KR20240159448A - Method for preparing novel antibody-drug conjugate having a homogeneous DAR - Google Patents

Abstract

The present invention provides a method for producing a novel antibody-drug polymer. According to the method of the present invention, there is a useful effect that an ADC having a uniform DAR can be provided.

Description

{Method for preparing novel antibody-drug conjugate having a homogeneous DAR}

The present invention relates to a method for producing a novel antibody-drug polymer.

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, and they use a bond using cysteine obtained by reducing the inter-chain disulfide bond of native antibodies.

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.

Kadcyla is an early ADC that uses DM1 as a drug and is a heterogeneous ADC with a DAR of approximately 3.5. Enhertu uses deruxtecan as a drug and has relatively high homogeneity with a DAR of approximately 7.7, but is still heterogeneous.

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 ADC can maintain a high DAR while controlling toxicity and stability, the higher and more uniform the DAR, the better the ADC can be evaluated.

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 we chose the strategy of conjugating DM1 to cysteine residues of antibodies.

Accordingly, the inventors of the present invention synthesized an ADC having a highly uniform DAR (DAR = 8) by using a novel linker including a maleimide group in a manner of binding DM1 to a cysteine residue of an antibody to develop an ADC having a uniform DAR. This novel ADC can be produced through an extremely simple synthetic process, and it was confirmed that a single ADC can be produced with a high conjugation yield.

Republic of Korea Patent No. 10-2442906 Republic of Korea Publication Patent No. 10-2023-0008723 Republic of Korea Publication Patent No. 10-2019-0038579

Chemical Science (2022), 13(10), 2909-2918 Bioconjugate Chem.　2021, 32, 8, 1525-1534 Clin Cancer Res (2004) 10 (20): 7063-7070 J. Med. Chem.　2014, 57, 16, 6949-6964 Mol. Pharmaceutics 12, 3986-3998

The present invention seeks to provide a method for producing a novel antibody-drug conjugate (ADC).

In one embodiment of the present invention for achieving such a task, a method for producing an antibody-drug polymer is provided, comprising: 1) a step of preparing a linker including a di-maleimide group, including a step of reacting diamine and maleic anhydride by dissolving them in a solvent, removing the solvent, and purifying the remaining mixture; 2) a step of preparing a linker-drug conjugate by contacting the linker prepared in step 1) with a drug at room temperature and optionally isolating the linker-drug conjugate; and 3) a step of reacting an antibody or an antigen-binding fragment thereof with the linker-drug conjugate prepared in step 2) and purifying it.

In addition, in another embodiment of the present invention, a method for preparing an antibody-drug polymer is provided, comprising the steps of: a) contacting a drug with a linker comprising any one structure selected from the following chemical formulas II to IV at room temperature and optionally isolating the linker-drug conjugate, thereby preparing a linker-drug conjugate; and b) reacting an antibody or an antigen-binding fragment thereof with the linker-drug conjugate prepared in step a) and purifying it.

[Chemical formula II]

,

[Chemical Formula III]

,

[Chemical Formula IV]

.

The method for producing a novel ADC compound according to the present invention is homogeneously linked to a cysteine residue with respect to positional specificity and has a remarkably high synthesis yield. In addition, it provides a technology for securing a single ADC by using a native antibody as it is without requiring antibody engineering, and at the same time provides a novel ADC compound, so that it can be usefully used for effective single ADC synthesis.

Figure 1 illustrates the structure of an antibody (Trastuzumab)-drug (DM1) polymer manufactured according to one embodiment of the present invention (Example 3).
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 Experimental Example 1 of the present invention.
Figure 4 shows the results of DAR analysis of an antibody-drug polymer (ABC-002) according to Experimental Example 2 of the present invention.
Figure 5 shows the results of measuring the yield of ADC according to Experimental Example 3 of the present invention.
Figure 6 shows the HIC and SEC analysis results of ABC-002 according to Experimental Example 4 of the present invention.
Figure 7 shows Q-TOF data of trastuzumab and ABC-002 according to Experimental Example 5 of the present invention.
Figure 8 shows the results of purity analysis of a single antibody-drug polymer using SDS-PAGE according to Experimental Example 6 of the present invention.

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 similar regulatory body for use in animals, and more particularly in humans, by avoiding significant toxic effects when used at typical 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 comprises the steps of: 1) preparing a linker including a di-maleimide group, comprising the steps of dissolving diamine and maleic anhydride in a solvent, reacting them, removing the solvent, and purifying the remaining mixture;

2) a step of preparing a linker-drug conjugate by contacting the drug with the linker prepared in step 1) at room temperature and optionally isolating the linker-drug conjugate; and

3) a step of reacting and purifying an antibody or an antigen-binding fragment thereof with the linker-drug conjugate prepared in step 2);

A method for producing an antibody-drug polymer is provided.

In one specific embodiment of the present invention, the reaction of step 1) may be performed at a temperature condition of 30 to 100°C. Specifically, the temperature may be 30 to 100°C, 30 to 95°C, 30 to 90°C, 30 to 85°C, 30 to 80°C, 30 to 75°C, 30 to 70°C, 35 to 70°C, 40 to 70°C, 45 to 70°C, 50 to 70°C, more specifically, 51 to 69°C, 52 to 68°C, 53 to 67°C, 54 to 66°C, 55 to 65°C, 56 to 64°C, 57 to 63°C, 58 to 62°C, 59 to 61°C, and even more specifically, 60°C, but is not limited thereto.

In one specific embodiment of the present invention, the solvent of step 1) may be selected from the group consisting of acetic acid, dichloromethane, chloroform, DMF (dimethyl formamide), THF (tetrahydrofuran), ethyl acetate, alcohol, toluene, diethyl carbonate, acetone, acetonitrile, DMSO, dimethylacetamide, cyclohexane, N-cyclohexylpyrrolidinone, distilled water, and a mixed solvent of two or more thereof, but is not limited thereto.

In one specific embodiment of the present invention, the linker of step 1) may include any one structure selected from the following chemical formulas II to IV, and may be prepared by the process of the following reaction formula I:

[Chemical formula II]

,

[Chemical Formula III]

,

[Chemical Formula IV]

.

[Reaction Formula I]

.

In one specific example of the present invention, the drug of step 2) may be DM1 of the following chemical formula I, and the linker-drug conjugate may be prepared by the process of the following reaction scheme II.

[Chemical formula I]

.

[Reaction Formula II]

.

In one specific embodiment of the present invention, the antibody of step 3) may be trastuzumab.

In one specific embodiment of the present invention, the reaction of step 3) can be performed at a temperature condition of 30 to 50°C. Specifically, the temperature may be 30 to 50°C, 30 to 49°C, 30 to 48°C, 30 to 47°C, 30 to 46°C, 30 to 45°C, 30 to 44°C, 30 to 43°C, 30 to 42°C, 30 to 41°C, 30 to 40°C, 31 to 40°C, 32 to 40°C, 33 to 40°C, 34 to 40°C, 35 to 40°C, 36 to 40°C, 36 to 39°C, 36 to 38°C, and more specifically, 37°C, but is not limited thereto.

In one specific embodiment of the present invention, the antibody-drug polymer may have a structure as shown in Chemical Formula V below:

[Chemical Formula V]

Here, n is an integer independently of the other, 3, 4, or 5.

Another aspect of the present invention comprises the steps of: a) preparing a linker-drug conjugate by contacting a drug with a linker comprising any one structure selected from the following chemical formulae II to IV at room temperature and optionally isolating the linker-drug conjugate; and

b) a step of reacting and purifying an antibody or an antigen-binding fragment thereof with the linker-drug conjugate prepared in step a);

Provided is a method for preparing an antibody-drug polymer:

[Chemical formula II]

,

[Chemical Formula III]

,

[Chemical Formula IV]

.

An antibody-drug polymer produced by the method for producing an antibody-drug polymer of the present invention is characterized by having a uniform DAR.

The above DAR may be 1 to 10, specifically 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 7 to 9, 7 to 8, but is not limited thereto.

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

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 white strands were generated, which were redissolved with continuous stirring. Acetic anhydride (1.23 g, 12 mmol) was then 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 purified 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 (100 MHz, CDCl ₃ ) δ 170.74, 134.30, 35.46, 27.55.

<1-2> 1,1'-(butane-1,4-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). 1,4-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.

<1-3> 1,1'-(pentane-1,5-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). 1,5-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. 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 6.68 (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.

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

DM1 and the maleimide linker manufactured in Example 1 were added in a ratio of 1:1 to 1:2, and then 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'-(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 , the linker prepared in Example 1-1 (47.6 mg, 0.203 mmol) 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.

<2-2> 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 , the linker prepared in Example 1-2 (50.4 mg, 0.203 mmol) 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.

<2-3> 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 , the linker prepared in Example 1-3 (53.3 mg, 0.203 mmol) 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.

<Example 3> Preparation of antibody (Trastuzumab)-drug (DM1) polymer

3-1. Antibody (Trastuzumab)-Linker 1-Drug (DM1) Polymer: ABC-002

1 ul of 50 mM TCEP solution was added to 1 ml of phosphate buffer containing 1 mg of trastuzumab, reduced at room temperature, and about 6 to 20 equivalents of the drug-linker prepared in Example 2-1 were added and reacted at room temperature for 30 minutes. After the reaction was completed, centrifugation was performed using a centrifuge (4000 rpm) and the supernatant was collected.

3-2. Antibody (Trastuzumab)-Linker 2-Drug (DM1) polymer

Using the drug linker manufactured in Example 2-2, an antibody-drug polymer was manufactured in the same manner as in 3-1.

3-3. Antibody (Trastuzumab)-Linker 2-Drug (DM1) polymer

Using the drug linker manufactured in Example 2-3, an antibody-drug polymer was manufactured in the same manner as in 3-1.

Example 3 (3-1 to 3-3) Antibody Trastuzumab (anti-HER2 Ab) Drugs DM1 Binding site Cysteine Linker Maleimide (Examples 2-1 to 2-3)

The structure is as shown in Fig. 1.

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

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

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

<Experimental Example 1> DAR Analysis by 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 3-1, Comparative Example 1, and Comparative Example 2 was analyzed.

Table 4 below shows the results.

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

Figure 4 shows the results of measuring DAR of ADC (ABC-002) manufactured in Example 3-1.

<Experimental Example 3> Yield Measurement of ADC

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

The results are shown in Figure 5.

As a result of the analysis, it was shown that the ADC (ABC-002) manufactured in Example 3-1 of the present invention had a higher yield than ^Enhertu® .

<Experimental Example 4> HIC, SEC Analysis of ABC-002

The analysis results are as shown in Fig. 6.

<Experimental Example 5> Q-TOF Analysis of Antibody and Linker-Drug Conjugates

Figure 7 shows Q-TOF data of trastuzumab and ADC (ABC-002) manufactured in Example 3-1.

The analysis results showed that there were only two peaks: one peak with one linker-DM1 attached to the light chain of the antibody and one peak with three linker-DM1 attached to the heavy chain.

<Experimental Example 6> Purity Analysis of Single Antibody-Drug Conjugate Using SDS-PAGE

To confirm the purity and molecular weight of the novel antibody-drug polymer, SDS-PAGE analysis was performed on reduced trastuzumab and the ADC (ABC-002) prepared in Example 3-1. Specifically, SDS-PAGE was performed using Bolt Bolt ^TM 4-12% Bis-Tris gradient gel.

Figure 8 shows the results. (H: heavy chain, L: light chain)

As a result, as shown in Fig. 8, the purity suitable for evaluating breast cancer cell proliferation inhibition activity was confirmed.

In addition, it was confirmed that the molecular weights of the heavy chain and light chain of the novel single antibody-drug polymer were higher than those of the heavy and light chains of the antibody trastuzumab.

In conclusion, the manufacturing method of the present invention is expected to be useful in future ADC synthesis because it can produce an antibody-drug polymer having a uniform DAR (DAR = 8) in high yield.

Claims (15)

1) A step for producing a linker including a di-maleimide group, comprising the steps of dissolving diamine and maleic anhydride in a solvent, reacting them, removing the solvent, and purifying the remaining mixture;
2) a step of preparing a linker-drug conjugate by contacting the drug with the linker prepared in step 1) at room temperature and optionally isolating the linker-drug conjugate; and
3) a step of reacting and purifying an antibody or an antigen-binding fragment thereof with the linker-drug conjugate prepared in step 2);
Method for producing an antibody-drug polymer.
In the first paragraph,
A method for producing an antibody-drug polymer, wherein the reaction of step 1) above is performed at a temperature of 30 to 100°C.
In the first paragraph,
A method for producing an antibody-drug polymer, wherein the solvent of step 1) is a solvent selected from the group consisting of acetic acid, dichloromethane, chloroform, DMF (dimethyl formamide), THF (tetrahydrofuran), ethyl acetate, alcohol, toluene, diethyl carbonate, acetone, acetonitrile, DMSO, dimethylacetamide, cyclohexane, N-cyclohexylpyrrolidinone, distilled water, and a mixed solvent of two or more thereof.
In the first paragraph,
A method for producing an antibody-drug polymer, wherein the reaction of step 3) above is performed at a temperature of 30 to 50°C.
In the first paragraph,
A method for producing an antibody-drug polymer, wherein the antibody of step 3) above is trastuzumab.
In the first paragraph,
A method for producing an antibody-drug polymer, wherein the drug of step 2) is DM1 of the following chemical formula I:
[Chemical formula I]
.
In the first paragraph,
A method for producing an antibody-drug polymer, wherein the linker comprises any one structure selected from the following chemical formulas II to IV:
[Chemical formula II]
,
[Chemical Formula III]
,
[Chemical Formula IV]
.
In the first paragraph,
The above antibody-drug polymer has a structure as shown in the following chemical formula V, a method for producing an antibody-drug polymer:
[Chemical Formula V]

Here, n is an integer independently of the numbers 3, 4, or 5.
In the first paragraph,
A method for producing an antibody-drug polymer, characterized in that the antibody-drug polymer has a uniform DAR.
In Article 9,
A method for producing an antibody-drug polymer, wherein the above DAR is a uniform value of 5 to 10.
In Article 9,
A method for producing an antibody-drug polymer, wherein the above DAR is a uniform value of 7 to 9.
a) a step of preparing a linker-drug conjugate by contacting a drug with a linker comprising any one structure selected from the following chemical formulas II to IV at room temperature and optionally isolating the linker-drug conjugate; and
b) a step of reacting and purifying an antibody or an antigen-binding fragment thereof with the linker-drug conjugate prepared in step a);
Method for preparing antibody-drug polymer:
[Chemical formula II]
,
[Chemical Formula III]
,
[Chemical Formula IV]
.
A method for producing an antibody-drug polymer, characterized in that in claim 12, the antibody-drug polymer has a uniform DAR.
A method for producing an antibody-drug polymer, wherein in claim 12, the DAR is a uniform value of 5 to 10.
A method for producing an antibody-drug polymer, wherein in claim 12, the DAR is a uniform value of 7 to 9.