An anti_ITGA2 antibody drug conjugate

Abstract

The present application discloses an antibody drug conjugate, which comprises the structure of the following formula 1 wherein, Ab is an antibody or antibody fragment which targets ITGA2, and j is 1 to 8, preferably 4 to 8. The antibody-drug conjugate of the application has a high specificity binding ability to tumor cells expressing ITGA2 protein, thereby achieving an excellent killing effect on tumor cells.

Description

[0199] . Example 8 Study on the efficacy of ADC on human cholangiocarcinoma cell line HuCCTl CDX model

[0200] . BALB / c nude mice, aged 4-6 weeks, were acclimated to the SPF environment for a week in advance. Subsequently, these mice were subcutaneously inoculated with 5* 106 human cholangiocarcinoma cells of the HuCCTl line (Zhejiang Meisen, CTCC-003-0103). On the 40th day following cell inoculation, each group was administered injections of PBS, A36-Dxd (10 mpk) as prepared in Example 3, and A36-LD-38 (10 mpk) drug molecules as prepared in Example 2. Thereafter, body weight and tumor size were measured twice weekly. The formula for calculating tumor volume was:

[0201] . Tumor volume (mm3) = 1 / 2 * (a x b2)

[0202] . (wherein ‘a’ represents the long diameter and ‘b’ represents the short diameter).

[0203] . The results are presented in Figure 5. Compared to the PBS control, the growth of subcutaneous tumors treated with a single dose of A36-Dxd and A36-LD-38 was significantly inhibited. The anti-tumor efficacy of A36-LD-38 was slightly superior to that of the A36-Dxd drug molecule. This result demonstrates that the A36-LD-38 drug molecule exhibits good efficacy in the human cholangiocarcinoma line HuCCTl CDX model.

[0204] . Example 9 Study on the efficacy of ADC on human pancreatic cancer cell line BxPC-3 CDX model

[0205] . BALB / c nude mice, aged 4-6 weeks, were acclimated to the SPF environment for one week prior to the experiment. The mice were then subcutaneously inoculated with 5* 106 human pancreatic cancer cells of the BxPC-3 line (Zhejiang Meisen, CTCC-001-0360). On the 38th day after cell inoculation, each group was administered injections of PBS, A36-Dxd (2.5 mpk, 5 mpk, 10 mpk) as prepared in Example 3, and A36-LD-38 (10 mpk) as prepared in Example 2. Body weight and tumor size were measured twice weekly. The formula for calculating tumor volume was:

[0206] . Tumor volume (mm3) = 1 / 2 * (a x b2)

[0207] . (wherein ‘a’ represents the long diameter and ‘b’ represents the short diameter).

[0208] . The results are depicted in Figure 6. Compared to the PBS control, the tumor growth was significantly inhibited by A36-Dxd, with the drug effect becoming more pronounced as the dose increased. The efficacy of A36-LD-38 at 10 mpk was significantly superior to that of the same dose of A36-Dxd. This result demonstrates that the A36-LD-38 drug molecule exhibits good efficacy in the human pancreatic cancer cell line BxPC-3 CDX model, and is more effective than A36-Dxd.

[0209] . Example 10 Study on the efficacy of ADC on human gastric cancer cell line NCI-N87 CDX model

[0210] . BALB / c nude mice, aged 4-6 weeks, were acclimated to the SPF environment for one week prior to the experiment. The mice were then subcutaneously inoculated with 5* 106 human gastric cancer cells of theNCI-N87 line (Procell, CL-0169). On the 4th, 7th, and 18th days after cell inoculation, each group was administered injections of PBS, A36-Dxd (5 mpk) as prepared in Example 3, and A36-LD-38 (5 mpk) as prepared in Example 2. Body weight and tumor size were measured twice weekly. The formula for calculating tumor volume was:

[0211] . Tumor volume (mm3) = 1 / 2 x (a x b2)

[0212] . (wherein ‘a’ represents the long diameter and ‘b’ represents the short diameter).

[0213] . The results are presented in Figure 7. Compared to the PBS control, the growth of subcutaneous tumors treated with A36-Dxd and A36-LD-38 was significantly inhibited. The antitumor efficacy of A36-LD-38 was notably superior to that of A36-Dxd. This result demonstrates that A36-LD-38 exhibits good efficacy in the human gastric cancer cell line NCI-N87 CDX model, and is more effective than A36-Dxd.

[0214] . Example 11 Study on the efficacy of ADC on human colon cancer cell line COLO205 CDX model

[0215] . BALB / c nude mice, aged 4-6 weeks, were acclimated to the SPF environment for one week prior to the experiment. The mice were then subcutaneously inoculated with 5* 106 human colon cancer cells of the COLO205 line (Procell, CL-0066). On the 8th and 15th days after cell inoculation, each group was administered injections of PBS, A36-Dxd (5 mpk) and IgG-Dxd (5 mpk) as prepared in Example 3, and A36-LD-38 (5 mpk), A36mut-LD-38 (1 mpk, 2.5 mpk, 5 mpk), and IgG-LD-38 (5 mpk) as prepared in Example 2. Body weight and tumor size were measured twice weekly. The formula for calculating tumor volume was:

[0216] . Tumor volume (mm3) = 1 / 2 * (a x b2)

[0217] . (wherein ‘a’ represents the long diameter and ‘b’ represents the short diameter).

[0218] . The results, as shown in Figure 8, indicated that the tumor volume treated with A36-LD-38 gradually decreased or even disappeared, while the tumor volume treated with A36-Dxd gradually increased, albeit slightly smaller than that in the PBS group. On days 50, 57, and 64, the mice in the A36-Dxd treatment group were switched to A36-LD-38 treatment (5 mpk) for three times, after which the tumor volume showed a significant decrease. This result demonstrates that A36-LD-38 has good efficacy in the human colon cancer cell line COLO205 CDX model, and is much better than the efficacy of A36-Dxd. A further observation was that when the tumors in the A36-Dxd treatment group grew to 1000 mm3 and became drug-resistant, three more doses of A36-LD-38 could reverse the growth trend of the tumors and shrink them, indicating that the A36-LD-38 molecule could overcome the resistance of Dxd in the COLO205 colorectal cancer model. Similarly, different doses of A36mut-LD-38 also inhibited the tumor growth of COLO205 (Figure 9), indicating that both the A36 antibody with wild-type IgGl (A36) and the A36 antibody with mutant IgGl (A36mut) could be conjugated with LD-38 to form ADCs, and they have great efficacies in the human colon cancer cell line COLO205 CDX model.

[0219] . Example 12 Study on the in vivo anti-tumor efficacy of ADC in the intestinal cancer PDX model

[0220] . Tumor tissue was collected from HuPrime® intestinal cancer C0170 xenograft tumorbearing mice models, and the tumors were cut into pieces with a diameter of 2-3 mm3. These pieces were then subcutaneously inoculated in the right front scapula of BALB / c nude mice. When the average tumor volume of the tumor-bearing mice reached approximately 100 mm3, the mice were randomly divided into groups according to the "experimental design." The mice were randomly assigned to four groups, including a control group (PBS) and drug administration groups (A36mut-Dxd and IgG-Dxd prepared in Example 3, A36mut-LD-38 prepared in Example 2). The mice were dosed once a week, with a dose of 5 mpk in the first three weeks and a dose of 10 mpk in the last week. Body weight and tumor size were measured twice weekly. The formula for calculating tumor volume was:

[0221] . Tumor volume (mm3) = 1 / 2 * (a x b2)

[0222] . (wherein ‘a’ represents the long diameter and ‘b’ represents the short diameter).

[0223] . The statistical efficacy results are presented in Figure 10 below. Compared to A36-Dxd, A36-LD-38 has a superior effect on the intestinal cancer PDX model.

[0224] . The above are merely the preferred embodiments of the present application and are not intended to limit the application to other forms. Any person skilled in the art may make changes or modifications, or equivalent alterations, using the technical content disclosed herein. For example, however, any simple modifications, equivalent changes, and modifications made to the aforementioned embodiments based on the technical essence of the present application, without departing from the content of the technical solution of the present application, still fall within the scope of protection of the technical solution of the present application.

Claims

1. An antibody drug conjugate, which comprises the structure of the following formula 1Formula 1wherein Ab is an antibody or antibody fragment which targets ITGA2;j is 1 to 8, preferably 4 to 8.

2. The antibody drug conjugate according to claim 1, wherein the antibody or antibody fragment which targets ITGA2 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:the amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1;the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2;the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3;the amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4;the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5;the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6.

3. The antibody drug conjugate according to claim 1, wherein the antibody or antibody fragment which targets ITGA2 comprises a heavy chain variable region and a light chain variable region, wherein, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7 or has at least 90 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the amino acid sequence of SEQ ID NO: 7;the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the amino acid sequence of SEQ ID NO: 8.

4. The antibody drug conjugate according to any one of claims 1 to 3, wherein the Ab is an antibody which targets ITGA2, and the heavy chain of the antibody is an IgGl subtype or an IgGl subtype with mutations, and the light chain of the antibody is of the Kappa type.

5. The antibody drug conjugate according to claim 4, wherein the antibody which targets ITGA2 comprises a heavy chain and a light chain, wherein,the amino acid sequence of the heavy chain is shown in SEQ ID NO: 9 or SEQ ID NO: 11 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11;the amino acid sequence of the light chain is shown in SEQ ID NO: 10 or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 10.

6. A method for preparing an antibody drug conjugate, which comprises the following steps: providing LD-38;constructing an antibody or an antibody fragment Ab which targets ITGA2;conjugating LD-38 with the antibody or antibody fragment Ab which targets ITGA2 to obtain an antibody drug conjugate;the structure of LD-38 iswherein, the antibody or antibody fragment Ab which targets ITGA2 is the one as defined in claims 2-6.

7. A pharmaceutical composition, which comprises the antibody drug conjugate of any one of claims 1 to 5, or tautomers, mesomers, racemates, enantiomers, diastereoisomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs, or solvates thereof.

8. Use of the antibody drug conjugate according to any one of claims 1 to 5, or tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs or solvates thereof in the preparation of a medicament for the treatment and / or prevention of tumors.

9. The use according to claim 8, wherein the tumors are selected from the group consisting of solid tumors, hematological tumors and metastatic, refractory or recurrent lesions of cancers.

10. The use according to claim 8, wherein the tumors are selected from the group consisting of esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (eg non-small cell lung cancer), liver cancer, stomach cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, cholangiocarcinoma, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (such as multiple myeloma), sarcoma, glioblastoma, and leukemia.

11. Use of the antibody drug conjugate according to any one of claims 1 to 5, or tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs or solvates thereof in combination with other therapeutic drugs in the preparation of a medicament for the treatment and / or prevention of tumors.

12. The use according to claim 11, wherein the other therapeutic drugs are selected from immune checkpoint drugs, drugs for chemotherapy or drugs for radiotherapy.

13. The antibody drug conjugate according to any one of claims 1 to 5, or tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs or solvates thereof for use in the treatment and / or prevention of tumors.5    14. The antibody drug conjugate according to any one of claims 1 to 5, or tautomers, mesomers,racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts, prodrugs or solvates thereof in combination with other therapeutic drugs for use in the treatment and / or prevention of tumors.10    15. The use according to claim 14, wherein the other therapeutic drugs are selected from immunecheckpoint drugs, drugs for chemotherapy or drugs for radiotherapy.

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