A diabody drug conjugate targeting pd-l1 and vegf, and a preparation method and application thereof

Abstract

The present application relates to a kind of double antibody drug conjugate targeted to PD-L1 and VEGF and its preparation method and application.The general formula of the double antibody drug conjugate is Ab-(L-D) x;In general formula Ab, it is the double antibody targeted to PD-L1 and VEGF, includes PD-L1 antigen binding portion and VEGF antigen binding portion;In general formula L-D, it is connection unit-drug compound, L is connection unit, D is drug molecule, x is selected from the integer in 1~8.The double antibody drug conjugate includes PD-L1 antigen binding portion, VEGF antigen binding portion and drug molecule, and the synergistic efficacy between anti-angiogenesis, PD-L1 immune target and antibody conjugate is realized by reasonable structure design.

Description

[0001] This application claims priority to Chinese Patent Application No. 202510209997.X, filed on February 24, 2025, entitled "A Biantibody Drug Conjugate Targeting PD-L1 and VEGF and Its Preparation Method and Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of antibody-drug conjugate technology, specifically to a dual antibody-drug conjugate targeting PD-L1 and VEGF, its preparation method, and its application. Background Technology

[0003] Immune checkpoints are the brakes of the immune system, composed of receptor-ligand molecules. They negatively regulate immune responses and play a crucial role in maintaining immune tolerance and modulating the strength of the immune response. Tumor cells exploit the negative regulation of immune responses by immune checkpoints to evade the surveillance of the body's immune system.

[0004] Programmed death ligand 1 (PD-L1) is a member of the type I transmembrane protein receptor B7 family and is encoded by the CD274 gene. Programmed death protein 1 (PD-1) is the PD-L1 receptor expressed on the surface of T cells. PD-L1 is widely expressed on the surface of tumor cells and interacts with PD-1 expressed on T cells. The binding of PD-L1 ligand to its receptor PD-1 is an important immune checkpoint mediating tumor immunosuppression. It prevents T cell activation, inhibits tumor-infiltrating T cells from killing tumor cells, and leads to tumor immune escape, making it one of the main targets of tumor immunotherapy.

[0005] Vascular endothelial growth factor (VEGF) is a key substance promoting vascular endothelial cell formation. VEGF induces angiogenesis in vivo. Under the hypoxic environment of the tumor microenvironment, VEGF released by tumor cells binds to VEGF receptors on the endothelial cell membrane, causing autophosphorylation of VEGF receptors, further activating mitogen-activated protein kinase (MAPK) and inducing endothelial cell proliferation. Simultaneously, under hypoxic conditions, VEGF can increase the mRNA expression of plasma prozyme activator (PA) and plasma prozyme activator inhibitor-1 (PAI-1), thereby increasing the activity of plasma prozyme activator, promoting extracellular protein hydrolysis, and thus promoting the formation of new capillaries at the tumor site.

[0006] CN117062841A discloses a bispecific antibody targeting PD-1 and VEGF, which can effectively block the binding of VEGFA to VEGFR2, specifically relieving the immunosuppressive effect of VEGFA on the body and its promoting effect on angiogenesis; at the same time, it can bind to PD-1 specifically and effectively block the binding of PD-1 to PD-L1, specifically relieving the immunosuppression of the body by PD-1 and activating the immune response.

[0007] However, there are still very few publicly available bispecific antibody drug conjugates. How to achieve synergistic efficacy among anti-angiogenesis, the PD-L1 immune target, and the drug molecule through rational design, thereby inhibiting tumor activity, has become a pressing technical problem to be solved. If synergistic efficacy exists, the design must first focus on maintaining the effectiveness and safety of the dosage of all three in the human body; a reasonably high-performance payload and a well-matched linker are crucial. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a dual antibody-drug conjugate targeting PD-L1 and VEGF, its preparation method, and its applications. This dual antibody-drug conjugate comprises a PD-L1 antigen-binding moiety, a VEGF antigen-binding moiety, and a drug molecule. Through rational structural design, it achieves synergistic pharmacodynamics among the anti-angiogenic, PD-L1 immune target, and drug molecule.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a dual antibody drug conjugate targeting PD-L1 and VEGF, wherein the general formula of the dual antibody drug conjugate is Ab-(LD)x.

[0011] In the general formula, Ab is a biantibody targeting PD-L1 and VEGF, containing a PD-L1 antigen-binding part and a VEGF antigen-binding part; in the general formula, LD is a linker-drug compound, L is a linker, D is a drug molecule, and x is an integer selected from 1 to 8, for example, it can be 1, 2, 4, 6, 8.

[0012] The amino acid sequences of the heavy chains CDR1, CDR2, and CDR3 of the PD-L1 antigen-binding region include SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0013] The amino acid sequences of the light chains CDR1, CDR2, and CDR3 of the PD-L1 antigen-binding moiety include SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0014] The amino acid sequences of the heavy chains CDR1, CDR2, and CDR3 of the VEGF antigen-binding region include SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

[0015] The amino acid sequences of the light chains CDR1, CDR2, and CDR3 of the VEGF antigen-binding portion include SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0016] The specific sequence of SEQ ID NO: 1 is: GFTFSSYAMS; the specific sequence of SEQ ID NO: 2 is: GIGSGGGFTYYADSVKG; the specific sequence of SEQ ID NO: 3 is: PPRGYNYGPFDY; the specific sequence of SEQ ID NO: 4 is: GGNNIGSKSVH; the specific sequence of SEQ ID NO: 5 is: DDSDRPS; and the specific sequence of SEQ ID NO: 6 is: QVWDSSSDHVV.

[0017] The specific sequence of SEQ ID NO: 7 is: GYTFTNYGMN; the specific sequence of SEQ ID NO: 8 is: WINTYTGEPTYAADFKR; the specific sequence of SEQ ID NO: 9 is: YPHYYGSSHWYFDV; the specific sequence of SEQ ID NO: 10 is: SASQDISNYLN; the specific sequence of SEQ ID NO: 11 is: YFTSSLHS; and the specific sequence of SEQ ID NO: 12 is: QQYSTVPWT.

[0018] This dual antibody drug conjugate contains a PD-L1 antigen-binding moiety, a VEGF antigen-binding moiety, and a drug molecule. Through a rational structural design, it achieves synergistic efficacy among anti-angiogenesis, the PD-L1 immune target, and the drug molecule, and has excellent PD-L1 and VEGF binding affinity.

[0019] Where x is selected from integers from 1 to 8, it can enable biantibodies and drug molecules targeting PD-L1 and VEGF to exert their efficacy better, reduce conjugate toxicity, and balance the relationship between efficacy and toxicity of biantibody-drug conjugates.

[0020] Preferably, the drug molecule is selected from any one of alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, immunomodulators, or chromatin function inhibitors.

[0021] Preferably, the chromatin function inhibitor is selected from camptothecin or camptothecin derivatives.

[0022] Camptothecin is a cytotoxic quinoline alkaloid that can inhibit DNA topoisomerase. It is a plant-based anticancer drug. Although camptothecin has secured a place among anticancer drugs due to its good efficacy, its physical properties, such as poor water solubility, make it difficult to administer and result in poor patient tolerance.

[0023] This invention chemically modifies camptothecin or camptothecin derivatives to increase the targeting, solubility and stability of camptothecin. At the same time, it works in combination with dual antibodies targeting PD-L1 and VEGF to improve the antitumor effect of dual antibody drug conjugates.

[0024] Preferably, the structure of the camptothecin derivative is as shown in Formula I or Formula ⅠⅠ:

[0025] Formula I;

[0026] Formula IⅠⅠ.

[0027] This invention creatively discovers that camptothecin derivatives can better synergize with dual antibodies targeting PD-L1 and VEGF, thereby enhancing the antitumor activity of the conjugates.

[0028] Preferably, the structure of the connecting unit-drug compound is as shown in Formula I or II:

[0029] Formulas I and II.

[0030] Preferably, the structure of the connecting unit-drug compound is shown in Formula IV:

[0031] Formula IV.

[0032] Preferably, the amino acid sequence of the heavy chain of the PD-L1 antigen-binding moiety and the VEGF antigen-binding moiety includes SEQ ID NO: 13, and the amino acid sequence of the light chain includes SEQ ID NO: 14.

[0033] Among them, the specific sequence of SEQ ID NO: 13 is: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGSGGFTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPPRGYNYGPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0034] The specific sequence of SEQ ID NO: 14 is: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0035] Preferably, in the general formula of the dual antibody drug conjugate, x is selected from any integer from 4 to 8.

[0036] In a second aspect, the present invention provides a method for preparing a biantibody-drug conjugate targeting PD-L1 and VEGF as described in the first aspect, the method comprising the following steps:

[0037] A dual antibody targeting PD-L1 and VEGF is mixed with a reducing agent to undergo a reduction reaction. After the reaction, it is mixed with a linker-drug compound to undergo a coupling reaction, thus obtaining a dual antibody-drug conjugate targeting PD-L1 and VEGF.

[0038] Preferably, the molar ratio of the dual antibody targeting PD-L1 and VEGF, the reducing agent, and the linker-drug compound is 1:(1~15):(5~25).

[0039] Among them, the specific point values ​​in 1 to 15 can be 1, 3, 5, 7, 9, 11, 13, 15, etc., and the specific point values ​​in 5 to 25 can be 5, 10, 15, 20, 25, etc. Other specific point values ​​in this range can be selected, which will not be elaborated here.

[0040] Preferably, the reducing agent comprises tris(2-hydroxyethyl)phosphonic acid hydrochloride.

[0041] Thirdly, the present invention provides the use of a biantibody drug conjugate targeting PD-L1 and VEGF as described in the first aspect, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for treating cancer.

[0042] Preferably, the cancer includes solid tumors and hematologic tumors, including any one of breast cancer, lymphoma, head and neck cancer, gastric cancer, colorectal cancer, ovarian cancer, cervical cancer, prostate cancer, myeloma, sarcoma, pancreatic cancer, or glioblastoma.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention creatively develops a dual antibody drug conjugate targeting PD-L1 and VEGF. The dual antibody drug conjugate contains a PD-L1 antigen-binding moiety, a VEGF antigen-binding moiety, and a drug molecule. Through rational structural design, it achieves synergistic pharmacodynamics among anti-angiogenesis, the PD-L1 immune target, and the drug molecule.

[0045] Furthermore, when the drug molecule in the biantibody drug conjugate is selected as a camptothecin derivative, it can better exert a synergistic effect with the biantibody and enhance its killing power against tumor cells.

[0046] Furthermore, selecting a value of 4 to 8 for x in the general formula Ab-(LD)x of the biantibody drug conjugate can better leverage the synergistic effect between the biantibody and the drug molecule, thereby enhancing the conjugate's ability to kill tumor cells. Attached Figure Description

[0047] Figure 1 This is the synthetic route diagram for the linker unit-drug compound CTW52;

[0048] Figure 2 This is the synthetic route diagram for the linker unit-drug compound CTW46;

[0049] Figure 3 It is the antitumor activity of the conjugate against a tumor model constructed from RKO cells;

[0050] Figure 4 It is the antitumor activity of the conjugate against a tumor model constructed from MDA-MB-231 cells. Detailed Implementation

[0051] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0052] Example 1

[0053] This embodiment provides a synthesis of a linker-drug compound (linker-loaded compound), referring to structure J2-1 in Chinese Patent Application No. 202311327506.9 and PCT / CN2024120991, whose structure is shown in Formula III, and is also named CTW52, with the molecular formula: C 91 H 122 FN9O 35 The molecular weight is 1921 Da;

[0054] Formula III.

[0055] Recommended synthesis route as follows Figure 1 As shown.

[0056] Example 2

[0057] This embodiment provides a synthesis of a linker-drug compound (linker load compound), referring to Chinese Patent Application No. 202311327506.9 and PCT / CN2024120991, whose structure is shown in Formula IV, named CTW46, and whose molecular formula is: C 90 H 123 N9O 35 The molecular weight is 1891 Da;

[0058] Formula IV.

[0059] Its nuclear magnetic resonance (NMR) spectral data are as follows:

[0060] 1H NMR (400 MHz, DMSO) δ 12.76 (s, 1H), 9.13 (s, 1H), 8.78 – 8.43 (m,1H), 8.40 – 8.23 ​​(m, 2H), 8.23 ​​– 8.02 (m, 1H), 7.93 (t, J = 5.7 Hz, 1H), 7.84(d, J = 8.1 Hz, 1H), 7.77 (dd, J = 13.2, 7.6 Hz, 2H), 7.50 (d, J = 9.4 Hz, 2H), 7.32 (s, 1H), 7.26 – 7.09 (m, 2H), 6.99 (s, 2H), 5.55 – 5.28 (m, 6H), 4.92 (d, J = 7.5 Hz, 1H), 4.82 – 3.64 (m, 26H), 3.56 (t, J = 6.5 Hz, 23H), 3.47 – 3.26 (m, 17H), 3.23 (s, 3H), 3.01 (dq, J = 40.3, 7.0 Hz, 4H), 2.58 (d,J = 7.1 Hz, 2H), 2.27 (t, J = 6.5 Hz, 2H), 2.07 (h, J = 7.1 Hz, 2H), 1.99 –1.78 (m, J = 7.3 Hz, 2H), 1.62 – 1.41 (m, 6H), 1.22 (dddd, J = 38.9, 22.7,15.2, 7.7 Hz, 10H), 0.88 (t, J = 7.2 Hz, 3H).

[0061] Recommended synthesis route as follows Figure 2 As shown.

[0062] Example 3

[0063] This embodiment provides a bispecific antibody targeting PD-L1 and VEGF. The amino acid sequences of the heavy and light chains of its antigen-binding portion are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. The sequences are derived from patent CN117062841A, and the antibody name has been renamed 2201. The preparation process is in accordance with patent CN117062841A.

[0064] The control antibody ISOTYPE used in this embodiment has the amino acid sequences of its heavy chain and light chain as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and its preparation process is the same as that of antibody 2201.

[0065] Among them, SEQ ID NO: The specific amino acid sequence of 15 is QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYITYSGSTIYYADSVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCARDRGTTM VPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0066] SEQ ID NO: The specific amino acid sequence of 16 is DIQMTQSPSSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYAASNLETGVPSRFSGSGSGTDFTFTISGLQPEDIATYYCQQYDNLPLTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0067] Example 4

[0068] This embodiment provides a method for preparing a dual antibody-drug conjugate targeting PD-L1 and VEGF, as well as other antibody-drug conjugates.

[0069] Take 20 mg of antibody and dilute it to 3 mg / ml with 10 mM sodium phosphate buffer (pH 7.4). Add tris(2-hydroxyethyl)phosphonic acid hydrochloride TCEP stock solution (TCEP manufacturer: Sigma, catalog number C4706-2G, CAS. 51805-45-9), ensuring the final molar concentration ratio of TCEP to the final molar concentration of the biantibody is 3:1. Incubate the antibody at 37°C for 45 minutes for reduction. Add CTW52 stock solution, which is dissolved in dimethyl sulfoxide (DMSO) to 5 mM, ensuring the final molar concentration ratio of CTW52 to the final molar concentration of the biantibody is 20:1. Incubate the antibody at 4°C for 2 hours for coupling. Terminate the reaction with 20 mM cysteine, then transfer to a 30 KD ultrafiltration tube (manufacturer: Merck, catalog number UFC9030), centrifuge at 3000g, and change the medium at 4°C to reduce residual small molecules to 0.1% of the reaction concentration. Then, aseptically filter, determine the concentration, aliquot, and freeze.

[0070] Biantibody drug conjugates are named as "biantibody-linking unit-drug compound". In this example, the biantibody drug conjugate prepared by further processing the linking unit-drug compound from Example 1 and the biantibody targeting PD-L1 and VEGF from Example 3 is named 2201-CTW52. Referring to the method in Chinese Patent 202311327506.9, the reaction time and molar ratio can be adjusted according to the reaction steps to control the conjugate ratio. Following the above steps, the following biantibody drug conjugates were prepared sequentially: 2201-CTW46 and ISOTYPE-CTW52.

[0071] Example 5

[0072] The bispecific antibody-drug conjugate was detected using the SEC-HPLC molecular sieve method. The analytical column was a TSKgel G3000SWXL (7.8 mm × 30 cm, 5 μm, catalog number 08541). Approximately 0.3 mg of the sample was centrifuged at 10000g for 5 minutes in a 300 μL volume, and the supernatant was collected. The mobile phase was 50 mM sodium phosphate + 0.1 M sodium chloride, pH 6.8. The analytical column was connected to an Agilent 1260 HPLC system at a flow rate of 0.8 mL / min. The mobile phase was eluted for at least 30 minutes until the 280 nm UV baseline stabilized. 100 μg of sample was injected, and the mobile phase was eluted for 20 minutes. The percentage of each peak (polymer, monomer, and low molecular weight) was calculated based on the peak area. The monomer concentration was used as the sample purity value.

[0073] The average number of linker loads conjugated to each antibody molecule is the coupling ratio (DAR), which is detected by hydrophobic chromatography-high-performance liquid chromatography (HIC-HPLC). The analytical column is a TSKgel Butyl-NPR, 4.6 mm × 10 cm, 2.5 μm, catalog number 042168. Approximately 0.3 mg of the sample to be tested was taken in a volume of 150 μL and added to 150 μL of mobile phase A. The mixture was centrifuged at 10000 g for 5 minutes, and the supernatant was collected. Mobile phase A consisted of 20 mM sodium phosphate + 1.5 M ammonium sulfate at pH 7.0, and mobile phase B consisted of mobile phase A consisting of 20 mM sodium phosphate + 20% (v / v) acetonitrile at pH 7.0. The analytical column was connected to an Agilent 1260 high-performance liquid chromatograph at a flow rate of 0.6 ml / min. Mobile phase A was used for rinsing for at least 30 minutes until the 280 nm UV baseline stabilized. 50 μg of sample was injected, followed by rinsing with mobile phase A for 2 minutes, then gradient elution (0% B – 100% B) for 18 minutes, and finally rinsing with 100% mobile phase B for 5 minutes. The percentage of each DAR value was calculated based on the peak area of ​​each DAR value. The average DAR value was obtained by multiplying the percentage of each peak by the DAR value and summing the results. The conjugates of this invention do not limit the DAR to 4 or 8, but can be extended to 2–8. As in Example 4, in a specific experiment, using a molar ratio of 1:15:25 for the biantibody, reducing agent, and linker-drug compound (CTW46) increased the product DAR value to 8, which is significantly higher than the DAR value obtained with a molar ratio of 1:3:20 as described in Example 4 for CTW52.

[0074] The average DAR value and purity data of the biantibody drugs prepared in the above embodiments are shown in Table 1.

[0075] Table 1

[0076]

[0077] Example 6

[0078] This embodiment tests the in vitro activity of the conjugate.

[0079] Human tumor cell lines Calu6 and MCF7, overexpressing human PD-L1, were revived to the logarithmic growth phase. When the viability was greater than 90%, they were seeded into 96-well culture plates with 3000 cells per well (150 μl volume). The plates were incubated at 37°C in a 5% CO2 incubator. After 20 h, 50 μl of culture medium containing different conjugates was added to achieve 12 dilutions between 0 and 1000 nM (1000 nM in the first well, followed by 10 five-fold dilutions, with the last dilution containing 0 nM; each sample was replicated). After co-culturing for 5 days, the culture plates were removed, and 50 μl of CTG detection reagent (CellTiterGlo, Promega, catalog number G7575) was added to each well. The reaction was allowed to proceed for 2 min according to the kit recommendations. The fluorescence value (MFI) was then measured using Tecan Spark. The average value of MFI in the duplicate wells was taken as the Y-axis, and the log10 value of the dilution gradient (nM) was taken as the X-axis. A smooth curve was plotted, and the IC50 value of the killing activity was calculated according to the four-parameter fitting.

[0080] The binding activity of the conjugate with human tumor cells RKO and MDA-MB-231 (FACS) was determined: The conjugate was serially diluted with flow cytometry dilution buffer. 50 μl of the dilution buffer was mixed with 50 μl of tumor cells (200,000 cells / well) and gently pipetted to mix. The mixture was then added to a 96-well U-bottom cell plate and incubated for 1 hour. The cells were washed with FACS buffer (PBS + 3% FBS) and centrifuged twice. 100 μl of PE-labeled goat anti-human secondary antibody (eBioscience, Cat. No.: 12-4998-82) diluted 400 times was added, and the cells were incubated in the dark for 40 min. The cells were washed with FACS buffer and centrifuged twice. The signal value of cell particles in the PE channel was detected using a flow cytometer (SinoCyte). The EC50 value of the binding activity was calculated by fitting the data.

[0081] The endocytic activity of human tumor cells RKO and MDA-MB-231 with conjugates was measured (FACS): The conjugates were serially diluted with flow cytometry dilution buffer to concentrations ranging from 0 to 1000 nM. 50 μl of the serial dilution buffer was mixed with 50 μl of cells (20,000 cells / well) and gently pipetted into 96-well U-bottom cell plates and incubated at 4°C for 1 hour. The cells were washed with FACS buffer (PBS + 3% FBS) and centrifuged twice. 100 μl of pH-dependent fluorescent dye CypHer5E (Cytiva, Cat. No.: PA15401) labeled with Goat anti-hFc (Jackson, Cat. No.: Secondary antibody (109-006-098) was incubated at 4°C in the dark for 40 minutes. Cells were washed with FACS buffer and centrifuged twice. Complete culture medium (RPMI 1640 + 10% FBS) was added, and the cells were incubated at 37°C for about 3 hours. The culture medium was removed by centrifugation, and the cells were resuspended in PBS (pH 9.0). The signal value of cell granules in the APC channel (CypHer5E) was detected by flow cytometry (SinoCyte). The EC50 value of endocytosis activity was calculated by fitting the data.

[0082] The results of the conjugates’ cytotoxicity, cell binding and endocytosis activities on overexpressing cells are shown in Table 2.

[0083] Table 2

[0084]

[0085] Example 7

[0086] This embodiment measures the in vivo antitumor activity of the conjugate.

[0087] Human tumor cell lines RKO and MDA-MB-231 were resuscitated to the logarithmic growth phase with a viability greater than 90%. Immunodeficient mice (NCG or equivalent) were inoculated with varying numbers of 10,000 to 1,000,000 cells. When tumors grew to approximately 100-300 square millimeters, mice were divided into groups of 3 or 4. The drugs were administered, and tumor size was measured twice weekly. All groups received a single intravenous dose, the first dose on the day of grouping, and the second dose one week later, for a total of two doses. The tumor size of each drug group was compared with that of the saline group to determine the inhibitory activity of each drug group on tumor cells. The changes in tumor volume in each experimental group are shown in the figures below. Figure 3 and Figure 4 As shown in the figure, the horizontal axis represents the number of days of tumor inoculation for each group of drugs, and the vertical axis represents the tumor size. Each group is named as antibody-linker payload compound-dose. Figure 3 and Figure 4The data show that the biantibody-drug conjugates involved in this invention can significantly inhibit tumor growth. The tumor size in the Isotype control group and the PBS group was basically the same, with no significant antitumor activity. CTW52 activity was slightly better than CTW46.

[0088] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0089] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0090] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A biantibody-drug conjugate targeting PD-L1 and VEGF, characterized in that, The general formula of the dual antibody drug conjugate is Ab-(LD)x; In the general formula, Ab is a biantibody targeting PD-L1 and VEGF, containing a PD-L1 antigen-binding part and a VEGF antigen-binding part; in the general formula, LD is a linker-drug compound, L is a linker, D is a drug molecule, and x is an integer selected from 1 to 8. The amino acid sequences of the heavy chains CDR1, CDR2, and CDR3 of the PD-L1 antigen-binding region include SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; The amino acid sequences of the light chains CDR1, CDR2, and CDR3 of the PD-L1 antigen-binding moiety include SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; The amino acid sequences of the heavy chains CDR1, CDR2, and CDR3 of the VEGF antigen-binding region include SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; The amino acid sequences of the light chains CDR1, CDR2, and CDR3 of the VEGF antigen-binding portion include SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively.

2. The dual antibody drug conjugate according to claim 1, characterized in that, The drug molecule is selected from any one of alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, immunomodulators, or chromatin function inhibitors. Preferably, the chromatin function inhibitor is selected from camptothecin or camptothecin derivatives.

3. The dual antibody drug conjugate according to claim 2, characterized in that, The structure of the camptothecin derivative is shown in Formula I or Formula ⅠⅠ: Formula I; Formula IⅠⅠ.

4. The biantibody drug conjugate according to any one of claims 1 to 3, characterized in that, The structure of the connecting unit-drug compound is shown in Formula I and II: Formulas I and II.

5. The biantibody drug conjugate according to any one of claims 1 to 3, characterized in that, The structure of the connecting unit-drug compound is shown in Formula IV: Formula IV.

6. The biantibody drug conjugate according to any one of claims 1 to 5, characterized in that, The amino acid sequence of the heavy chain of the PD-L1 antigen-binding moiety and the VEGF antigen-binding moiety includes SEQ ID NO: 13, and the amino acid sequence of the light chain includes SEQ ID NO:

14.

7. The biantibody drug conjugate according to any one of claims 1 to 6, characterized in that, The x is selected from any integer from 4 to 8.

8. A method for preparing a biantibody-drug conjugate targeting PD-L1 and VEGF as described in any one of claims 1 to 7, characterized in that, The preparation method includes the following steps: A dual antibody targeting PD-L1 and VEGF is mixed with a reducing agent to undergo a reduction reaction. After the reaction, it is mixed with a linker-drug compound to undergo a coupling reaction, thus obtaining a dual antibody-drug conjugate targeting PD-L1 and VEGF.

9. The preparation method according to claim 8, characterized in that, The molar ratio of the dual antibody, reducing agent, and linker-drug compound targeting PD-L1 and VEGF is 1:(1~15):(5~25).

10. The use of a biantibody-drug conjugate targeting PD-L1 and VEGF as described in any one of claims 1 to 7, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for treating cancer; Preferably, the cancer includes solid tumors and hematologic tumors, including any one of breast cancer, lymphoma, head and neck cancer, gastric cancer, colorectal cancer, ovarian cancer, cervical cancer, prostate cancer, myeloma, sarcoma, pancreatic cancer, or glioblastoma.

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