An antibody-drug conjugate targeting human IGSF9, its preparation method and application

Abstract

The present invention provides an antibody-drug conjugate targeting human IGSF9, its preparation method and application, belonging to the technical field of biomedicine. The antibody-drug conjugate is obtained by conjugating a monoclonal antibody targeting IGSF9 and a DNA topoisomerase I inhibitor with a linker maleimide-GGFG tetrapeptide. The purity of the antibody-drug conjugate targeting human IGSF9 prepared in the present invention can reach 99.7%, and it can target and kill THP-1 tumor cells positive for IGSF9. Mouse experiments show that the antibody-drug conjugate targeting human IGSF9 can significantly inhibit tumor growth and metastasis and improve the survival rate of mice. It solves the problem in the prior art that the monoclonal antibody targeting IGSF9 needs to be used in combination with a PD-1 antibody to significantly inhibit tumor growth.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an antibody-drug conjugate targeting human IGSF9, a preparation method thereof, and an application thereof. Background Art

[0002] Antibody-drug conjugates (ADCs) are a class of antibody-modified drugs that combine antibodies with cytotoxic small molecule drugs, integrating the targeting of antibodies and the killing effect of chemotherapeutic drugs, and can specifically kill tumor cells. Due to their strong targeting property, they are developing vigorously at a rapid pace. In addition to selecting appropriate targeting antibody molecules and highly active cytotoxic small molecules, an ideal ADC drug must also select a suitable linker to connect the cytotoxic small molecule and the antibody, ensuring that the antibody after connecting the cytotoxic small molecule still has good stability and activity. Therefore, the design and production process of ADC drugs are more complex than those of monoclonal antibodies. In 2000, the first ADC drug approved by the FDA, Mylotarg, was launched for the treatment of acute myeloid leukemia. Due to the heterogeneity of the drug and the toxic side effects brought by high doses (9 mg / m 2 ), this drug was withdrawn from the market in 2010; however, in 2017, the FDA re-approved this drug for the treatment of patients with CD33 + positive acute myeloid leukemia (3 mg / m 2 ).

[0003] In recent years, although ADC drugs have developed vigorously, the homogenization phenomenon of ADC drugs is serious, and they are basically concentrated on the Her2 target. In addition, the combination of ADC drugs with immune checkpoint inhibitors such as Anti-PD-1 / PD-L1 can further improve the treatment effect of cancer. However, at present, there is no ADC drug specifically targeting immune checkpoints on the market.

[0004] Meanwhile, Chinese Patent 《202211000509.7》 records a monoclonal antibody targeting IGSF9 that can inhibit tumor growth. However, this monoclonal antibody needs to be combined with a PD-1 antibody to achieve a significant tumor suppression effect. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an antibody-drug conjugate targeting human IGSF9, a preparation method thereof, and an application thereof. The antibody-drug conjugate targeting human IGSF9 provided by the present invention has a significant effect of killing, inhibiting growth and metastasis on IGSF9-positive tumor cells, and can solve the problem that the monoclonal antibody targeting IGSF9 in the prior art needs to be combined with a PD-1 antibody to achieve a significant inhibition of tumor growth.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an antibody-drug conjugate targeting human IGSF9, which is obtained by conjugating a monoclonal antibody targeting IGSF9 and a DNA topoisomerase I inhibitor with a linker maleimide-GGFG tetrapeptide.

[0008] Preferably, the linker maleimide-GGFG tetrapeptide is linked to the cysteine thiol group on the monoclonal antibody targeting IGSF9.

[0009] Preferably, the full-length amino acid sequence of the light chain of the antibody-drug conjugate targeting human IGSF9 is shown in SEQ ID No.1, and the full-length amino acid sequence of the heavy chain of the antibody-drug conjugate targeting human IGSF9 is shown in SEQ ID No.2.

[0010] The present invention also provides a method for preparing the antibody-drug conjugate targeting human IGSF9, comprising the following steps:

[0011] In a conjugation buffer system of a monoclonal antibody targeting IGSF9, add TCEP, wash and filter with a conjugation buffer to obtain a reduced antibody, add Deruxtecan, and wash, filter and displace with a conjugation buffer to obtain the antibody-drug conjugate.

[0012] Preferably, the conjugation buffer is PBS buffer.

[0013] Preferably, the molar ratio of TCEP to the monoclonal antibody targeting IGSF9 is 15-20:1, and TCEP is trichloroacetic acid phosphate; the molar ratio of Deruxtecan to the reduced antibody is 10-15:1.

[0014] Preferably, after adding TCEP, stirring treatment is carried out, the temperature of the stirring is 20-30 °C, and the time of the stirring is 1-2 h.

[0015] The present invention also provides the use of the antibody-drug conjugate in the preparation of a drug for targeting and killing IGSF9-positive tumor cells.

[0016] The present invention also provides the use of the antibody-drug conjugate in the preparation of a drug for inhibiting tumor growth, invasion and metastasis.

[0017] Preferably, the tumor is acute myeloid leukemia cells.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] By adopting the method of site-directed conjugation, the interchain disulfide bond of the monoclonal antibody targeting IGSF9 was cleaved, and MC-GGFG-DXd was site-directed conjugated to the sulfhydryl group of the cysteine of anti-IGSF9, thereby preparing an antibody-drug conjugate specifically targeting IGSF9. The purity of the antibody-drug conjugate Anti-IGSF9-IgG2b-DXd can reach 99.7%, the average DAR value is 10, and the payload is relatively high.

[0020] The antibody-drug conjugate Anti-IGSF9-IgG2b-DXd prepared by the present invention can target and kill IGSF9-positive THP-1 tumor cells; after treating THP-1 tumor-bearing mice with this antibody-drug conjugate, it can significantly inhibit the growth, invasion and metastasis of tumors, which is beneficial to expanding tumor immunotherapy strategies and is expected to bring new therapies for tumor patients. Description of the Drawings

[0021] Figure 1 Structural diagram of the antibody-drug conjugate targeting human IGSF9 prepared in Example 1;

[0022] Figure 2 Mass spectrometry diagram of the antibody-drug conjugate targeting human IGSF9 prepared in Example 1 measured by the SEC method;

[0023] Figure 3 Superimposed mass spectrometry diagram of the monoclonal antibody targeting IGSF9 and the antibody-drug conjugate prepared in Example 1 measured by the SEC method;

[0024] Figure 4 Mass spectrometry diagram of the antibody-drug conjugate measured by the HIC method;

[0025] Figure 5 Superimposed mass spectrometry diagram of the incompletely reduced sample, the monoclonal antibody targeting IGSF9 and the antibody-drug conjugate prepared in Example 1 measured by the HIC method;

[0026] Figure 6 Cell survival rates after treating THP1-IGSF9-WT and THP1-IGSF9-KO cells with different drugs;

[0027] Figure 7 Microscopic observation results after treating THP1-IGSF9-WT and THP1-IGSF9-KO cells with different drugs;

[0028] Figure 8 Cell survival rates after treating MV4-11 cells with Anti-IGSF9-IgG2b and Anti-IGSF9-IgG2b-DXd;

[0029] Figure 9Cell viability after treatment of MV4-11 cells with Anti-IGSF9-IgG2b-DXd and DXd drugs;

[0030] Figure 10 Microscopic observation results after treatment of MV4-11 cells with different drug treatment groups;

[0031] Figure 11 Flow chart of treatment strategy for mouse experiments;

[0032] Figure 12 Status diagram of monitoring tumor growth by in vivo imaging;

[0033] Figure 13 Survival results of mice after treatment with different drugs. Detailed implementation mode

[0034] The present invention provides an antibody-drug conjugate targeting human IGSF9, which is obtained by conjugating a monoclonal antibody targeting IGSF9 and a DNA topoisomerase I inhibitor with a linker maleimide-GGFG tetrapeptide.

[0035] In the present invention, the linker maleimide-GGFG tetrapeptide is connected to the cysteine thiol group on the monoclonal antibody targeting IGSF9; the full-length amino acid sequence of the light chain of the antibody-drug conjugate targeting human IGSF9 is shown in SEQ ID No.1, and the full-length amino acid sequence of the heavy chain of the antibody-drug conjugate targeting human IGSF9 is shown in SEQ ID No.2.

[0036] Table 1 Amino acid sequence of monoclonal antibody targeting IGSF9

[0037]

[0038]

[0039] The C 214 、C 134 、C 228 、C 231 、C 234 、C 237 represent the modified cysteine sites.

[0040] The present invention also provides a method for preparing the antibody-drug conjugate targeting human IGSF9, comprising the following steps:

[0041] In a coupling buffer system of a monoclonal antibody targeting IGSF9, add TCEP, wash and filter with a coupling buffer to obtain a reduced antibody, add Deruxtecan, and wash, filter and displace with a coupling buffer to obtain an antibody-drug conjugate.

[0042] In the present invention, the coupling buffer is preferably PBS buffer; the molar ratio of TCEP to the monoclonal antibody targeting IGSF9 is preferably 15-20:1, more preferably 16-18:1, and even more preferably 17:1; the TCEP is preferably trichloroacetic acid phosphate; the trichloroacetic acid phosphate is purchased from Sinopharm Chemical Reagent Co., Ltd.; the molar ratio of Deruxtecan to the reduced antibody is preferably 10-15:1, more preferably 12-14:1, and even more preferably 13:1; after adding TCEP, stirring treatment is preferably carried out, the temperature of the stirring is preferably 20-30 °C, more preferably 22-28 °C, and even more preferably 24-26 °C; the stirring time is preferably 1-2 h, more preferably 1.5 h, and the Deruxtecan is purchased from MCE Company.

[0043] The present invention also provides the use of the antibody-drug conjugate in the preparation of a drug for targeting and killing IGSF9-positive tumor cells.

[0044] The present invention also provides the use of the antibody-drug conjugate in the preparation of a drug for inhibiting tumor growth, invasion and metastasis.

[0045] In the present invention, the tumor is preferably acute myeloid leukemia.

[0046] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] Example 1 Preparation of an Antibody-Drug Conjugate Targeting Human IGSF9

[0048] The monoclonal antibody targeting IGSF9 (the monoclonal antibody targeting IGSF9 is prepared according to the preparation method described in the Chinese patent "202211000509.7") is added to PBS buffer, and after mixing, it is washed and filtered using an ultrafiltration tube with a size of 30 kD to obtain a coupling buffer system. Trichloroacetic acid phosphate is added at a molar ratio of 20:1 (trichloroacetic acid phosphate: monoclonal antibody targeting IGSF9), and it is stirred at 25 °C for 2 h, and then washed and filtered with PBS buffer to remove the excess trichloroacetic acid phosphate (reducing agent) to obtain the reduced antibody. Deruxtecan (purchased from MCE Company) is added at a molar ratio of 15:1 (Deruxtecan: reduced antibody), and it is stirred at 25 °C for 0.5 h, and then washed and filtered with PBS buffer for replacement to obtain the antibody-drug conjugate.

[0049] Test Example 1 Purity Detection

[0050] The purity of the sample was detected by size exclusion chromatography (SEC) method, specifically:

[0051] Mobile phase buffer: 200 mM PBS, 10% isopropanol, pH 7.0.

[0052] The monoclonal antibody targeting IGSF9 and the antibody-drug conjugate prepared in Example 1 were respectively diluted to 1 mg / mL with the mobile phase buffer. 10 μL was taken and injected into the high performance liquid chromatograph, and separation was carried out using a TSK gel G3000SWXL 5 μm × 7.8 mm × 300 mm chromatographic column.

[0053] Parameter settings: detection wavelength 280 nm, column temperature 25 °C, flow rate 0.8 mL / min, isocratic elution, analysis time 20 min. The results are as Figure 2 shown.

[0054] The purity of the antibody-drug conjugate was calculated by the area normalization method. The results showed that the purity of the antibody-drug conjugate was 99.7%.

[0055] Meanwhile, the polymer content of the monoclonal antibody targeting IGSF9 and the antibody-drug conjugate prepared in Example 1 was determined. The results are as Figure 3 shown.

[0056] The results showed that compared with the monoclonal antibody targeting IGSF9, the peak elution position of the antibody-drug conjugate prepared in Example 1 of the present invention shifted forward, and the conjugation reaction did not significantly increase the polymer content of the antibody.

[0057] Test Example 2 Determination of DAR value

[0058] The DAR value (drug / antibody ratio) of the sample was detected by hydrophobic interaction chromatography (HIC). The specific steps are as follows:

[0059] Mobile phase buffer A: 25 mM PBS, 1.5 M ammonium sulfate, pH 7.0;

[0060] Mobile phase buffer B: 25 mM PBS, 25% isopropanol, pH 7.0.

[0061] The incomplete reduction sample, the monoclonal antibody targeting IGSF9 (Anti-IGSF9-IgG2b), and the antibody-drug conjugate prepared in Example 1 (Anti-IGSF9-IgG2b-DXd) were diluted to 1 mg / mL with mobile phase buffer A. 10 μL was taken and injected into the high performance liquid chromatograph, and separation was carried out using a TSK gel Buty-NPR 2.5 μm × 4.6 mm × 35 mm chromatographic column.

[0062] Parameter settings: detection wavelength 280 nm, column temperature 30 °C, flow rate 0.8 mL / min, gradient elution, analysis time 20 min. The results are as Figure 4and Figure 5 as shown

[0063] The incompletely reduced sample is a sample in which the disulfide bonds in the monoclonal antibody targeting IGSF9 are not completely broken (i.e., 1 / 2 / 3 / 4 disulfide bonds are broken) under the action of different concentrations of trichloroacetic acid phosphate (reducing agent) when preparing the antibody-drug conjugate.

[0064] Use the incompletely reduced product (average DAR value around 4) to determine the positions of the DAR0 to DAR12 components in the chromatogram. By comparing with the positions of the respective component peaks of the DAR4 product, it can be determined that the main components of the final product are DAR8 - DAR12.

[0065] Calculation of the average DAR value in Test Example 3

[0066] Use a UV detector (UV) to assist in the detection and calculation of the average DAR value. Specifically:

[0067] Dilute the monoclonal antibody targeting IGSF9 and the antibody-drug conjugate prepared in Example 1 to 0.3 mg / mL with ultrapure water. Take 200 μL and add it to a cuvette, and use a UV-visible spectrophotometer to measure the absorbance values of the monoclonal antibody targeting IGSF9 and the antibody-drug conjugate prepared in Example 1 at 280 nm and 350 nm respectively. According to the ratio of A 280 and A 350 and the extinction coefficients of the monoclonal antibody targeting IGSF9 and the antibody-drug conjugate prepared in Example 1, calculate the average DAR value.

[0068] The results show that the average DAR value is 10.

[0069] Test Example 4 Determination of the killing ability of samples with different concentrations on THP1-IGSF9-WT and THP1-IGSF9-KO cells

[0070] Centrifuge, resuspend and count THP1-IGSF9-WT and THP1-IGSF9-KO cells (the construction method is the same as the method described in Chinese Patent "202211000509.7"). Seed 2000 cells into a 96-well U-bottom cell culture plate at 50 μL / well.

[0071] Dilute the monoclonal antibody targeting IGSF9 and the antibody-drug conjugate prepared in Example 1 with RPMI-1640 medium respectively. Take 50 μL and add it to the U-bottom cell culture plate to make the total volume of each well reach 100 μL, and the final concentrations are 100 ng / mL, 1 μg / mL, 5 μg / mL and 10 μg / mL respectively. Set three replicates for each concentration, and at the same time set the drug-free group as the control group and the single medium group as the blank group.

[0072] After culturing at 37°C for 72 h, 10 μL of CCK8 (Cell Counting Kit-8 for cell proliferation and cytotoxicity detection, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) was added, and the cells were cultured for another 1 h. The absorbance at 450 nm was measured. The survival rate of tumor cells was calculated according to the formula: cell survival rate (%) = (experimental group - blank well) / (control group - blank well) × 100%. The results are as Figure 6 and Figure 7 shown.

[0073] As Figure 6 and Figure 7 shown, in THP1-IGSF9-WT cells, compared with Anti-IGSF9-IgG2b, when the concentration of Anti-IGSF9-IgG2b-DXd reached 5 μg / mL, obvious killing effect appeared, and with the increase of concentration, the killing intensity increased. Anti-IGSF9-IgG2b-DXd had a weak killing effect on THP1-IGSF9-KO cells, indicating that the IGSF9-IgG2b-DXd drug could specifically kill IGSF9-positive cells, had a weak killing effect on IGSF9-negative cells, and had good targeting.

[0074] Test Example 5 Killing ability of samples with different concentrations on MV4-11 cells

[0075] The MV4-11 cells (purchased from ATCC) were centrifuged, resuspended and counted, and 2000 cells / 50 μL / well were seeded in a 96-well U-shaped cell culture plate.

[0076] RPMI-1640 medium was used to dilute the drugs Anti-IGSF9-IgG2b, Anti-IGSF9-IgG2b-DXd and DXd respectively. 50 μL of each was taken and added to the U-shaped cell culture plate to make the total volume of each well reach 100 μL, so that the final concentrations of Anti-IGSF9-IgG2b and Anti-IGSF9-IgG2b-DXd were 100 ng / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL and 50 μg / mL respectively, and the final concentration of DXd was 50 nM. Three replicates were set for each concentration, and at the same time, the group without adding drugs was set as the control group, and the group with only medium was set as the blank group.

[0077] After culturing at 37°C for 72 h, 10 μL of CCK8 (Cell Counting Kit-8 for cell proliferation and cytotoxicity detection, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) was added, and the cells were cultured for another 1 h. The absorbance at 450 nm was measured, and the survival rate of tumor cells was calculated according to the formula: cell survival rate (%) = (experimental group - blank well) / (control group - blank well) × 100%. The results are as Figure 8 、 Figure 9 and Figure 10as shown

[0078] As Figures 8 - 10 shown, in MV4-11 cells, compared with Anti-IGSF9-IgG2b, when the concentration of Anti-IGSF9-IgG2b-DXd reached 5 μg / mL, obvious killing effect appeared, and with the increase of concentration, the killing intensity increased. When the concentration of Anti-IGSF9-IgG2b-DXd reached 50 μg / mL, the killing ability of tumor cells was consistent with that of 50 nM DXd, indicating that Anti-IGSF9-IgG2b-DXd could be endocytosed by MV4-11 cells.

[0079] Test Example 6 Mouse Experiment

[0080] NSG mice (purchased from Shanghai Model Organisms Center, Inc.) were numbered. Among them, mice numbered 1-5 were in the Anti-IGSF9-IgG2b treatment group, and mice numbered 6-10 were in the Anti-IGSF9-IgG2b-DXd treatment group.

[0081] THP-1-LUC cells (THP-1 cells expressing luciferase, constructed in the laboratory. Specifically: the luciferase vector was packaged into a virus and used to infect THP-1 cells to obtain THP-1-LUC cells) were collected, centrifuged, counted, and the cells were diluted to 1×10⁶ cells / 200 μL. 1×10⁶ cells were injected into the tail vein of each mouse, and immediately 100 μL of luciferase substrate (purchased from Abcam) was injected subcutaneously. After reacting for 3 min, small animal in vivo imaging was performed to record data. And on the 7th and 14th days respectively, the grouped mice were injected with the corresponding 10 mg / kg Anti-IGSF9-IgG2b and Anti-IGSF9-IgG2b-DXd for treatment, and in vivo imaging was performed on the same day after injection to record the results. The mice were sacrificed after in vivo imaging on the 21st day. The results are as Figure 11 、 Figure 12 and Figure 13 as shown

[0082] As Figures 11 - 13 shown, on the 21st day, there was no obvious tumor growth in the Anti-IGSF9-IgG2b-DXd group, while in the Anti-IGSF9-IgG2b group, the tumor grew rapidly on the 14th and 21st days, and on the 21st day, two mice died due to excessive tumor burden. It can be seen that Anti-IGSF9-IgG2b-DXd can significantly inhibit tumor growth, invasion and metastasis.

[0083] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. An antibody-drug conjugate targeting human IGSF9, characterized in that, The antibody-drug conjugate is obtained by conjugating a monoclonal antibody targeting IGSF9 and a DNA topoisomerase I inhibitor with a linker maleimide-GGFG tetrapeptide; The full-length amino acid sequence of the light chain of the antibody-drug conjugate targeting human IGSF9 is shown in SEQ ID No.1, and the full-length amino acid sequence of the heavy chain of the antibody-drug conjugate targeting human IGSF9 is shown in SEQ ID No.2; The DNA topoisomerase I inhibitor is DXd.

2. The antibody-drug conjugate according to claim 1, wherein The linker maleimide-GGFG tetrapeptide is linked to the cysteine sulfhydryl group on the monoclonal antibody targeting IGSF9.

3. A method for preparing the antibody-drug conjugate targeting human IGSF9 according to claim 1 or 2, characterized in that, It includes the following steps: In the conjugation buffer system of the monoclonal antibody targeting IGSF9, TCEP is added, washed and filtered with the conjugation buffer to obtain the reduced antibody, Deruxtecan is added, and washed, filtered and replaced with the conjugation buffer to obtain the antibody-drug conjugate.

4. The method according to claim 3, wherein The conjugation buffer is PBS buffer.

5. The method according to claim 3, characterized in that, The molar ratio of TCEP to the monoclonal antibody targeting IGSF9 is 15-20:1, and TCEP is trichloroacetic acid phosphate; the molar ratio of Deruxtecan to the reduced antibody is 10-15:

1.

6. The method according to claim 3, wherein After adding TCEP, stirring treatment is carried out, the temperature of the stirring is 20-30 °C, and the time of the stirring is 1-2 h.

7. Use of the antibody-drug conjugate according to claim 1 or 2 in the preparation of a drug for treating acute myeloid leukemia.

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