Preparation and application of antibody coupling drug LZU-F001

Abstract

The invention discloses preparation and application of an antibody-conjugated drug LZU-F001, and a preparation method comprises the following steps: step 1, taking a TCEP solution as a reducing agent, and reacting at 37 DEG C for 2 hours to reduce a humanized monoclonal antibody LY3076226; and step 2, mixing the GGFG-DXD with the reduced humanized monoclonal antibody LY3076226, and carrying out a reaction at a temperature of 0 to 37 DEG C for 30 to 120 min so as to obtain the ADC, compared with the prior art, the ADC has the advantages that the tumor inhibition effect of the ADC designed by the invention is obviously superior to that of gemcitabine + cis-platinum in a common chemotherapy regimen, and is also obviously superior to that of an FGFR3 monoclonal antibody. The safety of the pharmaceutical composition is better than that of a chemotherapy regimen.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted anti-tumor drug development, and in particular to the preparation and application of an antibody-coupled drug LZU-F001. Background Art

[0002] In recent years, the strategy of antibody-drug conjugates (ADC) has become one of the most promising anti-tumor drugs because of its breakthrough clinical benefits. At present, the development of ADC is mainly focused on star targets such as HER2, and homogeneity is serious. FGFR3, as a tyrosine kinase receptor closely related to many malignant tumors represented by bladder cancer, has not received much attention in the development field of ADC. Although small molecule inhibitors for FGFR3 mutations have been successful, the advancement process of clinical trials for FGFR3 monoclonal antibodies has not been completed, and its actual efficacy data is not clear. In order to obtain an effective anti-tumor drug based on FGFR3 antibodies, the present invention adopts the design scheme of ADC to develop LZU-F001.

[0003] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the above technical defects and provide the preparation and application of antibody-drug conjugate LZU-F001. The ADC antibody part involved is the human anti-FGFR3 monoclonal antibody LY3076226 (original patent US8043618B2), the linker is GGFG, and the payload is the camptothecin derivative DXD.

[0005] The ADC of the present invention is used to treat malignant tumors in which FGFR3 target is expressed. Including blood cancer, multiple myeloma, leukemia, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, lymphoma, Hodgkin's disease, non-Hodgkin's disease or multiple cancers; and solid tumors, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, pseudomyxoma peritonei, lymphangioendotheliosarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer , breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, head and neck cancer, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular tumor, lung cancer, small cell lung cancer, epithelial cancer, glioma, glioblastoma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma, thymoma, or any combination thereof.

[0006] Preferably, the ADC of the present invention is used to treat bladder cancer.

[0007] In order to solve the above problems, the technical solution of the present invention is the preparation and application of antibody-drug conjugate LZU-F001: the preparation method comprises the following steps:

[0008] Step 1: using TCEP solution as a reducing agent, reacting at 37° C. for 2 h to reduce the humanized monoclonal antibody LY3076226;

[0009] Step 2: GGFG-DXD is mixed with the reduced humanized monoclonal antibody LY3076226, and reacted at 0-37° C. for 30-120 min to obtain the ADC.

[0010] Furthermore, the molar equivalent of the TCEP solution is 30-50 times that of the humanized monoclonal antibody LY3076226.

[0011] Furthermore, the step 2 also includes adding 15 times the molar equivalent of LP (GGFG-DXD) (10 mM dissolved in DMSO) to the reduced antibody solution, and supplementing DMSO to a final DMSO concentration of 15% (V / V); using a 30Kd ultrafiltration tube to remove excess small molecules and replacing the coupled antibody solution to 100 mM Pro; using UV-Vis to detect ADC concentration; HIC to detect DAR (Drug-to-Antibody Ratio, drug-antibody ratio); HPLC-SEC to detect ADC purity; end-point colorimetric method to detect endotoxin; ELISA to determine the binding ability to the target FGFR3.

[0012] Furthermore, the application of the antibody-drug conjugate LZU-F001 includes an activity assay under in vitro conditions, and the activity assay under in vitro conditions includes: evaluating the therapeutic activity in bladder cancer T24 and UMUC-3 cell lines and organoid tumors derived from clinical patients, determining the inhibition curve and IC50 through the CCK-8 experiment, performing a comprehensive evaluation through clone formation experiments, scratch experiments, transwell invasion experiments, flow cytometry to determine apoptosis, etc., and using pre-constructed and flow cytometry-verified FGFR3 knockdown cells to evaluate the relationship between efficacy and target.

[0013] Furthermore, the application of the antibody-drug conjugate LZU-F001 also includes the mechanism of action and stability evaluation of LZU-F001, and the mechanism of action and stability evaluation of LZU-F001 includes the following steps: determining the mechanism of action of LZU-F001 through experiments such as confocal imaging and flow cytometry, evaluating the stability through UV-vis and hemolysis experiments, and evaluating the bystander effect using in vitro co-culture and nude mouse subcutaneous tumor experiments.

[0014] Furthermore, the application of the antibody-drug conjugate LZU-F001 also includes an in vivo tumor inhibition assay, which uses a umuc3 cell nude mouse subcutaneous tumor model and a PDX tumor model derived from multiple patients to verify the in vivo efficacy.

[0015] Furthermore, the application of the antibody-drug conjugate LZU-F001 also includes the effect of LZU-F001 in the PDX model.

[0016] The advantages of the present invention compared with the prior art are:

[0017] 1. The tumor inhibition effect of the ADC designed by the present invention is significantly better than the commonly used chemotherapy regimen of gemcitabine + cisplatin, and is also significantly better than the inhibitory effect of FGFR3 monoclonal antibody. The present invention is also better than the chemotherapy regimen in terms of safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A is the optimization result diagram of the coupling conditions of LZU-F001 of the present invention, and the product is most stable under the preferred 40X TCEP reduction equivalent and the DAR is 8; Figure 1 B is the purity report graph; Figure 1 C. is a graph showing the endotoxin test results of FGFR3 monoclonal antibody; Figure 1 D is the ELISA binding result of the product LZU-F001 with FGFR3 protein.

[0019] Figure 2 A is a dose-response curve of LZU-001 in wild-type and knockdown T24 cells as determined by cck-8; Figure 2 B is a dose-response curve of LZU-001 in wild-type and knockdown umuc-3 cells as determined by cck-8; Figure 2 C is a graph of FGFR3 protein expression of LZU-001 in wild-type and knockdown T24 and umuc3 cells determined by flow cytometry; Figure 2 D is a graph showing the effect of 0.5 μM LZU-001 intervention on the clone formation ability of wild-type and knockdown T24 cells; Figure 2 E is a graph showing the effect of 0.5 μM LZU-001 intervention on the clone formation ability of wild-type and knocked-down umuc3 cells; Figure 2 F is the effect of 0.5 μM LZU-001 intervention on the cell morphology of wild-type T24 and umuc3 cells observed under a light microscope; Figure 2 G is the effect of 0.5 μM LZU-001 intervention on two FGFR3-positive bladder cancer organoids observed under light microscopy; Figure 2 H is the effect of 0.5 μM LZU-001 intervention on the migration ability of wild-type and knockdown T24 cells; Figure 2 I is a graph showing the effect of 0.5 μM LZU-001 intervention on the migration ability of wild-type and knocked-down umuc3 cells; Figure 2 J is a graph showing the effect of 0.5 μM LZU-001 intervention on the transwell invasion ability of wild-type and knockdown T24 cells; Figure 2 K is a graph showing the effect of 0.5 μM LZU-001 intervention on the transwell invasion ability of wild-type and knockdown umuc3 cells.

[0020] Figure 3 A is a graph showing the effect of 0.5 μM LZU-001 intervention on apoptosis of wild-type and knockdown T24 and umuc3 cells; Figure 3 B is the sub-localization of LZU-001 in cells and the co-localization with lysosomes during 0-24h intervention; Figure 3C is a graph showing the proportion of LZU-001 internalized after binding to FGFR3 as detected by flow cytometry; Figure 3 D is to determine the content of monomeric DXD released from LZU-F001 added to human, mouse, and fetal bovine serum at 37°C for 0-96h based on the optimal absorption spectrum and standard curve of DXD determined by UV-visible spectrophotometer, and calculate the proportion of undecomposed LZU-F001; Figure 3 E is a graph showing the effects of different concentrations of LZU-F001 on red blood cells in an in vitro hemolysis experiment; Figure 3 F is the results of the culture intervention model and detection of the in vitro bystander effect of LZU-F001.

[0021] Figure 4 A is a diagram showing the in vivo bystander effect and calculation results of LZU-F001 observed in nude mouse subcutaneous tumor models constructed with wild-type and knockdown umuc3 cells; Figure 4 B is the anti-tumor effect of LZU-F001 in the nude mouse subcutaneous tumor model constructed with wild-type umuc3 cells and the comparison with GC regimen, naked antibody and DXD;

[0022] Figure 4 C is a graph showing the anti-tumor effect of LZU-F001 in the GC+tislelizumab-ineffective PDX1 model reconstituted with hPBMC; Figure 4 D is a graph showing the anti-tumor effect of LZU-F001 in the GC-ineffective PDX2 model; Figure 4 E is a graph showing the protection of mouse survival by LZU-F001 in the highly lethal PDX3 model with GC null; Figure 4 F is the expression diagram reflected by the IHC results of FGFR3 in three PDX tumors. The black arrows indicate the timing and frequency of drug administration.

[0023] Figure 5 It is the structural diagram of ADC. DETAILED DESCRIPTION

[0024] In order to make the contents of the present invention more clearly understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0025] like Figure 1-5 As shown in the figure, the preparation and application of antibody-drug conjugate LZU-F001, taking bladder cancer as an example, the preparation method of ADC is as follows:

[0026] A 30-50 times molar equivalent TCEP solution (TCEP aqueous solution) is used as a reducing agent, and the mixture is reacted at 37° C. for 2 hours to reduce the humanized monoclonal antibody LY3076226. GGFG-DXD is mixed with the reduced humanized monoclonal antibody LY3076226, and the mixture is reacted at 0-37° C. for 30-120 minutes to obtain the ADC. The specific steps are as follows: coupling: add 15-fold molar equivalent of LP (GGFG-DXD) (10 mM dissolved in DMSO) to the reduced antibody solution, and add DMSO to a final DMSO concentration of 15% (V / V); purification: use a 30Kd ultrafiltration tube to remove excess small molecules and replace the coupled antibody solution to 100 mM Pro; quality inspection: use UV-Vis to detect ADC concentration; HIC to detect DAR (Drug-to-Antibody Ratio); HPLC-SEC to detect ADC purity; end point colorimetric method to detect endotoxin; ELISA to determine the binding ability to the target FGFR3.

[0027] Activity determination under in vitro conditions: The therapeutic activity was evaluated in bladder cancer T24 and UMUC-3 cell lines and organoid tumors derived from clinical patients. The inhibition curve and IC50 were determined by CCK-8 experiment. A comprehensive evaluation was performed through clone formation experiment, scratch experiment, transwell invasion experiment, flow cytometry to determine apoptosis, etc., and the relationship between efficacy and target was evaluated using pre-constructed and flow cytometry-verified FGFR3 knockdown cells.

[0028] Results: In vitro experiments were performed to evaluate the inhibitory ability of LZU-F001 in T24 and umuc3 cell lines. In both cell lines, LZU-F001 showed an IC50 of 1-10 μM. Figure 2 A, B). To further explore whether the inhibitory ability depends on the strength of target expression, two FGFR3 knockdown sequences (sh2 and sh3) were selected in T24 and umuc3 cells to knock down the FGFR3 protein in both cells. Figure 2 The flow cytometry results in C show the expression of FGFR3 in each cell type and the efficiency of knockdown. After the knockdown cells were treated with different concentrations of LZU-F001, the IC50 increased significantly ( Figure 2 A, B), this phenomenon indicates that ADC drugs rely on the expression of FGFR3 to recognize and kill cells. The results of the clone formation experiment also confirmed the above viewpoint. The use of 0.5μM concentration of LZU-F001 can produce significant clone formation inhibition on T24 WT and umuc3 WT, but has no inhibitory ability on knockdown cells ( Figure 2 D, E). After 72h of intervention with WT T24 and umuc3, the cells will die completely ( Figure 2 F).

[0029] LZU-F001 also produced significant pharmacological effects in rapidly established FGFR3-positive bladder cancer organoids. After one week of intervention at 0.5 μM, it was observed that the organoids were obviously fragmented and dead and no longer had a 3D shape ( Figure 2 G). The results of the cell scratch assay showed that ( Figure 2 H, I), 0.5 μM LZU-F001 intervention for 24 h can make wild-type T24 and umuc3 cells unable to migrate, while knockdown cells can retain most of their migration ability. Similarly, in the Transwell invasion assay ( Figure 2 J, K), 0.5 μM LZU-F001 can prevent wild-type T24 and umuc3 cells from decomposing matrix gel and shuttling, while knockdown cells can shuttle normally to the bottom of the Transwell chamber. In summary, LZU-F001 can significantly inhibit the migration and invasion of BCa cells, and this inhibitory ability is also dependent on target expression. Figure 3 As shown in A, after 24 hours of treatment with 0.5 μM LZU-F001 on T24 and umuc3 cells, the apoptosis level of wild-type cells increased significantly (early apoptosis: Annexin V+, PI-; late apoptosis or necrosis: Annexin V+, PI+), while the number of apoptotic cells in the knockdown cell line increased only slightly. This experimental result shows that LZU-F001 is dependent on the expression of FGFR3 to kill cells and induce apoptosis in vitro. It can be inferred that the process of LZU-F001 binding to the FGFR3 target on the cell surface and then being internalized into the cell and releasing toxic payload to produce killing may be its main mechanism of action.

[0030] Evaluation of the mechanism of action and stability of LZU-F001: The mechanism of action of LZU-F001 was determined by confocal imaging and flow cytometry, and the stability was evaluated by UV-vis and hemolysis experiments. The bystander effect was evaluated by in vitro co-culture and nude mouse subcutaneous tumor experiments.

[0031] Results: In order to clarify the process of LZU-F001 binding and entering cells, cy5-labeled LZU-F001 was incubated with T24 cells for 0, 0.5, 4, and 24 hours, and the nucleus (DAPI) and lysosome (Lyso-Tracker) were stained and localized. Figure 3As shown in B, LZU-F001 did not bind to the cells at 0h. After 0.5h, it was mainly bound and distributed on the cell membrane and co-localized with some lysosomes. After 4h, a large amount of LZU-F001 entered the cytoplasm and was closely localized with the lysosomes. After 24h, a small amount of LZU-F001 still entered the cells. The results of this experiment confirmed from one side that LZU-F001 binds to FGFR3 on the cell membrane to produce an antigen-antibody complex and is internalized into the cytoplasm. After being degraded by lysosomes, the toxic load is released to cause cell death. Flow cytometry was used to detect the number of FGFR3 receptors on the cell membrane (without the cell membrane permeabilization step) after co-incubation of FGFR3 with LZU-F0010.5μM and T24 and umuc3 cells for 0h, 0.5h, 1h, and 2h. The relative amount of FGFR3 protein internalized from the membrane into the cytoplasm was calculated by measuring the difference in fluorescence intensity between cells incubated on ice and cells incubated at 37°C for a certain period of time. Figure 3 The average fluorescence intensity of FGFR3 on the membrane of the two cells shown in C decreased significantly, and the endocytosis gradually increased with the extension of time. After 2 hours of intervention, the endocytosis ratio exceeded 30%. This experimental result shows that FGFR3, as a membrane receptor with good endocytosis ability, is a high-quality ADC target option. On the other hand, it shows that LZU-F001 can form a complex by targeting FGFR3 and being endocytosed into cells to exert its efficacy.

[0032] The stability of LZU-F001 in serum in vivo was simulated in vitro using human, bovine, and mouse sera involved in our study. LZU-F001 0.5 μM was added to each serum and placed at 37°C for 0 h, 4 h, 8 h, 24 h, 48 h, 72 h, and 96 h. According to the pre-determined UV optimal absorption wavelength of the released monomer DXD ( Figure 3 D) and the standard curve, the content of DXD monomer in the serum after different time was determined by UV-vis and the proportion of remaining LZU-F001 was calculated. Figure 3 The calculation results of D show that LZU-F001 maintained good serum stability during the observed period. At least 80% of the undecomposed LZU-F001 was retained in all types of serum after 96 hours.

[0033] At the same time, hemolysis experiments were performed to evaluate the effect of LZU-F001 on red blood cells in the blood to determine whether it could produce a fatal hemolytic reaction. Figure 3 The comparison of the effects of PBS and H2O on red blood cells shown in E shows that different concentrations of LZU-F001 will not cause obvious hemolysis.

[0034] The bystander effect allows ADCs to exert additional killing power on cells that weakly express the target through cells that strongly express the target. Figure 3 The co-culture model shown in F evaluates whether LZU-F001 has a significant bystander effect. In the experiment, the specific knockdown cells planted in the lower chamber were used as the detection target, and LZU-F0010.5μM or LZU-F0010.5μM + WT cells corresponding to the lower chamber cells were added to the upper chamber. After co-culture, the difference between the CCK-8 detection values ​​of the two groups of knockdown cells in the lower chamber was the additional killing caused by the bystander effect. Figure 3 The calculation results of F show that LZU-F001 has a significant bystander effect.

[0035] Then, the bystander effect was verified in the nude mouse subcutaneous tumor model to confirm whether there is still a significant bystander effect in the in vivo model. UMUC3 WT, UMUC31 / 2WT+1 / 2sh3, and UMUC3 sh3 were implanted subcutaneously in 3 groups of nude mice from the same batch, with 106 cells implanted in each mouse. When the subcutaneous tumor was about 100mm3 in size, LZU-F0015mg / kg was given once. By observing the changes in tumors in different groups of mice ( Figure 4 A), we found that the inhibition of tumors by LZU-F001 mainly depends on the expression of the overall FGFR3 target in the tumor. In addition, through the calculation of tumor volume, we found that the degree of tumor inhibition in the umuc31 / 2WT+1 / 2sh3 group was significantly higher than that in the 1:1 cell implantation ratio. Based on the data, it is speculated that in the in vivo tumor model, LZU-F001 still has a significant bystander effect and can cause additional killing of surrounding low-expressing cells through cells with high target expression.

[0036] In vivo tumor inhibition assay: The in vivo efficacy was verified using the UMUC3 cell nude mouse subcutaneous tumor model and multiple patient-derived PDX tumor models.

[0037] Results: In order to determine whether LZU-F001 has significant tumor inhibition or safety advantages compared with the GC (gemcitabine + cisplatin) regimen and its own components, efficacy and safety tests were performed in the umuc3 WT 106 / nude mouse subcutaneous tumor model. Figure 4 The results shown in B are the gross images of tumor size, estimated tumor volume, and changes in mouse weight after a dose of the corresponding drug was given after the tumor of the same batch grew to about 100mm3. According to the results shown in the figure, LZU-F001 has the strongest tumor inhibition ability and has no effect on the weight of mice; naked antibody has no obvious tumor inhibition ability; GC regimen and DXD have moderate tumor inhibition ability, but the GC regimen has a significant effect on the weight of mice after administration.

[0038] The effects of LZU-F001 in multiple PDX models are as follows:

[0039] PDX1: While using this patient-derived tumor to mimic clinical tumors, an hPBMC model and tislelizumab dosing regimen were incorporated into this experiment. Figure 4 The experimental results shown in C show that LZU-F001 still has a considerable tumor inhibitory effect in this case of GC+tislelizumab clinically ineffective tumor. LZU-F001 failed to produce the effect of immune ignition and sensitization to tislelizumab in this experiment.

[0040] PDX2: Figure 4 The experimental results shown in D show that LZU-F001 also has a significant tumor inhibitory effect in this case of bladder cancer where GC is clinically ineffective.

[0041] PDX3: This specimen is also from bladder cancer that has not responded to clinical treatment with the GC regimen. Figure 4 The survival data shown in E show that LZU-F001 can significantly increase the survival of mice bearing this PDX tumor (p=0.0206).

[0042] The present invention and its embodiments are described above, which is not restrictive. The drawings are only one of the embodiments of the present invention. In short, if ordinary technicians in the field are inspired by it and design structural methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. Preparation of antibody-drug conjugate LZU-F001, characterized in that: The method for preparing the same comprises the following steps: Step 1: using TCEP solution as a reducing agent, reacting at 37° C. for 2 h to reduce the humanized monoclonal antibody LY3076226; Step 2: GGFG-DXD is mixed with the reduced humanized monoclonal antibody LY3076226, and reacted at 0-37° C. for 30-120 min to obtain the ADC.

2. The preparation of the antibody-drug conjugate LZU-F001 according to claim 1, characterized in that: The molar equivalent of the TCEP solution is 30-50 times that of the humanized monoclonal antibody LY3076226.

3. The preparation of the antibody-drug conjugate LZU-F001 according to claim 1, characterized in that: The step 2 further comprises adding 15 times the molar equivalent of LP (GGFG-DXD) (10 mM dissolved in DMSO) to the reduced antibody solution, and adding DMSO to a final DMSO concentration of 15% (V / V); using a 30Kd ultrafiltration tube to remove excess small molecules and replacing the coupled antibody solution to 100 mM Pro; Use UV-Vis to detect ADC concentration; HIC to detect DAR (Drug-to-Antibody Ratio); HPLC-SEC to detect ADC purity; endpoint colorimetric method to detect endotoxin; ELISA to determine the binding ability to the target FGFR3.

4. The use of the antibody-drug conjugate LZU-F001 according to claims 1-3, characterized in that: The method comprises an activity assay under in vitro conditions, wherein the activity assay under in vitro conditions comprises: evaluating the therapeutic activity in bladder cancer T24 and UMUC-3 cell lines and organoid tumors derived from clinical patients, determining the inhibition curve and IC50 by means of a CCK-8 experiment, performing a comprehensive evaluation by means of a clone formation experiment, a scratch experiment, a transwell invasion experiment, and apoptosis determination by flow cytometry, and evaluating the relationship between the efficacy and the target by using FGFR3 knockdown cells pre-constructed and verified by flow cytometry.

5. The use of the antibody-drug conjugate LZU-F001 according to claim 4, characterized in that: The method also includes an evaluation of the mechanism of action and stability of LZU-F001, which includes the following steps: determining the mechanism of action of LZU-F001 through experiments such as confocal imaging and flow cytometry, evaluating stability through UV-vis and hemolysis experiments, and evaluating the bystander effect using in vitro co-culture and nude mouse subcutaneous tumor experiments.

6. The use of the antibody-drug conjugate LZU-F001 according to claim 5, characterized in that: Also included is an in vivo tumor inhibition assay that uses a umuc3 cell nude mouse subcutaneous tumor model and multiple patient-derived PDX tumor models to validate the in vivo efficacy.

7. The use of the antibody-drug conjugate LZU-F001 according to claim 6, characterized in that: Also included is the effect of LZU-F001 in PDX models.

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