(1H-pyrrole-2,5-dione)-DM1

In a round bottom flask, DM1 (100 mg, 0.135 mmol) dissolved in CH ₂ Cl _{2 ,} linker 1 (44.7 mg, 0.203 mmol) prepared in Example 1-1, and triethylamine were added, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was washed with distilled water and dried over sodium sulfate. The organic layer was collected, evaporated using a rotary evaporator, and then purified by silica column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 30: 1) to obtain the product.

Obtained form: white solid; ^1H NMR (400MHz, CDCl ₃ ) δ ^1H NMR (400MHz, chloroform-d) δ 6.79 (d, J = 9.9 Hz, 1H), 6.64 (t, J = 4.6 Hz, 3H), 6.59 (d, J = 4.3 Hz, 1H), 6.40 (dd, J = 15.0, 11 .4 Hz, 1H), 6.31 (s, 1H), 5.62 (dd, J = 15.2, 9.1 Hz, 1H), 5.38 - 5.31 (m, 1H), 4.73 (dd, J = 11.9, 2.4 Hz, 1H), 4.24 (t, J = 11.1 Hz, 1H), 3. 96 (s, 3H), 3.70 - 3.58 (m, 5H), 3.46 (d, J = 10.2 Hz, 1H), 3.32 (s, 3H), 3.17 (s, 3H), 3.00 (dddd, J = 34.7, 24.6, 17.7, 8.4 Hz, 6H), 2.80 (s, 4H), 2.58 (dt, J = 14.8, 8.6 Hz, 2H), 2.28 (ddd, J = 18.6, 7.4, 4.1 Hz, 1H), 2.14 (d, J = 14.2 Hz, 1H), 2.01 (s, 1H), 1.60 (s, 3H), 1.51 (d, J = 13.4 Hz, 1H), 1.47 - 1.40 (m, 1H), 1.25 (q, J = 10.8, 9.3 Hz, 7H), 0.77 (s, 3H). ¹³ C NMR (101MHz, Chloroform-d) 176.83, 176.66, 174.64, 170.72, 168.81, 155.94, 152.35, 142.10, 141.13, 139.36, 134.26, 133.26, 127.77, 125.36, 122.17, 118.64, 113.14, 88.53, 80.81, 78.18, 74.10, 67.24, 59.99, 56.72, 56.62, 52.49, 46.64, 39.81, 39.68, 38.83, 38.04, 37.92, 36.23, 35.63, 35.59, 34.26, 33.94, 32.42, 30.78, 27.29, 27.19, 15.57, 14.62, 13.40, 12.13.

<2-2> 1,1'-(propane-1,3-diyl)bis(1H-pyrrole-2,5-dione)-DM1

In a round bottom flask, DM1 (100 mg, 0.135 mmol) dissolved in CH ₂ Cl _2, linker 2 (47.6 mg, 0.203 mmol) prepared in Example 1-2, and triethylamine were added, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was washed with distilled water and dried over sodium sulfate. The organic layer was collected and evaporated using a rotary evaporator, and then purified by silica column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 30: 1) to obtain the product.

Obtained form: white solid; ^1H NMR (600 MHz, CDCl ₃ ) δ 6.81 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 3.5 Hz, 3H), 6.62 (d, J = 3.8 Hz, 1H), 6.42 (dd, J = 15.2, 11.1 Hz, 1H), 6.24 (s, 1H), 5.64 (dd, J = 15.4, 9.4 Hz, 1H), 5.38 (q, J = 6.9 Hz, 1H), 4.79 - 4.73 (m, 1H), 4.27 (t, J = 11.5 Hz, 1H), 3.98 (s, 3H), 3.75 - 3.63 (m, 2H), 3.52 - 3.45 (m, 4H), 3.44 - 3.39 (m, 1H), 3.35 (s, 3H), 3.19 (s, 3H), 3.04 (tdd, J = 18.2, 13.5, 7.7 Hz, 5H), 2.84 (s, 4H), 2.67 - (m, 2H), 2.37 (td, J = 18.5, 3.8 Hz, 1H), 2.17 (d, J = 14.7 Hz, 1H), 1.88 (dq, J = 13.4, 7.1 Hz, 2H), 1.65 (d, J = 12.9 Hz, 4H), 1.55 (d, J = 1) 3.7 Hz, 1H), 1.46 (td, J = 10.0, 5.7 Hz, 1H), 1.32 - 1.18 (m, 7H), 0.79 (s, 3H). ¹³ C NMR (151MHz, Chloroform-d) δ 176.66, 174.46, 170.79, 170.72, 168.87, 156.13, 152.30, 142.31, 141.20, 139.43, 134.32, 133.43, 127.7 8, 125.49, 122.33, 118.91, 113.29, 88.62, 81.00, 78.29, 74.22, 67.29, 60.05, 56.76, 56.71, 52.54, 46.73, 39.89, 39.74, 39.00, 36.60, 36.26, 35.72, 35.66, 35.29, 34.25, 34.05, 32.54, 30.83, 27.22, 26.57, 26.54, 15.63, 14.68, 13.50, 12.26.

<2-3> 1,1'-(butane-1,4-diyl)bis(1H-pyrrole-2,5-dione)-DM1

In a round bottom flask, DM1 (100 mg, 0.135 mmol) dissolved in CH ₂ Cl ₂ , linker 3 (50.4 mg, 0.203 mmol) prepared in Example 1-3, and triethylamine were added, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was washed with distilled water and dried over sodium sulfate. The organic layer was collected and evaporated using a rotary evaporator, and then purified by silica column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 30: 1) to obtain the product.

Obtained form: white solid; ^1H NMR (400MHz, CDCl ₃ ) δ 6.82 (d, J = 8.2 Hz, 1H), 6.72 - 6.68 (m, 3H), 6.62 (d, J = 4.0 Hz, 1H), 6.42 (dd, J = 15.3, 11.2 Hz, 1H), 6.24 (s, 1H), 5.63 (dd, J = 15.1, 9.1 Hz, 1H), 5.41 - 5.34 (m, 1H), 4.76 (dd, J = 11.9, 2.7 Hz, 1H), 4.28 (t, J = 11.0 Hz, 2H), 3.98 (s, 3H), 3.74 - 3.63 (m , 3H), 3.52 - 3.48 (m, 5H), 3.41 - 3.34 (m, 5H), 3.24 - 3.17 (m, 4H), 3.13 - 2.92 (m, 6H), 2.86 - 2.78 (m, 5H), 2.66 - 2.55 (m, 3H), 2.33 (ddd, J = 18.7, 9.9, 3.8 Hz, 1H), 2.17 (d, J = 14.3 Hz, 1H), 1.67 (s, 2H), 1.63 (s, 3H), 1.56 - 1.43 (m, 7H), 1.33 - 1.18 (m, 8H), 0.79 (s, 3H) . ¹³ C NMR (101 MHz, Chloroform-d) δ 176.91, 176.74, 174.59, 174.56, 170.87, 170.83, 170.80, 170.76, 170.69, 168.86, 168.84, 156.02, 155. 99, 152.37, 142.15, 142.12, 141.14, 141.06, 139.42, 139.38, 134.21, 133.32, 127.75, 125.35, 122.19, 118.72, 113.23, 113.17, 88.54, 80.87, 78.29, 78.23, 74.15, 67.28, 60.01, 56.75, 56.67, 56.66, 52.61, 52.52, 46.66, 39.79, 39.57, 38.88, .40, 38.34, 37.14, 36.22, 35.76, 35.65, 35.59, 34.27, 33.94, 33.42, 32.46, 30.82, 27.36, 27.20, 25.80, 25.78, 24.72, 14.66, 13.46, 13.42, 12.18, 12.16.

<2-4> 1,1'-(pentane-1,5-diyl)bis(1H-pyrrole-2,5-dione)-DM1

In a round bottom flask, DM1 (100 mg, 0.135 mmol) dissolved in CH ₂ Cl _{2 ,} linker 4 (53.3 mg, 0.203 mmol) prepared in Example 1-4, and triethylamine were added, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was washed with distilled water and dried over sodium sulfate. The organic layer was collected and evaporated using a rotary evaporator, and then purified by silica column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 30: 1) to obtain the product.

Obtained form: white solid; ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.80 (d, J = 8.9 Hz, 1H), 6.70 - 6.65 (m, 3H), 6.61 (d, J = 5.0 Hz, 1H), 6.41 (dd, J = 15.2, 11.4 Hz, 1H), 6.28 (s, 1H), 5.63 (dd, J = 15.3, 8.5 Hz, 1H), 5.37 (q, J = 6.3 Hz, 1H), 4.74 (dd, J = 12.0, 2.6 Hz, 1H), 4.30 - 4.22 (m, 1H), 3.97 (s, 3H), 3.73 - 3.61 (m, 2H), 3.51 - 3.41 (m, 5H), 3.37 - 3.29 (m, 4H), 3.18 (s, 3H), 3.12 - 2.90 (m, 5H), 2.87 - 2.77 (m, 4H), 2.59 (tt, J = 14.6, 11.8, 5.1 Hz, 2H) , 2.31 (ddd, J = 18.0, 14.0, 3.6 Hz, 1H), 2.16 (d, J = 14.2 Hz, 1H), 1.84 (s, 1H), 1.62 (s, 3H), 1.59 - 1.39 (m, 7H), 1.31 - 1.17 (m, 9H), 0.78 (s, 3H). ¹³ C NMR (101 MHz, Chloroform-d) δ 176.95, 176.78, 174.62, 170.96, 170.86, 170.81, 170.70, 168.86, 156.04, 152.37, 142.18, 141.14, 141. 07, 139.45, 139.40, 134.19, 133.35, 127.75, 125.36, 122.23, 118.76, 113.23, 88.54, 80.90, 78.31, 74.17, 67.30, 60.02, 56.76, 56.68, 46.68, 39.82, 39.60, 38.91, 38.84, 38.74, 37.50, 36.22, 35.76, 35.66, 35.59, 33.96, 32.48, 30.85, 29.80, 28. 10, 28.07, 27.40, 27.26, 27.06, 23.90, 15.62, 14.67, 13.47, 12.19.

<2-5> 1,1'-(hexane-1,6-diyl)bis(1H-pyrrole-2,5-dione)-DM1

In a round bottom flask, DM1 (100 mg, 0.135 mmol) dissolved in CH ₂ Cl ₂ , Linker 5 (56.1 mg, 0.203 mmol) prepared in Example 1-5, and triethylamine were added, and the reaction mixture was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was washed with distilled water and dried over sodium sulfate. The organic layer was collected and evaporated using a rotary evaporator, and then purified by silica column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 30: 1) to obtain the product.

Obtained form: white solid; ^1H NMR (400MHz, CDCl ₃ ) δ 6.81 (d, J = 8.9 Hz, 1H), 6.71 - 6.67 (m, 3H), 6.62 (d, J = 5.6 Hz, 1H), 6.46 - 6.37 (m, 1H), 6.27 (s, 1H), 5.63 (dd, J = 15.0, 9.1 Hz, 1H), 5.40 - 5.33 (m, 1H), 4.75 (d, J = 10.8 Hz, 1H), 4.26 (t, J = 10.9 Hz, 1H), 3.97 (s, 3H), 3.72 - 3.62 (m, 2H), 3.49 - 3. 41 (m, 5H), 3.32 - 3.30 (m, 4H), 3.19 (s, 3H), 3.11 - 2.92 (m, 5H), 2.88 - 2.77 (m, 4H), 2.65 - 2.55 (m, 2H), 2.37 - 2.26 (m, 1H), 2.16 (d, J = 14.1 Hz, 1H), 1.79 (s, 1H), 1.62 (s, 3H), 1.58 - 1.39 (m, 7H), 1.33 - 1.17 (m, 11H), 0.78 (s, 3H). ¹³ C NMR (101 MHz, Chloroform-d) δ 176.78, 174.61, 174.58, 171.00, 170.76, 170.69, 168.86, 156.06, 152.35, 142.22, 141.15, 141.07, 139. 43, 134.19, 134.17, 133.39, 127.74, 125.43, 118.79, 88.53, 80.94, 78.29, 74.19, 67.32, 60.02, 56.76, 56.70, 39.85, 39.62, 37.80, 37.71, 36.19, 35.68, 35.59, 34.34, 33.96, 32.50, 30.86, 28.46, 28.37, 27.45, 27.26, 26.28, 26.23, 26.20, 15. 63, 14.69, 13.49, 12.21.

<2-6> Mal-Mal-SPDP-DM1 (14R,16R,32R,33R,2S,4R,10E,12E,14S)-86-chloro-14-hydroxy-85,14-dimethoxy-2,7,10-trimethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecapan-10,12-dien-4-yl N-(4-((3-((2-((1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)-2,5-dioxopyrrolidin-3-yl)thio)ethyl)amino)-3-oxopropyl)disulfanyl)-2-oxobutyl)-N-methyl-L-alaninate

SPDP-DM1 (50 mg, 0.053 mmol) dissolved in CH ₂ Cl ₂ , the linker prepared in Example 1-6 (23.6 mg, 0.203 mmol) and triethylamine were added to a round-bottomed flask, and the reaction mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was washed with distilled water and dried with sodium sulfate. The organic layer was collected and evaporated using a rotary evaporator, and then purified by silica column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 30: 1) to give the product (Mal-Mal-SPDP-DM1 (14R, 16R, 32R, 33R, 2S, 4R, 10E, 12E, 14S)-86-chloro-14-hydroxy-85, 14-dimethoxy-2, 7, 10-trimethyl-12, 6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(4-((3-((2-((1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)-2,5-dioxopyrrolidin-3-yl)thio)ethyl)amino)-3-oxopropyl)disulfanyl)-2-oxobutyl)-N-methyl-L-alaninate) was obtained.

<2-7> Mal-Mal-SPDB-DM1 (14R,16R,32R,33R,2S,4R,10E,12E,14S)-86-chloro-14-hydroxy-85,14-dimethoxy-2,7,10-trimethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecapan-10,12-dien-4-yl N-(4-((3-((2-((1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)-2,5-dioxopyrrolidin-3-yl)thio)ethyl)amino)-3-oxobutyl)disulfanyl)-2-oxobutyl)-N-methyl-L-alaninate

SPDB-DM1 (50 mg, 0.053 mmol) dissolved in CH ₂ Cl ₂ , the linker prepared in Example 1-6 (23.6 mg, 0.203 mmol) and triethylamine were added to a round-bottomed flask, and the reaction mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was washed with distilled water and dried with sodium sulfate. The organic layer was collected and evaporated using a rotary evaporator, and then purified by silica column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 30: 1) to give the product (Mal-Mal-SPDB-DM1 (14R, 16R, 32R, 33R, 2S, 4R, 10E, 12E, 14S)-86-chloro-14-hydroxy-85, 14-dimethoxy-2, 7, 10-trimethyl-12, 6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(4-((3-((2-((1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)-2,5-dioxopyrrolidin-3-yl)thio)ethyl)amino)-3-oxobutyl)disulfanyl)-2-oxobutyl)-N-methyl-L-alaninate) was obtained.

N=1 (Mal-Mal-SPDP-DM1)

Obtained form: white solid; ^1H NMR (400 MHz, CDCl ₃ ) δ 6.83 (s, 1H), 6.71 (s, 2H), 6.68 - 6.61 (m, 2H), 6.55 (d, J = 8.9 Hz, 1H), 6.42 (dd, J = 15.6, 11.0 Hz, 1H), 6.27 (s, 1H), 5.64 (dd, J = 15.6, 8.4 Hz, 1H), 5.25 (s, 1H), 4.81 (d, J = 12.7 Hz, 1H), 4.34 - 4.25 (m, 1H), 3.98 (s, 3H), 3.79 (dt, J = 6.6, 2.4 Hz, 1H), 3.66 - 3.47 (m, 9H), 3.35 (s, 3H), 3.22 (s, 3H), 3.19 - 3.07 (m, 4H), 3.04 - 2.91 (m, 3H), 2.90 - 2.77 (m, 8H), 2.72 - 2.48 (m, 6H), 2 .18 (d, J = 14.3 Hz, 1H), 2.06 - 2.01 (m, 1H), 1.94 (td, J = 13.1, 6.1 Hz, 3H), 1.70 - 1.57 (m, 7H), 1.51 - 1.42 (m, 2H), 1.35 - 1.19 (m, 9H) ), 0.80 (s, 3H).

N=2 (Mal-Mal-SPDB-DM1)

Obtained form: white solid; ^1H NMR (400 MHz, CDCl ₃ ) δ 6.83 (s, 1H), 6.70 (t, J = 3.6 Hz, 2H), 6.64 (d, J = 16.2 Hz, 2H), 6.47 - 6.38 (m, 2H), 6.26 (s, 1H), 5.65 (dd, J = 14. 8, 9.1 Hz, 1H), 5.33 (d, J = 7.2 Hz, 1H), 4.78 (d, J = 11.9 Hz, 1H), 4.28 (t, J = 12.0 Hz, 1H), 3.98 (s, 3H), 3.77 (ddt, J = 9.2, 3.7, 1.6 Hz, 1H), 3.66 - 3.43 (m, 9H), 3.34 (s, 3H), 3.22 (s, 3H), 3.19 - 3.06 (m, 4H), 3.04 - 2.89 (m, 3H), 2.88 - 2.77 (m, 6H), 2.70 - 2.48 (m, 6H), 2 .26 (t, J = 7.3 Hz, 2H), 2.17 (dd, J = 14.0, 2.9 Hz, 1H), 1.94 (ddd, J = 14.4, 7.9, 4.3 Hz, 4H), 1.74 (s, 2H), 1.63 (s, 3H), 1.57 (d, J = 13.6 Hz, 1H), 1.46 (d, J = 7.6 Hz, 2H), 1.34 - 1.19 (m, 8H), 0.80 (s, 3H). HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₅₂ H ₆₉ ClN ₆ O ₁₅ S ₃ Na 1171.3569, found 1171.35589

<Example 3> Preparation of antibody (Trastuzumab)-drug (DM1) polymer: ADC 1-5

To prepare ADCs utilizing the linker payloads manufactured in Example 2 above, trastuzumab was completely reduced using TCEP to generate eight available thiols. Subsequently, the linker-payloads dissolved in DMSO were fused and conjugated at an appropriate temperature to prepare ADCs.

Specifically, 5 equivalents of tris(2-carboxyethyl)phosphine (TCEP, 50 mM stock solution in distilled water, pH 7.0) solution was added to a solution of trastuzumab (2 mg/mL) dissolved in phosphate-buffered saline (PBS), pH 7.5. The mixture was incubated at 37°C for 1 h. Then, solutions of linker-payloads 1-5 prepared in Example 2 were each added to a DMSO solution (10 mM in DMSO), and additional DMSO was added to a final concentration of 10%. The reaction mixture was incubated at 37°C for 1 h. Excess reagents were removed by repeated purification with PBS using an Amicon ^® Ultra-15 centrifugal filter device (10 KDa MWCO, 15 mL). The resulting conjugates were subjected to the following HPLC, MS, and SEC analyses.

Example 3 Antibody Trastuzumab (anti-HER2 Ab) Drugs DM1 Binding site Cystein Linker Maleimide

<Comparative Example 1> Antibody (Trastuzumab)-Drug (DXd) Polymer (Enhertu)

Comparative Example 1 ( ^Enhertu® ) Antibody Trastuzumab (anti-HER2 Ab) Drugs DXd (topoisomerase inhibitor) Binding site Cystein Linker Maleimide

The structure is as shown in Fig. 2 (a).

<Comparative Example 2> Antibody (Trop-2)-Drug (SN38) Polymer (Trodelvy)

Comparative Example 2 ( ^Trodelvy® ) Antibody Trop-2 Drugs SN38 (topoisomerase inhibitor) Binding site Cystein Linker Maleimide

The structure is as shown in Fig. 2 (b).

<Experimental Example 1> DAR analysis according to conjugation method

To confirm that the antibody-drug polymer of the present invention has a uniform DAR, the DAR according to the conjugation method was analyzed. Specifically, the DAR was analyzed for each of lysine linkage, cysteine linkage, and Ab engineering.

The results are as shown in Fig. 3.

<Experimental Example 2> DAR Analysis of Antibody-Drug Polymers

The DAR of each of the antibody-drug polymers manufactured by Example 3, Comparative Example 1, and Comparative Example 2 was analyzed.

Table 4 below shows the results.

DAR Example 3 8.0 Comparative Example 1 7.7 Comparative Example 2 7.6

Figure 4 shows the results of measuring DAR in Example 3.

<Experimental Example 3> Yield Measurement of ADC

To confirm the high yield of the ADC (ABC-002) of the present invention, the yields of each of Example 3 and Comparative Example 1 ( ^Enhertu® ) were measured.

The results are as shown in Fig. 5.

As a result of the analysis, Example 3 of the present invention has a higher yield than ^Enhertu® . Appeared.

Experimental Method General

All reactions were monitored by thin layer chromatography on 0.25 mm silica plates (F-254), and reaction spots were visualized under a UV light lamp (254 nm, 365 nm) or by staining with potassium permanganate and liquid chromatography-mass spectrometry (LC-MS).

Column chromatography was performed on silica gel (230-400 mesh). ^1H and ^13C NMR spectra were recorded on 400 MHz (Agilent) and 600 MHz (Jeol) NMR spectrometers and expressed as multiplicities (s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; m, multiplet; or a combination thereof (e.g., dd, dt, etc.)), coupling constants in hertz (Hz), and chemical shifts reported in parts per million (ppm). NMR solvents used were purchased from Eurisotop ^® . Spectra were analyzed by MestReNova. HRMS was measured using electrospray ionization (ESI) and Q-TOF mass spectrometry (Agilent 6530). Analytical high-performance liquid chromatography (HPLC) and protein MS were performed using a WuXi App Tec. Size exclusion chromatography (SEC) was performed using an Agilent Technologies 1260 Infinity Ⅱ. All chemical reagents and solvents were of analytical or HPLC grade from commercial sources. Trastuzumab (Herceptin ^® ) was purchased from Roche Pharma and DM1 was purchased from Angene. Room temperature (RT) indicates ambient temperature. Unless otherwise stated, yields refer to spectroscopically and chromatographically pure compounds.

<Experimental Example 4> Optimization of reaction conditions

Trastuzumab (2 mg/ml) 1 equivalent, 37°C 1 h, buffer pH 7.5; The conjugation ratio was confirmed by hydrophobic interaction chromatography (HIC). Table 5 below shows the results.

Entry linker-payload Equivalent ratio
(mAb:TCEP:Linker-Payload) ADC Conjugation ratio ^b (%) 1 1 1 : 5 : 10 ADC 1 Heterogeneous (<90%) 2 1 1:5:20 ADC 1 Heterogeneous (<90%) 3 1 1:5:30 ADC 1 Heterogeneous (<90%) 4 1 1:5:40 ADC 1 Heterogeneous (<90%) 5 2 1 : 5 : 10 ADC 2 Heterogeneous (<90%) 6 2 1:5:20 ADC 2 > 90% 7 3 1:5:20 ADC 3 > 90% 8 4 1:5:20 ADC 4 > 90% 9 5 1:5:20 ADC 5 Heterogeneous (<90%)

Figure 6 shows (a) trastuzumab; (b)-(f) hydrophobic interaction chromatography (HIC) data of ADC 1-5, respectively, with all optimization test results for ADC 1 overlaid.

As a result, it was shown that most trastuzumab molecules participated in the conjugation process through the use of a hydrophobic interaction chromatography (HIC) column, and no unreacted trastuzumab existed.

<Experimental Example 5> MS Analysis

LC-MS analysis was performed on ADC 2-4 manufactured in Example 3 above. Specifically, MS analysis was performed at Wuxibiologics, and the manufactured ADC was treated with pH8.0 Tris and 0.1 M DTT, incubated at 37°C for 15 minutes, and then subjected to MS analysis.

Figure 7 shows one linker-payload detected in the light chain (left) and three linker-payloads detected in the heavy chain (right): (a) linker-payload 2 (MW ~972); (b) linker-payload 3 (MW ~986); and (c) linker-payload 4 (MW ~1,000).

Figure 8 shows the mass spectrometry results of ADC 2-4; TIC spectra and DAR calculation table. (a) ADC 2; (b) ADC 3; and (c) ADC 4.

As a result, the homogeneity of ADCs was found to vary depending on the alkyl chain length of the linker while maintaining the same conjugation conditions. SDS-PAGE and LC-MS data identified linker payloads with a DAR close to 8, which in turn confirmed that the optimal alkyl chain length was 3–5 carbon atoms.

<Experimental Example 6> SEC Analysis

Aggregates were analyzed using a Size Exclusion Chromatography (SEC) column. Specifically, the ratios of ADC and aggregates prepared using a phosphate buffer were calculated using an Agilent AdvanceBio SEC, 300Å 2.7μm, 4.6*300mm column.

Table 6 below shows the results of size exclusion chromatography (SEC) analysis for the aggregates of ADC 2-4 manufactured in Example 3.

ADC ADC 2 ADC 3 ADC 4 DAR 7.89 7.82 7.81 Aggregates (%) 3.03 5.19 12.45

Figure 9 shows size exclusion chromatography (SEC) spectra for aggregates of ADC 2-4: (a) ADC 2; (b) ADC 3; and (c) ADC 4.

As a result, it was found that the aggregation rate increased as the carbon chain length of the linker increased.

<Experimental Example 7> in vitro Testing and Combination Analysis

In vitro experiments were performed using SK-BR-3 cell lines that overexpress HER2. Specifically, 5,000 cells/well (10% FBS media 96-well plate) were seeded and the cells were cultured for 24 hours at 37°C and 5% _CO2 before drug treatment. To prevent evaporation of the medium, PBS was added to all wells next to the cell seed wells.

100 μL of medium containing the drug to be tested (drug by concentration + 1% FBS) was added. PBS was added to the surrounding wells to prevent evaporation. The culture was performed at 37°C and 5% CO ₂ for the set time (payload: select appropriate culture conditions by observing the culture conditions for 48 hr, 72 hr, and 96 hr after initial observation). For antibodies and ADCs, appropriate culture conditions were selected by observing the culture conditions for 96 hr, 120 hr, and 144 hr. For the 120 hr and 144 hr culture conditions, additional medium was added after 72 hr of culture. The drug concentration was set to 3 or less and 3 or more based on the IC ₅₀ value used as a standard. The positive control generally used etoposide 10 μM or 24 μM.

Add 10 μl of CCK-8 solution (Cell counting Kit-8, Dojindo Laboratories, Japan) to each well and incubate for 2.5 hours at 37°C and 1 to 4 hours in 5% _CO2 , and then incubate for 2.5 hours according to the instructions of the CCK-8 solution kit. After shaking for 15 seconds, the absorbance was measured at 450 nm (Tecan microplate reader). Table 7 below shows the results.

SK-BR-3 IC ₅₀ CI ₉₅ Concentration mAb Trastuzumab > 10 - μg/ml ADC ADC 2 2.41 1.29 - 4.10 ng/ml ADC 3 2.54 1.20 - 4.75 ADC 4 4.66 2.35 - 8.74

As a result, it was confirmed that the IC ₅₀ value was reduced compared to the intact antibody. ADC 2-4, which had a DAR close to approximately 8, all showed the ability to inhibit cancer cells.

ADC 2, which showed the lowest IC ₅₀ and aggregation rate, was selected as the optimal candidate for further analysis. Since the linker has the non-cleavable property, the linker-payload efficacy and the payload itself were also evaluated (Figure 10). Table 8 below shows the results of evaluating the cytotoxicity of ADC 2, trastuzumab, DM1, and linker-payload 2 against tumor cells SK-BR-3.

SK-BR-3 IC ₅₀ CI ₉₅ Concentration mAb Trastuzumab > 10,000 - ng/ml ADC ADC 2 2.41 1.29 - 4.10 Payload DM1 1.00 0.87 - 1.15 nM linker-payload linker-payload2 18.14 11.43 - 28.77

Figure 10 compares the cytotoxicity against tumor cells SK-BR-3: (a) DM1 and linker-payload 2; (b) trastuzumab and ADC 2.

The IC ₅₀ value of T-DM1 is known to be 7–18 ng/ml, which is higher than that of ADC 2–4. This may be due to the difference in DAR (~3.5 for T DM1 and ~8 for ADC 2–4). When comparing the efficacy of ADC 2–4, it was found that the cytotoxicity decreased as the length of the linker increased. This can be inferred to be related to the fact that as the linear carbon chain of the linker increases, the hydrophobicity increases, the number of aggregates increases, and the ADC concentration reaching the cell decreases.

<Experimental Example 8> ELISA analysis for binding affinity

A test was conducted on the binding affinity of ADC. First, an ELISA test was performed to confirm the binding affinity of ADC 2 stored for 1 week. The experiment was conducted with reference to the paper reported in Qin et al., 2022. Specifically, all trastuzumab-conjugated ADCs, including partially conjugated or unconjugated trastuzumab that immunoreacts with the HER2 extracellular domain, were measured. Microtiter plates (96 wells, Nunc, NY, USA) were coated with 0.5 μg/ml of recombinant HER2 ECD, p105HER-2 (Genetech, Inc., CA, USA) in coating buffer (0.05 M carbonate/bicarbonate buffer, pH 9.6). After incubation at 2–8°C for 16–72 h, the coating solution was removed, and assay diluent (phosphate-buffered saline [PBS], 0.5% bovine serum albumin [BSA], 0.05% polysorbate 20, 0.05% Proclin 300) was added with agitation at ambient temperature for 1–2 h to block nonspecific binding sites. After washing, F(ab')2 goat anti-human IgG Fc conjugated to horseradish peroxidase (HRP; Jackson ImmunoResearch, PA, USA) was diluted in assay diluent and added for detection. The plates were incubated at ambient temperature for 1 h with agitation and then washed. The bound HRP conjugate was detected using tetramethyl benzidine peroxidase substrate (Moss, Inc., MD, USA). Color development was allowed for 10–20 min at ambient temperature without agitation. The enzyme reaction was stopped by adding 1 M phosphoric acid. Absorbance was measured at 450 nm against a reference wavelength of 620 or 630 nm using a microplate reader. Total trastuzumab concentrations were calculated by interpolation from a four-parameter fit to the standard curve. Table 9 below shows the results comparing the Bmax and Kd values of trastuzumab, T-Dxd, and ADC 2 over a one-week period.

Trastuzumab T-Dxd ADC 2 Day Day 1 Day 1 Day 7 Day 1 Day 7 Bmax 3.602 2.672 2.462 3.963 3.863 Kd 1.284 0.768 1.327 1.210 1.216

Figure 11 shows a graph comparing the binding affinity of trastuzumab, T-Dxd, and ADC 2 over a period of one week.

As a result, we confirmed that steady binding was maintained by comparing Bmax and Kd. In addition, the binding affinity of ADC 2 was juxtaposed to that of T-Dxd (DAR ~7.7) with similar DAR. Over the course of a week, the Kd value of T-Dxd increased from 0.768 to 1.327, whereas that of ADC 2 showed little change from 1.210 to 1.216. In addition, the Bmax value decreased from 2.672 to 2.462 for T-Dxd and from 3.963 to 3.863 for ADC2. When comparing the data of the two high DAR ADCs, we confirmed that ADC 2 had more stable binding than T-Dxd.

<Experimental Example 9> Purity Analysis of Single Antibody-Drug Conjugate Using SDS-PAGE

To confirm the purity and molecular weight of the novel antibody-drug conjugate, SDS-PAGE analysis was performed on reduced trastuzumab and ADC 2-4 prepared in Example 3. Specifically, the sample concentration was calculated based on the BCA Assay results, adjusted to a constant concentration of 0.2 mg/ml, mixed with 4x loading buffer (Thermo, #NP0007) and 10x reducing agent (Thermo, #B0009) in a ratio (PBS 3.75 ul, 4x loading buffer, 10x reducing buffer, 0.2 mg/ml protein), and incubated at 95°C. After heating for 10 minutes, cooled on ice, and when using a gradient gel, Bolt ^TM 4-12%, Bis-Tris, 1.0 mm, Mini Protein Gel 10-well (Invitrogen, #NW04120BOX) was used. Fill the mini gel tank (Thermo, #A25977) with the appropriate buffer for the gel, insert the gel, and load 3ul of Ladder (BIO-RAD, #1610375) and 10ul of sample (1.0ug/well). Connect to PowerEase ^TM Touch 350W power supply (Invitrogen, #PS0351) and run (200V for 30 min when using Bis-Tris gel). After the sample was properly unloaded, turn off the instrument and take out the gel. Place the gel in a lidded container, pour Coomassie Brilliant Blue R-250 staining solution (BIO RAD, #1610436), and shake at 25 rpm overnight on a digital rocker (DAIHAN, #RK-2D). After removing the dye solution, decolorization was performed using a decolorizing solution (distilled water 6: methanol 3: acetic acid 1), changing the solution three times in total once every hour. Figure 12 shows the results.

As a result, as shown in Fig. 12, the purity suitable for evaluating breast cancer cell proliferation inhibition activity was confirmed.

<Experimental Example 10> Evaluation of breast cancer cell proliferation inhibition activity in an animal model

Xenograft anticancer activity efficacy test using HER2 positive cell line SK-BR3 (Mean ± SEM)

To observe the efficacy of the novel antibody-drug conjugate, in vivo efficacy experiments were performed in a HER2-positive breast cancer cell line (SK-BR3) xenograft model using BALB/c-nude mice.

Specifically, the comparator Kadcyla® (T-DM1), which received FDA approval in 2013 and conjugated the same antibody (trastuzumab) and payload (DM1), and the single antibody-drug conjugate (ABC-002) were administered intravenously as a single dose of 3 mg/kg body weight to BALB/c-nude mice transplanted with SK-BR3 cells, and the degree of inhibition of transplanted tumor growth was compared.

Figure 14 (a) shows the result.

As a result, as shown in (a) of Fig. 14, it was confirmed that the single antibody-drug polymer according to the present invention had a superior anticancer effect compared to the results of the comparative group, Kadcyla® (T-DM1).

<Experimental Example 11> Toxicity evaluation in a xenograft model using HER2-positive cell line SK-BR3 (Mean ± SEM) - Weight change

A single-dose toxicity test was performed using BALB/c-nude mice to determine whether the stability of a novel antibody-drug polymer and its preparation method affect toxicity.

Figure 14 (b) shows the results for weight change.

As a result, as shown in (b) of Figure 14, the antibody-drug polymer group showed a significant weight loss compared to the Kadcyla® comparison group.

Additionally, the comparator group Kadcyla® (T-DM1), which was approved by the FDA in 2013 and conjugated with the same antibody (trastuzumab) and payload (DM1), and the single antibody-drug conjugate (ABC-002) were administered intravenously as a single dose of 3 mg/kg body weight to BALB/c-nude mice transplanted with SK-BR3 cells. Body weights were measured at 3-4 day intervals from the time of test substance administration to the end of the experiment (day 28).

As a result, as shown in (b) of Fig. 14, the single antibody-drug polymer according to the present invention showed a significant weight gain compared to the comparative group Kadcyla® (T-DM1), confirming that no significant signs of toxicity were observed.

The linker-payload of the present invention was prepared by a simple synthesis using a dimaleimide functional group, and various types of linker payloads were synthesized in favorable yields using inexpensive and commercially available reagents. In addition, ADCs with high DAR were synthesized using linker systems of various lengths with a simple process and high yield. It was shown that aggregation increased as the linker length increased. In vitro tests showed the expected anticancer effect in cell lines overexpressing HER2. Not only the linker-payload itself showed anticancer effect, but the ADC also showed good anticancer activity, showing stable binding ability despite the high DAR. In addition, when comparing the binding affinity between the ADC and T-Dxd, it was confirmed that the ADC 2 of the present invention showed superior binding affinity than T-Dxd. The linker payload of the present invention is expected to be useful for ADC developers in the future.

Claims (25)

A linker-payload conjugate represented by the following general formula Ⅰ: [General Formula Ⅰ] M ₁ -SU-M ₂ -XP In the above formula, M ₁ and M ₂ are maleimide moieties, SU is a first spacer unit represented by (SU ₁ ) _a -(SU ₂ ) _b -(SU ₃ ) _c , and SU ₁ , SU ₂ , and SU ₃ may be the same or different, and a, b, and c are each independently integers from 0 to 3, X is a second spacer unit expressed as (X ₁ ) _x -(X ₂ ) _y -(X ₃ ) _z , wherein X ₁ , X ₂ , and X ₃ may be the same or different, and x, y, and z are each independently integers from 0 to 3, P is the payload. In the first paragraph, A linker-payload conjugate, wherein the above SU ₁ , SU ₂ , and SU ₃ are each independently selected from the group consisting of a substituted or unsubstituted C _1-30 alkylene, a substituted or unsubstituted C _1-30 heteroalkylene, a substituted or unsubstituted C _2-30 alkenylene, a substituted or unsubstituted C _3-30 cycloalkylene, a substituted or unsubstituted C _6-30 arylene, and a substituted or unsubstituted C _6-30 heteroarylene. In the second paragraph, The above SU ₁ , SU ₂ , and SU ₃ may each be independently selected from the group consisting of -(CH ₂ ) _n -, -C _3-6 cycloalkyl-, -C _3-6 cycloaryl-, -C _3-6 heteroaryl-, and -(CH ₂ CH ₂ O) _m -, Here, n is an integer from 1 to 10, independently. A linker-payload conjugate, where m is an integer from 1 to 10. In the first paragraph, A linker-payload conjugate wherein the above SU is -(CH ₂ ) _n -, where n is an integer greater than or equal to 2. In the first paragraph, A linker-payload conjugate, wherein X ₁ , X ₂ , and X ₃ are each independently selected from the group consisting of a substituted or unsubstituted C _1-30 alkylene, a substituted or unsubstituted C _1-30 heteroalkylene, a substituted or unsubstituted C _2-30 alkenylene, a substituted or unsubstituted C _3-30 cycloalkylene, a substituted or unsubstituted C _6-30 arylene, and a substituted or unsubstituted C _6-30 heteroarylene. In the first paragraph, At least one of the above X ₁ , X ₂ , and X ₃ is a substituted C _1-10 heteroalkylene, when substituted, is substituted with a group selected from halo, =O, OH, NH ₂ , SH, NO ₂ , N ₃ , CN, OR ₁ , SR ₁ , OC(O)R ₁ , OC(O)NHR ₁ , OC(O)OR ₁ , CONHR ₁ , CON(R ₁ ) ₂ , NHC(O)R ₁ , C(O)R ₁ , NHR ₁ , N(R ₁ ) ₂ , C(O)R ₁ , OS(O) ₂ R ₁ , -OP(O)(OR ₁ )(OR ₁ ), OP(O)(NHR ₁ )(NHR ₁ ), and C _1-3 alkyl, A linker-payload conjugate wherein R ₁ is selected from the group consisting of H, OH, C _1-5 alkyl, C _1-5 heteroalkyl, C _3-8 aryl and C _3-8 heteroaryl. In the first paragraph, A _linker - _payload conjugate wherein the above X is selected from the group consisting of -S-(CH ₂ ) _a1 -NHC(O)-(CH ₂ ) _b1 -S-, -S-(CH ₂ ) _a1 OC(O)-(CH ₂ ) _b1 -S-, -S-(CH ₂ ) _a1 C(O)-(CH ₂ ) _b1 -S-, -S-(CH ₂ ) _a1 NH-(CH ₂ ) _b1 -S-, -S-(CH ₂ ) _a1 C(O)NH-(CH 2 ) b1 -S- and -S-(CH ₂ ) _a1 OC(O)NH-(CH ₂ ) _b1 -S-, wherein a1 and b1 are each independently an integer from 1 to 5. In Article 7, A linker-payload conjugate wherein X is -S-(CH ₂ ) _a1 -NHC(O)-(CH ₂ ) _b1 -S-, wherein a1 and b1 are each independently an integer from 1 to 3. A ligand-drug conjugate represented by the following general formula II: [General Formula II] Ab-(M ₁ -SU-M ₂ -XP) _n In the above formula, Ab is a ligand, M ₁ and M ₂ are maleimide moieties, SU is a first spacer unit represented by (SU ₁ ) _a -(SU ₂ ) _b -(SU ₃ ) _c , and SU ₁ , SU ₂ , and SU ₃ may be the same or different, and a, b, and c are each independently integers from 0 to 3, X is a second spacer unit expressed as (X ₁ ) _x -(X ₂ ) _y -(X ₃ ) _z , wherein X ₁ , X ₂ , and X ₃ may be the same or different, and x, y, and z are each independently integers from 0 to 3, P is the payload, n is an integer from 1 to 20. In Article 9, A ligand-drug conjugate, wherein the ligand is covalently bonded to a maleimide moiety by a thioether bond, wherein the thioether bond comprises a sulfur atom of a cysteine of the ligand. In claim 10, the ligand comprises a cysteine-terminal amino acid motif; and A ligand-drug conjugate wherein the thioether bond comprises a sulfur atom of cysteine of the amino acid motif. In Article 9, A ligand-drug conjugate wherein the ligand is a monoclonal antibody, a polyclonal antibody, an antibody fragment, a Fab, a Fab', a Fab-SH, F(ab')2, an Fv, a single-chain Fv ("scFv"), a diabody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody or a fusion protein comprising an antigen-binding portion of an antibody. In Article 12, The above ligands are muromonab-CD3 abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab, etanercept, basiliximab, gemtuzumab, alemtuzumab, ibritumomab, adalimumab, alefacept, omalizumab, efalizumab, tositumomab, cetuximab, ABT-806, bevacizumab, Natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab, romiplostim, AMG-531, golimumab, ustekinumab, ABT-874, belatacept, belimumab, atacicept, anti-CD20 antibodies, canakinumab, tocilizumab, atlizumab, mepolizumab, pertuzumab, HuMax CD20, tremelimumab, ticilimumab, A ligand-drug selected from the group consisting of ipilimumab, IDEC-114, inotuzumab, HuMax EGFR, aflibercept, HuMax-CD4, teplizumab, otelixizumab, catumaxomab, anti-EpCAM antibody IGN101, adecatumomab, oregovomab, dinutuximab, girentuximab, denosumab, bapineuzumab, motavizumab, efumgumab, raxibacumab, LY2469298, and veltuzumab. Conjugate. In Article 9, A ligand-drug conjugate wherein the payload is at least one active agent selected from a chemotherapeutic agent and a toxin. In Article 14, A ligand-drug conjugate wherein the active agent is an immunomodulating compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent or a combination thereof. In Article 15, A ligand-drug conjugate wherein the above active agent is independently selected from: (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, Carboquone, meturedopa, uredopa, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, calystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, Duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, Lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophores, aclacinomycin, actinomycin, antrmycin, azaserine, bleomycin, cactinomycin, carabicin, carninomycin, carzinophilin, Chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, Aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocin, mitoguazone, Mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, and anguidine, urethane, Vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate or acid of any of the foregoing; (b) monokines, lymphokines, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factor, interferon, interferon-α, interferon-β, interferon-γ, Colony stimulating factor (“CSF”), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (“IL”), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF, kit ligand, or a combination of any of the foregoing; (c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, amanitin derivatives, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, a dolastatin analogue, cryptophycin, camptothecin, camptothecin derivatives and metabolites, rhizoxin, a rhizoxin derivative, CC-1065, a CC-1065 analogue or derivative, a duocarmycin, an enediyne antibiotic, esperamicin, an epothilone, azonafide, apridine, a toxoid, or a combination of any of the foregoing; (d) an affinity ligand (wherein the affinity ligand is a substrate), an inhibitor, a stimulant, a neurotransmitter, a radioisotope, or a combination of any of the foregoing; (e) a radiolabel, ³² P, ³⁵ S, a fluorescent dye, an electron dense reagent, an enzyme, biotin, streptavidin, dioxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to the target, or a combination of any of the foregoing; (f) immunomodulating compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or a combination of any of the foregoing; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene; (h) 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole; (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine; (j) aromatase inhibitors; (k) protein kinase inhibitors; (l) lipid kinase inhibitors; (m) antisense oligonucleotides; (n) ribozyme; (o) vaccines; and (p) Anti-angiogenic agent. In Article 16, A ligand-drug conjugate wherein the above active agent is DM1. A ligand-drug conjugate of claim 9, characterized in that the ligand-drug conjugate has a DAR (Drug-to-Antibody Ratio) of 3 to 8. A ligand-drug conjugate of claim 18, characterized in that it has a DAR (Drug-to-Antibody Ratio) of 7 to 8. In Article 9, A ligand-drug conjugate, wherein the linkage between Ab and M ₁ and the linkage between X and P are each independently cleavable or non-cleavable. A pharmaceutical composition for treating cancer, comprising a ligand-drug conjugate of claim 9. A pharmaceutical composition for treating cancer, wherein in claim 21, the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, uterine corpus cancer, or melanoma. In Article 22, A pharmaceutical composition for treating cancer, wherein the cancer is a cancer associated with Her2 expression or underexpression. A pharmaceutical composition for treating cancer, wherein the cancer is breast cancer in claim 23. A method comprising the step of administering the pharmaceutical composition of claim 21 to a subject in need thereof.

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