A pharmaceutical composition and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a pharmaceutical composition and its application. The pharmaceutical composition provided by the present invention comprises an anti-PSMA antibody-drug conjugate and a focal adhesion kinase inhibitor; the anti-PSMA antibody-drug conjugate is obtained by coupling a humanized antibody J591 or a functional fragment thereof with a tubulin inhibitor through a linker. The present invention destroys the tumor microenvironment by a FAK inhibitor to increase the penetration of the anti-PSMA antibody-drug conjugate in the tumor tissue, further increases the killing activity against PSMA-positive tumor cells, and can effectively inhibit tumor growth and metastasis. Compared with the single administration of ADC drugs or FAK inhibitors, the present invention can break through the tumor microenvironment-mediated tumor resistance and metastasis, and can broaden the treatment window of ADC drugs to improve the safety of medication, which is expected to meet the clinical medication needs of PSMA-positive castration-resistant metastatic prostate cancer patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a pharmaceutical composition and application thereof. Background Art

[0002] Prostate cancer (PCa) is a malignant solid tumor with a high incidence in men worldwide. For the treatment of early localized prostate cancer, radical prostatectomy or combined with radiotherapy can be selected. For advanced or metastatic prostate cancer, androgen deprivation therapy (ADT) combined with second-generation anti-androgen drugs (abiraterone acetate, enzalutamide, apalutamide and darolutamide) is used for treatment. However, during disease progression, most PCa will become resistant to ADT and develop into castration-resistant prostate cancer (CRPC) or metastatic castration-resistant prostate cancer (mCRPC). Among them, prostate specific membrane antigen (PSMA) is significantly overexpressed in castration-resistant prostate cancer, and its overexpression is associated with high tumor grade, disease progression and recurrence, poor clinical prognosis and shortened patient survival.

[0003] mCRPC is the final stage of PCa progression and the main cause of death. Once it occurs, patients can only choose cytotoxic chemotherapy, radiotherapy, androgen receptor inhibitors or PARP inhibitors to prolong survival. However, these therapies have limitations such as poor tissue selectivity, high systemic toxicity and drug resistance, and the survival extension period is usually limited to less than 6 months. Therefore, new and more effective treatments are urgently needed for the treatment of mCRPC.

[0004] In recent years, targeted tumor therapy has shown significant advantages over traditional therapies. J591 is the first humanized monoclonal antibody targeting the extracellular domain of PSMA. It has the advantages of good targeting and low relative molecular weight, and avoids the immune effect of the human body on mice. Therefore, humanized J591 has good application prospects in the diagnosis and treatment of PCa. Compared with traditional cytotoxic drugs, antibody-drug conjugates (ADC) avoid systemic administration, reduce non-target organ toxicity, and are expected to improve the targeted therapeutic effect of PSMA-positive tumors. So far, three ADC molecules targeting PSMA have entered clinical trials, but they still have low killing activity against PSMA-positive tumor cells and cannot effectively inhibit tumor growth and metastasis. Summary of the invention

[0005] The object of the present invention is to provide a pharmaceutical composition and its application. The pharmaceutical composition provided by the present invention can improve the penetration of anti-PSMA antibody-drug conjugates in tumor tissues by destroying the tumor microenvironment through a focal adhesion kinase (FAK) inhibitor, further improve the killing activity against PSMA-positive tumor cells, and effectively inhibit tumor growth and metastasis.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate and a focal adhesion kinase inhibitor;

[0008] The anti-PSMA antibody-drug conjugate is obtained by coupling the humanized antibody J591 or a functional fragment thereof with a tubulin inhibitor via a dipeptide linker.

[0009] Preferably, the focal adhesion kinase inhibitor comprises defatinib and / or GSK2256098.

[0010] Preferably, the microtubule protein inhibitor is an auristatin-type cytotoxic molecule.

[0011] Preferably, the auristatin cytotoxic molecule comprises monomethyl auristatin peptide E and / or monomethyl auristatin peptide F.

[0012] Preferably, the dipeptide linker is a Val-Cit linker.

[0013] Preferably, the anti-PSMA antibody-drug conjugate has a structure as shown in Formula 1:

[0014]

[0015] In Formula 1, Ab is the humanized antibody J591 or a functional fragment thereof;

[0016] P is any integer from 1 to 16.

[0017] Preferably, the molar ratio of the anti-PSMA antibody-drug conjugate to the focal adhesion kinase inhibitor is (0.01-8):(1000-50000).

[0018] Preferably, in the pharmaceutical composition, the concentration range of the focal adhesion kinase inhibitor is 0.5 to 20 μM.

[0019] The present invention provides use of the pharmaceutical composition described in the above technical solution in preparing a drug for treating PSMA-positive solid tumors.

[0020] Preferably, the PSMA-positive solid tumor is a PSMA-positive castration-resistant metastatic prostate tumor.

[0021] The present invention provides a pharmaceutical composition, including an anti-PSMA antibody-drug conjugate and a focal adhesion kinase inhibitor; the anti-PSMA antibody-drug conjugate is obtained by coupling a humanized antibody J591 or a functional fragment thereof with a tubulin inhibitor through a dipeptide linker. The pharmaceutical composition provided by the present invention improves the penetration of the anti-PSMA antibody-drug conjugate in tumor tissue by destroying the tumor microenvironment through a focal adhesion kinase (FAK) inhibitor, further improves the killing activity against PSMA-positive tumor cells, and can effectively inhibit tumor growth and metastasis. Compared with the single administration of ADC drugs or (FAK) inhibitors, the pharmaceutical composition provided by the present invention can break through tumor microenvironment-mediated tumor resistance and metastasis, and can broaden the therapeutic window of ADC drugs to improve drug safety, which is expected to meet the clinical drug needs of PSMA-positive castration metastatic prostate cancer patients. The results of the examples show that, in PSMA-positive LNCaP cells, the combination of the pharmaceutical composition provided by the present invention has a higher killing activity on LNCap cells than ADC alone, wherein the killing effect of the drug combination of ADC and GSK2256098 is better than that of the drug combination with VS-6063; in C4-2B cells, the combination of the pharmaceutical composition provided by the present invention has a higher killing activity on LNCap cells than ADC alone, wherein there is no significant difference between the drug combinations of ADC and GSK2256098 and VS-6063; and the killing activity of the combination on tumor cells is PSMA-specific. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The preparation route of MC-VC-PAB-MMAE in the embodiment of the present invention is shown in FIG.

[0023] Figure 2 The SDS-PAGE electrophoresis diagram of the humanized J591 monoclonal antibody prepared in the embodiment of the present invention under reducing and non-reducing conditions;

[0024] Figure 3 The size exclusion chromatography analysis results of the humanized J591 monoclonal antibody prepared in the embodiment of the present invention at a wavelength of 214 nm;

[0025] Figure 4 The size exclusion chromatography analysis results of the humanized J591 monoclonal antibody prepared in the embodiment of the present invention at a wavelength of 280 nm;

[0026] Figure 5 It is a curve diagram of absorbance value and Biotin-PSMA recombinant protein concentration;

[0027] Figure 6 This is a comparison of the fluorescence intensity of humanized monoclonal antibody J591 after binding to different PSMA-positive tumor cell lines;

[0028] Figure 7 This is a graph showing the internalization activity of humanized monoclonal antibody J591 in different PSMA-positive tumor cells;

[0029] Figure 8 The killing IC values ​​of different FAK inhibitors on the prostate cancer tumor cell line C4-2 in the present invention are shown in FIG. 50 Determination curve;

[0030] Fig. 9 The killing IC values ​​of different FAK inhibitors on the prostate cancer tumor cell line LNCap in the embodiments of the present invention are shown in FIG. 50 Determination curve;

[0031] Fig.10 The killing IC values ​​of different FAK inhibitors on the prostate cancer tumor cell line CP-3 in the present invention are shown in FIG. 50 Determination curve;

[0032] Fig.11 The killing results of the combined use of the pharmaceutical composition provided in the embodiment of the present invention on the prostate cancer tumor cell line LNCap;

[0033] Fig.12 The killing results of the combined use of the pharmaceutical composition provided in the embodiment of the present invention on the prostate cancer tumor cell line C4-2B;

[0034] Fig.13 The killing results of the combined use of the pharmaceutical composition provided in the embodiments of the present invention on the prostate cancer tumor cell line CP-3 are shown. DETAILED DESCRIPTION

[0035] The present invention provides a pharmaceutical composition comprising an anti-PSMA antibody-drug conjugate and a focal adhesion kinase inhibitor;

[0036] The anti-PSMA antibody-drug conjugate is obtained by coupling the humanized antibody J591 or a functional fragment thereof with a tubulin inhibitor via a dipeptide linker.

[0037] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0038] The present invention provides a pharmaceutical composition comprising a focal adhesion kinase inhibitor.

[0039] In the present invention, focal adhesion kinase (FAK) is a tyrosine kinase with a molecular weight of 125kD, which plays a vital role in the cell signal transduction system. FAK is overexpressed and / or overphosphorylated in multiple cancer cells, leading to cell migration, survival, proliferation, and adhesion. In addition, FAK is closely related to the occurrence and development of tumors. The second-generation FAK inhibitor defactinib (VS-6063) is a FAK inhibitor with higher safety and has been shown to be well tolerated in Phase 1 clinical trials. GSK2256098 has been evaluated in several Phase I clinical trials and has good tolerance and anti-tumor activity as a single drug and in combination with other anticancer drugs.

[0040] In the present invention, the focal adhesion kinase inhibitor includes defatinib (VS-6063) and / or GSK2256098, more preferably VS-6063 or GSK2256098.

[0041] The pharmaceutical composition provided by the present invention comprises an anti-PSMA antibody-drug conjugate. The anti-PSMA antibody-drug conjugate is obtained by coupling a humanized antibody J591 or a functional fragment thereof with a tubulin inhibitor via a dipeptide linker.

[0042] In the present invention, the humanized antibody J591 preferably comprises a heavy chain and a light chain.

[0043] In the present invention, the light chain preferably comprises a variable region and a constant region; the variable region of the light chain has an amino acid sequence as shown in SEQ ID NO.1, or has a sequence with at least 80% sequence identity to SEQ ID NO.1, preferably 85-99%, specifically preferably 85%, 90%, 95%, 98% or 99%.

[0044] In the present invention, the amino acid sequence shown in SEQ ID NO.1 is:

[0045] DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLT FGAGTMLDLK.

[0046] In the present invention, the amino acid sequence shown in SEQ ID NO.1 is obtained from the nucleotide sequence shown in SEQ ID NO.2, and the nucleotide sequence shown in SEQ ID NO.2 is:

[0047] GACATTTGTGATGACCCAGAGCCACAAATTTATGAGCACCAGCGTGGGAGATAGAGTGAGTATTATCTGCAAGGCCAGCCAGGACGTGGGAACCGCCGTGGACTGGTATCAGCAGAAACCTGGCCAGAGCCCAAAACTGCTGATCTACTGGGCCAGCACCAG ACATACCGGAGTGCCCGACAGGTTCACAGGATCAGGCAGCGGCACAGACTTCACCCTGACCATCACCAACGTGCAGAGCGAGGACCTGGCCGACTATTTCTGCCAGCAGTACAACAGCTACCCCCTGACATTTGGAGCCGGCACCATGCTGGACCTGAAG.

[0048] In the present invention, the constant region of the light chain is preferably humanized or human, more preferably human.

[0049] In the present invention, the heavy chain preferably comprises a variable region and a constant region; the variable region of the heavy chain has an amino acid sequence as shown in SEQ ID NO.3, or a sequence having at least 80% sequence identity with SEQ ID NO.3, preferably 85-99%, specifically preferably 85%, 90%, 95%, 98% or 99%.

[0050] The amino acid sequence shown in SEQ ID NO.3 is:

[0051] EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEW IGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCA AGWNFDYWGQGTTLTVSS.

[0052] In the present invention, the amino acid sequence shown in SEQ ID NO.3 is obtained from the nucleotide sequence shown in SEQ ID NO.4, and the nucleotide sequence shown in SEQ ID NO.4 is:

[0053] GAGGTGCAGCTGCAGCAGAGCGGGCCAGAGCTGAAGAAGCCAGGGACAAGCGTGAGAATTAGCTGCAAGACAAGCGGGTACACATTTACAGAGTATACCATTCACTGGGTGAAACAGAGCCACGGTAAGAGCCTGGAATGGATTGGAAACATCAACCCCAATAACGGAGGAAC AACCTACAACCAGAAATTCGAGGACAAAGCCACCCTGACCGTGGACAAAAGCAGTAGCACCGCCTACATGGAGCTGAGAAGCCTGACCAGCGAAGACAGCGCCGTGTACTACTGCGCCGCCGGCTGGAACTTCGACTATTGGGGACAGGGCACCACCCTGACCGTGAGCAGC.

[0054] In the present invention, the constant region of the heavy chain is preferably humanized or human, more preferably human.

[0055] In the present invention, the subtype of the humanized antibody J591 preferably includes one or more of IgM, IgG1, IgG2, IgG3 and IgG4, and more preferably IgG1.

[0056] In the present invention, the microtubule inhibitor is preferably an auristatin-type cytotoxic molecule.

[0057] In the present invention, the auristatin cytotoxic molecule preferably includes monomethyl auristatin peptide E (MMAE) and / or monomethyl auristatin peptide F (MMAF), more preferably MMAE.

[0058] In the present invention, the dipeptide linker is preferably a Val-Cit linker.

[0059] In the present invention, the anti-PSMA antibody-drug conjugate preferably has a structure shown in Formula 1:

[0060]

[0061] In Formula 1, Ab is the humanized antibody J591 or a functional fragment thereof, preferably the humanized antibody J591;

[0062] P is an integer of 1 to 16, and preferably 4.

[0063] In the present invention, P in Formula 1 represents the molar ratio of the humanized antibody J591 or its functional fragment to the MMAE in the anti-PSMA antibody-drug conjugate molecule; in a specific embodiment of the present invention, in the anti-PSMA antibody-drug conjugate molecule: the molar ratio of the humanized antibody J591 to the MMAE is 1:4.

[0064] In the present invention, the method for preparing the PSMA antibody-drug conjugate preferably comprises the following steps:

[0065] Mixing a reducing agent, a protecting agent and a humanized antibody J591, and reducing the mixture to obtain a reduced humanized J591 antibody;

[0066] Mixing a linker, an active drug, a coupling reagent and a solvent, and performing a coupling reaction to obtain a linker-active drug conjugate;

[0067] The reduced humanized J591 antibody, the linker-active drug conjugate and a solvent are mixed to perform a coupling reaction to obtain a PSMA antibody-drug conjugate.

[0068] The present invention mixes a reducing agent, a protecting agent and a humanized antibody J591, performs reduction, and obtains the reduced humanized J591 antibody.

[0069] In the present invention, the method for preparing the humanized antibody J591 preferably comprises the following steps:

[0070] The variable region of the mouse monoclonal antibody J591 and the human IgG1κ constant region were connected to form a complete fragment by overlapping extension PCR, the complete fragment was subcloned into an expression vector, and the constructed plasmid was transfected into suspended host cells to express and produce the humanized antibody J591.

[0071] In the present invention, the expression vector is preferably the expression vector pcDNA3.0.

[0072] In the present invention, the host cell is preferably a CHO cell (Invitrogen).

[0073] The present invention preferably collects the expressed humanized antibody J591 from the culture supernatant of the transfected suspended host cells. In the present invention, the crude product of humanized antibody J591 is collected from the culture supernatant of the transfected suspended host cells. The present invention preferably purifies the crude product of humanized antibody J591 to obtain a pure product of humanized antibody J591. In the present invention, the purification is preferably performed using Protein A.

[0074] The present invention preferably analyzes the purity of the humanized antibody J591 by SDS-PAGE electrophoresis and SEC.

[0075] In the present invention, the purity of the pure product of the humanized antibody J591 is preferably ≥95%.

[0076] In the present invention, the reducing agent is preferably tris(2-carboxyethyl)phosphine hydrochloride (Tris-2-car rboxyethyl-phosphine, abbreviated as TCEP).

[0077] In the present invention, the protective agent is preferably diethylenetriaminepentacetate acid (DTPA for short).

[0078] In the present invention, the molar ratio of the reducing agent to the protecting agent is preferably 1-20:1-20.

[0079] In the present invention, the molar ratio of the reducing agent to the humanized antibody J591 is preferably 1 to 4:1, more preferably 3:1.

[0080] In the present invention, the reduction reaction is preferably carried out in a solution. The solvent for the reduction reaction is preferably a PBS buffer.

[0081] In the present invention, the mixing of the reducing agent, the protective agent and the humanized antibody J591 preferably includes the following steps: dissolving the reducing agent and the protective agent in a PBS buffer to obtain a mixed mother liquor; ultrafiltration and dialyzing the humanized antibody J591 into a PBS buffer using a 30KD membrane package, and concentrating to obtain a humanized antibody J591 solution; and mixing the mixed mother liquor with the humanized antibody J591 solution. In the present invention, in the mixed mother liquor, the molar concentration of the reducing agent is preferably 1 to 20 mM, and the molar concentration of the protective agent is preferably 1 to 20 mM; the mass concentration of the humanized antibody J591 solution is preferably 5 to 30 mg / mL; and the volume ratio of the mixed mother liquor to the humanized antibody J591 solution is preferably 1:1.

[0082] In the present invention, the temperature of the reduction reaction is preferably 37° C., and the time of the reduction reaction is preferably 30 to 120 min, more preferably 30 min.

[0083] The present invention mixes a linker, an active drug, a coupling reagent and a solvent, performs a coupling reaction, and obtains a linker-active drug conjugate.

[0084] In the present invention, the linker is preferably Fmoc-Val-Cit-PAB-PNP.

[0085] In the present invention, the active drug is preferably MMAE.

[0086] In the present invention, the linker-active drug conjugate is preferably MC-VC-PAB-MMAE.

[0087] In the present invention, the preparation method of MC-VC-PAB-MMAE preferably comprises the following steps:

[0088] Fmoc-Val-Cit-PAB-PNP and MMAE are dissolved in N,N-dimethylformamide (DMF), 1-hydroxybenzotriazole (HoBt) and pyridine are added in sequence, and the reaction is stirred overnight at room temperature; the reaction product is separated and purified by high performance liquid chromatography, the pure product is collected, and the reaction product is lyophilized to obtain the pure product of MC-VC-PAB-MMAE. In the present invention, the molar ratio of the Fmoc-Val-Cit-PAB-PNP and MMAE is preferably 0.125:0.083; the molar ratio of the Fmoc-Val-Cit-PAB-PNP and HoBt is preferably 0.125:0.074; the ratio of the amount of the Fmoc-Val-Cit-PAB-PNP to the volume of the pyridine is preferably 0.125mmol:0.5mL.

[0089] After obtaining the reduced humanized J591 antibody and the linker-active drug conjugate, the present invention mixes the reduced humanized J591 antibody, the linker-active drug conjugate and a solvent to perform a coupling reaction to obtain a PSMA antibody-drug conjugate.

[0090] In the present invention, the solvent is preferably dimethyl sulfoxide (DMSO) and water; the volume percentage of DMSO in the solvent is preferably 25%. In the present invention, the molar ratio of the linker-active drug conjugate to the reduced humanized J591 antibody is preferably 1 to 2:1, more preferably 1.5 to 2:1; the temperature of the coupling reaction is preferably 0 to 37°C, more preferably 0°C, and the time of the coupling reaction is preferably 30 to 120 min, more preferably 130 min; the coupling reaction is carried out under stirring. In the present invention, the reaction solution obtained by the coupling reaction is preferably subjected to centrifugal ultrafiltration 3 times using PBS buffer to purify and remove residual unreacted linker-active drug conjugates and free small molecules such as DMSO. In the present invention, the present invention preferably uses SDS-PAGE electrophoresis and hydrophobic high performance liquid chromatography (HIC-HPLC) to detect the coupling situation.

[0091] In the present invention, the PSMA antibody-drug conjugate is preferably Ab-MC-VC-PAB-MMAE (denoted as YC1605).

[0092] In the present invention, the molar ratio of the anti-PSMA antibody-drug conjugate to the focal adhesion kinase inhibitor is preferably (0.01-8):(1000-50000).

[0093] In a specific embodiment of the present invention, the molar ratio range of the anti-PSMA antibody-drug conjugate having killing activity against cancer cells and the focal adhesion kinase inhibitor is preferably: when the focal adhesion kinase inhibitor is VS-6063, the molar ratio of the antibody-drug conjugate to the VS-6063 is preferably 0.03125-8:4101-4625; when the focal adhesion kinase inhibitor is GSK2256098, the molar ratio of the antibody-drug conjugate to the GSK2256098 is preferably 0.0195-5:10000.

[0094] In the present invention, in the pharmaceutical composition, the concentration range of the focal adhesion kinase inhibitor is preferably 0.5 to 20 μM, more preferably 0.8 to 19.5 μM.

[0095] The present invention provides use of the pharmaceutical composition described in the above technical solution in preparing a drug for treating PSMA-positive solid tumors.

[0096] In the present invention, the PSMA-positive solid tumor is a PSMA-positive castration-resistant metastatic prostate tumor.

[0097] The present invention prepares an ADC drug by coupling the J591 monoclonal antibody to MMAE, and combines the ADC drug with the FAK inhibitors VS-6063 and GSK2256098 for the treatment of PSMA-positive tumors, which can break through the tumor microenvironment-mediated tumor resistance and metastasis, and can broaden the therapeutic window of the ADC drug to improve the safety of the drug, and is expected to meet the clinical drug needs of PSMA-positive castration-resistant metastatic prostate cancer patients.

[0098] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0099] In an embodiment of the present invention:

[0100] Types and sources of raw materials:

[0101] In the present invention, small molecule chemical reagents such as MC-MMAE and Fmoc-Val-Cit-PAB-PNP were purchased from Shanghai Haoyuan Biopharmaceutical Co., Ltd.; prostate cancer cell lines LNCap cells, 22RV1 cells and PC-3 cells were purchased from Wuhan Shanen Biotechnology; C4-2B cells were purchased from Beina Biotechnology. Cell Counting Kit-8 (CCK8) was purchased from Beyotime, and human PSMA recombinant protein was purchased from ACRO. Protein A antibody purification filler was purchased from GE, and the microplate reader was purchased from OMEGA.

[0102] Example 1

[0103] Recombinant expression and purification of humanized antibody J591:

[0104] The variable region of the mouse monoclonal antibody J591 and the human IgG1κ constant region were connected into a complete fragment by overlapping extension PCR, wherein the variable region of the light chain of the mouse monoclonal antibody J591 has the amino acid sequence shown in SEQ ID NO.1, and the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO.3.

[0105] The above amplified fragment was subcloned into the expression vector pcDNA3.0, and the constructed plasmid was transfected into the suspended host cells (Invitrogen) to produce the humanized antibody J591. The expressed antibody was collected from the host cell culture supernatant and purified by Protein A. The antibody purity was analyzed by size exclusion chromatography and SDS-PAGE under reducing and non-reducing conditions. The results showed that the purity of the expressed and purified humanized antibody J591 was greater than 95%, and the endotoxin content was less than 1EU / mg after filtration through a 0.22μm filter membrane. The SDS-PAGE analysis results of the purified humanized antibody J591 are shown in Figure 2 The size exclusion chromatography results are shown in Figure 3 and Figure 4 shown.

[0106] Figure 2 The SDS-PAGE electrophoresis diagram of the humanized antibody J591 under reducing and non-reducing conditions, wherein: Lane M: Marker; Lane R: reducing electrophoresis; Lane NR: non-reducing electrophoresis. Figure 3 SEC-HPLC spectrum at 214 nm wavelength; Figure 4 : SEC-HPLC spectrum under 280nm wavelength conditions.

[0107] Example 2

[0108] (1) Biological activity analysis of humanized monoclonal antibody J591:

[0109] ELISA analysis of the binding activity of the humanized antibody J591 prepared in Example 1 to the PSMA recombinant protein: The PSMA recombinant protein purchased from ACRO was used to verify the binding activity of the expressed humanized monoclonal antibody J591. The humanized monoclonal antibody J591 was diluted to 2 μg / mL, 100 μL / well was coated on the ELISA plate, and the coating was carried out overnight at 4°C. 2% BSA was blocked at 37°C for 2h. The Biotin-PSMA recombinant protein was diluted to 1 μg / mL, 2-fold gradient dilution was performed to 7 points, 100 μL / well was added to the ELISA plate, and incubated at 37°C for 1h. 100 μL / well was added with a 1:10000-fold diluted HRP-Streptavidin enzyme-labeled secondary antibody, and after incubation at 37°C for 1h, TMB colorimetric solution was added for color development reaction, and the absorbance value at a wavelength of 450nm was measured after terminating the color development with 2M H2SO4. The absorbance value and Biotin-PSMA recombinant protein concentration curve were plotted to analyze the biological activity of humanized monoclonal antibody J591. Figure 5 , Figure 5 This indicates that the humanized monoclonal antibody J591 prepared in Example 1 can bind to PSMA recombinant protein and has binding activity to PSMA protein.

[0110] (2) Flow cytometry analysis of the binding activity of J591 to PSMA-positive tumor cell lines:

[0111] The binding activity of humanized monoclonal antibody J591 to PSMA-positive prostate cancer cell lines LNCap cells, C4-2B cells and 22RV1 cells, and to PSMA-negative prostate cancer cell line PC-3 was detected. The above four cells in the logarithmic growth phase were collected after digestion, and the cell pellet was collected by centrifugation. The cells were resuspended in PBS and washed by centrifugation once. The supernatant after centrifugation was removed, the cells were resuspended in PBS, and 5×10 cells of each type were taken after counting. 5 Each tube was incubated with 5 μg / mL J591 monoclonal antibody for 1 h at 4°C, and the cells were washed twice with PBS. PE-labeled goat anti-human IgG was diluted 1:100 and added to each tube. The tubes were incubated at 4°C in the dark for 30 min, and then rinsed twice with PBS solution. The fluorescence intensity of each cell line was detected by flow cytometry. Figure 6 The humanized monoclonal antibody J591 prepared in Example 1 showed strong binding to the PSMA high-expressing cell lines C4-2B cells and LNCap cells, weak binding to the low-expressing cell line 22RV1 cells, and no binding to the PSMA negative cell line PC-3.

[0112] Example 3

[0113] Biacore determination of affinity of humanized monoclonal antibody J591:

[0114] To further characterize the anti-PSMA humanized monoclonal antibody J591, the affinity of the humanized monoclonal antibody J591 to the human PSMA protein was determined. The recombinant PSMA protein was immobilized on the CM5Biacore chip, and the analyte was bound to the protein in the mobile phase at a concentration of 0.125μg / mL, 0.25μg / mL, 0.5μg / mL, 1.0μg / mL, 2.0μg / mL, and 4.0μg / mL; the mobile phase flow rate was 30μL / min; the binding time was 180s; and the dissociation time was 300s. The binding affinity Kd of the humanized monoclonal antibody J591 prepared in Example 1 to human PSMA was measured to be 1.612nM.

[0115] Example 4

[0116] Flow cytometry was used to detect the internalization activity of humanized monoclonal antibody J591 in PSMA-positive tumor cells:

[0117] LNCap cells were incubated with 5 μg / mL humanized monoclonal antibody J591 and control antibody anti-hPSMA AF488 at 4°C for 30 minutes. After washing, the cells were incubated at 37°C to internalize the antibodies. The fluorescence intensity of each group of cells was detected by flow cytometry at 0 min, 30 min, 60 min, and 120 min in a 37°C environment to analyze the internalization activity of J591. The results are shown in Figure 7 The humanized monoclonal antibody J591 had a high internalization activity in the PSMA-positive tumor cells LNCap, and the internalization rates at 30min, 60min, and 120min were 18.71%, 27.37%, and 45.67%, respectively.

[0118] Example 5

[0119] according to Figure 1 The anti-PSMA antibody-drug conjugate (MC-VC-PAB-MMAE) was prepared by the following process:

[0120] Fmoc-Val-Cit-PAB-PNP (93 mg, 0.125 mmol) and MMAE (60 mg, 0.083 mmol) were dissolved in 4 mL DMF, and HoBt (10 mg, 0.074 mmol) and 0.5 mL pyridine were added in sequence, and the reaction was stirred overnight at 37 ° C. The reaction product was separated and purified using preparative liquid phase, the pure product was collected, and the reaction product was lyophilized to obtain MC-VC-PAB-MMAE.

[0121] Preparation of anti-PSMA antibody-drug conjugates:

[0122] (1) Purification and treatment of humanized antibody J591:

[0123] The humanized antibody J591 captured from the host cell culture supernatant by Protein A, the antibody purity reached more than 95% by SDS-PAGE electrophoresis and SEC analysis. The obtained antibody protein was ultrafiltered and dialyzed into PBS buffer using a 30KD membrane package, and after concentration, the concentration was calibrated with a UV spectrophotometer for subsequent coupling reactions;

[0124] (2) Preparation of anti-PSMA antibody-drug conjugates

[0125] The preparation of antibody-drug conjugates adopts a general coupling method: the reducing agent and protective agent are prepared in PBS buffer as follows: 1mM TCEP (Tris-2-carboxyethyl-phosphine), 1mM DTPA (Diethylenetriaminepentacetate acid) stock solution; mixed with a mass concentration of 30mg / mL monoclonal antibody in a volume ratio (1:1), the final molar ratio of TCEP to antibody is 0.5:1, and stirred at 25°C for 1h. The antibody after TCEP reduction can be directly coupled;

[0126] Take the MC-VC-PAB-MMAE prepared in Example 5 to prepare a linker-active drug conjugate with a molar concentration of 5mM, dissolve the MC-VC-PAB-MMAE prepared in Example 5 in a 25% DMSO aqueous solution (dimethyl sulfoxide), slowly add the drug according to the molar ratio of the active drug of the linker-active drug conjugate to the thiol group in the reduced antibody of 1.5:1, and stir the reaction at 0°C for 120min. After the reaction, centrifuge and ultrafilter with PBS buffer for 3 times to purify and remove residual unreacted drugs and free small molecules such as DMSO, and use SDS-PAGE electrophoresis and hydrophobic high performance liquid chromatography (HIC-HPLC) to detect the coupling status;

[0127] By the method provided in this example, an antibody-drug conjugate Ab-MC-VC-PAB-MMAE (ie, YC1605) was prepared, and the structural formula is shown in Formula 1:

[0128]

[0129] In Formula 1, A is the humanized antibody J591, and p is 4.

[0130] Example 6

[0131] IC of different FAK inhibitors against prostate cancer cell lines 50 Determination:

[0132] (1) Cell viability was determined using a cell counting kit 8 (CCK-8). Prostate cancer cell lines LNCap, C4-2B, 22RV1, and PC-3 cells were seeded into a 96-well plate at 5,000 cells / well and incubated overnight.

[0133] (2) The FAK inhibitors VS-6063 and GSK2256098 were diluted 2-fold in a culture medium containing 10% FBS and then added to each well. A blank control group (no cell and drug group) and a negative control group (with cells and no drug group) were set up at the same time. Each concentration gradient was set up with 3 replicate wells. The cell plate was incubated in a humidified box at 37°C and 5% CO2 for 72 hours.

[0134] (3) Add 10 μL of CCK8 reagent to each well and return the well to the incubator. After incubation for 1 h, measure the absorbance at 450 nm.

[0135] (4) According to the absorbance values ​​of each group of cells, the cell viability was calculated according to the formula shown in Formula 1. The data were analyzed using PrismGraphPad software to draw dose-response curves and calculate the IC values ​​of FAK inhibitors for the four cell lines. 50 The result is Figures 8 to 10 As shown, the inhibitor VS-6063 of FAK inhibits the proliferation of C4-2B, LNCap and PC-3 cells. 50 The values ​​were: 4.675μM, 4.101μM and 4.265μM respectively; the inhibition rate of GSK2256098 on the three cell lines reached 50%.

[0136] Cell survival rate = (group A 450 Value-Blank Group A 450 value) / (control group A 450 Value-Blank Group A 450 value)×100%Formula 1.

[0137] Example 7

[0138] Determination of the killing activity of ADC combined with FAK inhibitor against prostate cancer tumor cell lines:

[0139] (1) Cell viability was determined using a cell counting kit 8 (CCK-8). Prostate cancer cell lines LNCap, C4-2B, and PC-3 cells were seeded into a 96-well plate at 3,000 to 10,000 cells / well and incubated overnight.

[0140] (2) The antibody-drug conjugate prepared in Example 5 was diluted to 8 nM, 4 nM, 2 nM, 1 nM, 0.5 nM, 0.25 nM, 0.125 nM, 0.0625 nM and 0.03125 nM in a culture medium containing 10% FBS, and added to each cell well, respectively, with J591 naked antibody as a control; and the IC corresponding to each cell calculated in Example 6 was added 50 16μM VS-6063 or 16μM GSK2256098. Three parallel wells were set for each drug concentration. The control group was the group with cells but no drug, and the blank group was the group without cells and no drug. The cells were cultured in an incubator at 37℃ and 5% CO2 for 72h.

[0141] (3) Add 10 μL of CCK-8 reagent to each well and continue to culture for 1 to 2 hours. Observe the color reaction, and measure the absorbance value at a wavelength of 450 nm with an enzyme marker. According to the absorbance value of each group of cells, the cell viability is calculated according to Formula 1. The data are analyzed using Prism GraphPad software to draw a dose-response curve, and the killing activity of the antibody-drug conjugate on the four cell lines is analyzed. Among them, when the antibody-drug conjugate (ADC) prepared in Example 5 is used in combination with VS-6063 or GSK2256098, the concentration range of ADC is: 0.03125nM to 8nM, the concentration of FAK inhibitor GSK2256098 is 16μM, and the concentration range of VS-6063 is 4.101μM to 4.625μM. The molar ratio of ADC to FAK inhibitor VS-6063 is 0.03125-8:4101-4625; the molar ratio of ADC to FAK inhibitor GSK2256098 is 0.0195-5:10000.

[0142] The results are as follows Figures 11 to 13 The results showed that in PSMA-positive LNCaP cells, the killing activity of the combination therapy on LNCap cells was higher than that of ADC alone, and the killing effect of the drug combination of ADC and GSK2256098 was better than that of the drug combination with VS-6063; in C4-2B cells, the killing activity of the combination therapy on LNCap cells was higher than that of ADC alone, and there was no significant difference between the drug combinations of ADC and GSK2256098 and VS-6063; and the killing activity of the combination therapy on tumor cells was PSMA-specific.

[0143] Cell survival rate = (group A 450 Value-Blank Group A 450 value) / (control group A 450 Value-Blank Group A 450 value)×100%Formula 1.

[0144] The present invention utilizes J591 monoclonal antibody, adopts MMAE as active molecule, Val-Cit linker, and couples with VC-MMAE by reducing interchain disulfide bond of antibody to construct an antibody-drug conjugate YC1605 targeting PSMA for solid tumor treatment, which has high killing activity against PSMA-positive tumor cells.

[0145] The present invention provides a combination of ADC molecules and FAK inhibitors VS-6063 (Defactinib) and GSK2256098 for the treatment of PSMA-positive solid tumors. Compared with the single administration of ADC drugs or FAK inhibitors, the combined treatment method destroys the tumor microenvironment through FAK inhibitors to improve the penetration of drugs in tumor tissues, further improves the killing activity of PSMA-positive tumor cells, and can effectively inhibit tumor growth and metastasis.

[0146] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The invention comprises an anti-PSMA antibody-drug conjugate and a focal adhesion kinase inhibitor; the focal adhesion kinase inhibitor is GSK2256098; The anti-PSMA antibody-drug conjugate is obtained by coupling the humanized antibody J591 or its functional fragment with MMAE via a dipeptide linker and has a structure shown in Formula 1: Formula 1; In Formula 1, Ab is the humanized antibody J591; P in Formula 1 represents the molar ratio of the humanized antibody J591 or its functional fragment to the MMAE in the anti-PSMA antibody-drug conjugate molecule, and P is 4; The molar ratio of the anti-PSMA antibody-drug conjugate to the focal adhesion kinase inhibitor is (0.01-8):(1000-50000).

2. The pharmaceutical composition according to claim 1, characterized in that In the pharmaceutical composition, the concentration range of the focal adhesion kinase inhibitor is 0.5-20 μM.

3. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of a drug for treating PSMA-positive solid tumors; the PSMA-positive solid tumor is a PSMA-positive castration-resistant metastatic prostate tumor.

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