CN118105511A - Antibody coupling drug and preparation method and application thereof - Google Patents

Antibody coupling drug and preparation method and application thereof Download PDF

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CN118105511A
CN118105511A CN202310685347.3A CN202310685347A CN118105511A CN 118105511 A CN118105511 A CN 118105511A CN 202310685347 A CN202310685347 A CN 202310685347A CN 118105511 A CN118105511 A CN 118105511A
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antibody
drug
humanized monoclonal
monoclonal antibody
tumor
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潘利强
宁江涛
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Zhejiang University ZJU
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Abstract

The invention discloses an antibody coupling drug, a preparation method and application thereof, and relates to the technical field of biological pharmacy. The novel immune checkpoint-based multifunctional antibody coupling drug has the beneficial effects that the novel immune checkpoint-based multifunctional antibody coupling drug is further combined with the small molecular drug on the basis of improving the anti-tumor activity of an immune checkpoint inhibitor, and can effectively reduce the toxic and side effects caused by systemic administration of the chemotherapy small molecular drug. In vitro experiments show that the antibody-coupled drug provided by the invention has good killing effect on various CD24 positive cell lines. Further, compared with the naked antibody, the antibody-coupled drug shows better in-vivo anti-tumor activity, and effectively reduces the possibility of tumor recurrence.

Description

Antibody coupling drug and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to an antibody coupling drug, a preparation method and application thereof.
Background
The concept of antibody conjugated drugs (ADC) has been proposed since the beginning of the 20 th century, and ADC drug development has been mature over the course of development and precipitation. By 2022, 14 drugs are marketed in batches worldwide, involving 11 targets, and indications are contemplated to encompass solid tumors and hematological tumors; there are more than 400 drugs under investigation, of which more than 200 enter clinical stages, nearly 60 candidate products enter clinical stages domestically and most of the projects are in preclinical or early trial stages. However, from the point of view of the target, although there is a trend of diversification development, development heat is still focused on mature targets such as HER2, TROP2, claudin18.2, CD19, etc., which have high tumor specificity and have been sufficiently verified. Along with the continuous acceleration of the development of the domestic ADC drugs, the design and development of the ADC drugs from the viewpoint of target selection and the deep exploration of a brand new ADC drug activity mechanism are particularly important in the face of increasingly homogenization competition.
CD24, cluster of differentiation 24 (Cluster of differentiation 24), a small, highly glycosylated cell adhesion protein, also known as Heat-stable antigen (HSA), is highly expressed in a variety of tumor cells including ovarian cancer, breast cancer, cervical cancer, endometrial cancer, acute Lymphoblastic Leukemia (ALL), cholangiocarcinoma, bladder cancer, pancreatic cancer, gastric adenocarcinoma, and glioblastoma. And it has been reported that the degree of up-regulation of CD24 expression is highest in Triple Negative Breast Cancer (TNBC) and ovarian cancer patients. The high selectivity and specificity of CD24 expression (high expression of tumor tissue and low expression of normal tissue) enable the CD24 expression to serve as a better selection target point of ADC, and toxic small molecules are accurately brought to tumor parts so as to kill tumor cells. And the ADC design based on the target point is expected to realize broad-spectrum anti-tumor effect due to the high expression characteristics of the target point in various tumor cells.
CD24 and its ligand Siglec-10 are novel innate immune checkpoints. Siglec-10, a member of the sialic acid binding immunoglobulin-like lectin (Siglecs) family, is an inhibitory receptor that is widely expressed in immune cells such as macrophages, B cells, NK cells and activated T cells. Expression of Siglec-10 can inhibit T cell activation, BCR and NK cell receptor mediated signal transduction on B cells and NK cells. Siglec-10 expressed on the surface of macrophages can be bound by CD24 highly expressed on the surface of tumor cells, and generate a signal of 'do not eat me' through interaction. The inhibitory signal inhibits phagocytosis of cancer cells by macrophages, thereby allowing tumor cells to evade immune surveillance by macrophages. And it has been reported that blocking CD24-Siglec 10 interaction by antibodies causes reduced tumor growth via macrophages and prolongs the survival of ovarian and breast cancer tumor-bearing mice.
Since more and more researches show that CD24-Siglec 10 is a novel innate immune checkpoint, monoclonal antibodies, bispecific antibodies, CAT-T cell therapies and the like based on the target point in the global range are also developed gradually, and are in a preliminary stage in general. And both CD24 and CD47 are macrophage-related immune checkpoints, and compared with the side effect of red blood cell killing caused by a treatment strategy based on a CD47 target, the CD24 has proved that the target has better characteristics in a certain degree of low expression of normal tissues. Whether the design of the ADC drugs can be carried out aiming at immune checkpoints and the mechanism by which the ADC drugs can exert the anti-tumor curative effect are the problems to be solved at present. Meanwhile, through the deep research of the scientific problem, innovative theoretical basis and practical basis are expected to be provided for the design and development of the ADC drugs of the immune checkpoints.
In summary, CD24 is a vital innate immune checkpoint in anti-tumor immunity, and the highly expressed properties of various tumor cells indicate that CD24 is an excellent target for ADC drug development. While development for CD24 is still in an early stage. Therefore, the design of a novel broad-spectrum anti-tumor ADC drug based on immune checkpoints is particularly important for deeply exploring the anti-tumor activity mechanism behind the novel broad-spectrum anti-tumor ADC drug and establishing an evaluation system, solving the problem of bundling of ADC drug research and development targets and improving original innovation.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an Antibody coupling Drug (ADC) prepared by coupling an Antibody with macrophage immune checkpoint inhibitor function with small molecule toxin. By utilizing the high affinity of the antibody to CD24 expressed on the surface of tumor cells, the antibody can block the 'do-nothing' signal formed by CD24 and ligand Sigelc thereof, play the role of an immune checkpoint inhibitor and simultaneously combine with the effect of small molecule toxin to enhance the therapeutic effect of the antibody drug aiming at CD 24.
Therefore, the invention provides a design concept of the antibody coupling drug. Specifically, an antibody with an immune checkpoint inhibitor function is covalently coupled with a small molecule toxin to design a novel antibody coupled drug, wherein the antibody and the drug SN38 coupled with the antibody are conjugates with a general formula Ab- (L-U) n, and Ab represents a humanized monoclonal antibody of anti-CD 24 or a functional fragment thereof;
l represents a coupling linker;
U represents a therapeutic agent selected from a cytotoxic drug, an immunopotentiator or a radioisotope, and n is an integer of 1 to 8. When n is plural, L-U is not linked in a chain but is linked at a different position of Ab.
Preferably, the therapeutic agent is a cytotoxic drug; the cytotoxic drug is topoisomerase I inhibitor irinotecan or its derivative.
According to an embodiment of the invention, the humanized monoclonal antibody or functional fragment thereof against CD24 is humanized antibody hAb-L3H4 which has high affinity for the CD24 target as obtained earlier by the applicant. Comprises a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown as SEQ ID NO.5, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6.
In some embodiments, the small molecule drug is coupled to the antibody or functional fragment thereof via a linker. The coupling linkers used in the present invention may be attached to the antibodies by any means known in the art, preferably by sulfhydryl and/or amino groups. In a particularly preferred embodiment, the antibodies of the invention are linked to the linker through a thiol group. The linker used in the present invention may be a cleavable linker (i.e., a linker that can break in an in vivo environment) or a non-cleavable linker. In some embodiments, the linker of the invention is selected from cleavable linkers, preferably from peptide, hydrazone and disulfide linkers, such as maleimidoacetyl-valine-citrulline-p-aminobenzyloxycarbonyl (hereinafter abbreviated Mc-VC-PAB or VC, i.e. maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl). In other embodiments, the linker of the invention is selected from non-cleavable linkers, such as maleimidoacetyl (hereinafter abbreviated as Mc, maleimidocaproyl). In other embodiments, the linker may also be selected from those listed in table 1 below.
TABLE 1
According to an embodiment of the invention, the therapeutic agent selected is SN38, i.e. 7-ethyl-10-hydroxycamptothecin, an active metabolite of the topoisomerase I inhibitor irinotecan. The drug SN38 is coupled to the cysteine residue position of the antibody after the interchain disulfide bond is opened via a coupling linker. The coupling joint is Mc-VC-PAB, wherein one end of the PAB is coupled with the drug SN38, and one end of the Mc is coupled with the antibody. In the embodiment of the invention, the toxic small molecule SN38 containing a cleavable linker (Mc-VC-PAB) is an active metabolite of topoisomerase I inhibitor irinotecan, namely small molecule toxin Mc-VC-PAB-SN38, which is purchased from MCE company, and the chemical structural formula of the small molecule toxin is shown in figure 3, and the maleimide group and the disulfide bond between antibody chains undergo an addition Michael addition reaction to obtain the antibody coupled drug.
According to the embodiment of the invention, the antibody-coupled drug can further kill tumor cells by carrying the toxic small molecules while playing the function of an immune checkpoint inhibitor, so that the treatment effect of the CD24 monoclonal antibody is further enhanced.
In a second aspect of the invention, the invention provides a method of preparing an antibody drug conjugate. The method comprises the following steps:
(1) Preparing and obtaining a humanized monoclonal antibody of the CD 24;
(2) Treating the humanized monoclonal antibody against CD24 with a reducing agent to obtain an inter-chain disulfide bond opened humanized monoclonal antibody against CD 24;
(3) And (3) carrying out Michael addition reaction on the drug SN38 with the coupling joint of Mc-VC-PAB and the humanized monoclonal antibody of the anti-CD 24 with the disulfide bond opened between chains in the step (2) to obtain the antibody coupling drug.
Specifically, the molar ratio of humanized monoclonal antibody against CD24 to reducing agent is 1:50.
In the step (3), the molar ratio of the drug SN38 with the coupling joint of Mc-VC-PAB to the humanized monoclonal antibody of the anti-CD 24 with the disulfide bond between chains opened is 10-15:1.
According to the embodiment of the invention, the preparation method specifically comprises the following steps:
1) And (3) reduction: the concentration of the antibody solution is adjusted to 3mg/mL by using a reaction buffer solution, trichloroethyl phosphate (TCEP) with 50 times of the molar ratio of the antibody is added, and the mixture is reacted at 4 ℃ overnight;
2) Coupling: adjusting the concentration of the antibody solution to 3mg/mL by using a reaction buffer solution, adding a dimethyl sulfoxide solution with a linker drug with the molar ratio of 10-15 times of the antibody, and reacting the solution for 2 hours at room temperature;
3) Purifying: and (3) replacing the reacted solution with PBS (phosphate buffer solution) at 4 ℃ by using a centrifugal ultrafiltration tube, removing residual small molecules in the reaction solution, filtering, sterilizing, sub-packaging and freezing.
In a third aspect of the invention, the invention provides the use of the antibody-conjugated drug in the preparation of an anti-tumour drug. The medicament is for the treatment of CD24 related diseases, preferably tumors. Further preferred, the tumor is breast cancer, colon cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, renal cell carcinoma, cervical cancer, endometrial cancer, cholangiocarcinoma, gastric adenocarcinoma, or glioblastoma.
Compared with the prior art, the invention has the beneficial effects that: the novel immune checkpoint-based multifunctional antibody coupling drug is provided and designed, the beneficial effects brought by the small molecular drug are further combined on the basis of improving the anti-tumor activity of the immune checkpoint inhibitor, and the toxic and side effects brought by systemic administration of the chemotherapy small molecular drug can be effectively reduced. In vitro experiments show that the antibody-coupled drug provided by the invention has good killing effect on various CD24 positive cell lines. Further, compared with the naked antibody, the antibody-coupled drug shows better in-vivo anti-tumor activity, and effectively reduces the possibility of tumor recurrence. Finally, the antibody coupling drug designed by the invention provides innovative theoretical basis and practical basis for the design and development of the immune checkpoint ADC drug.
Drawings
FIG. 1 is a graph showing the result of analyzing the light and heavy chain bands of an anti-CD 24 monoclonal antibody in a reduced state by SDS-PAGE according to an embodiment of the present invention.
FIG. 2 is a schematic illustration of the preparation of an antibody-conjugated drug according to an embodiment of the present invention.
FIG. 3 is a schematic diagram of a toxic small molecule SN38 containing maleimide and cleavable dipeptide linker (Mc-VC-PAB) employed in the preparation of an antibody-conjugated drug according to an embodiment of the present invention.
FIG. 4 is a graph showing the results of SDS-PAGE analysis of the variation of the coupling ratio of small molecules to antibodies at different feed rates according to an embodiment of the present invention.
FIG. 5 is a graph showing the results of detecting changes in affinity between the naked antibody and the ADC before and after coupling using flow cytometry according to an embodiment of the present invention.
FIG. 6 is a graph showing the results of detecting CD24 expression on the surface of tumor cells of different species using flow cytometry according to an embodiment of the present invention.
FIG. 7 is a graph of co-localization results of ADC endocytosis into cells and with lysosomes over time under different temperature conditions using a laser confocal microscope according to an embodiment of the invention; wherein A is at 4deg.C, and B is at 37deg.C.
FIG. 8 is a graph showing the results of evaluating the in vitro killing activity of ADC against different tumor cells using CCK8 assay according to an embodiment of the present invention.
FIG. 9 is a graph of experimental results of evaluating anti-tumor activity in an antibody-conjugated drug and a naked antibody using a C57BL/6 mouse model transplanted with MC38-hCD24 tumor cells according to an embodiment of the present invention; wherein A is the experimental result of the inhibition capacity of the tumor growth; b is a weight data change chart of each group of mice in the experimental process; c is a graph of the increasing trend of the tumor volume of the hAb-L3H4-vcSN administration group; d is a graph showing the increasing trend of tumor volume in the naked anti-hAb-L3H 4 administration group.
Detailed Description
EXAMPLE 1 expression and purification of hAb-L3H4 antibody
The high affinity antibody hAb-L3H4 for recognizing CD24 target in the antibody coupling medicine is obtained through humanized modification of a murine antibody obtained in the early stage of the laboratory. The amino acid sequence of the light chain variable region is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; the constant region of the antibody light chain is derived from a Kappa light chain constant region of human origin, the amino acid sequence of the constant region is shown as SEQ ID NO.3, the constant region of the heavy chain is derived from a heavy chain constant region of human IgG1, and the amino acid sequence of the constant region is shown as SEQ ID NO. 4. And integrating the light and heavy chain variable region sequence with the constant region sequence to obtain the light chain amino acid sequence of the antibody hAb-L3H4 as shown in SEQ ID NO.5 and the heavy chain amino acid sequence as shown in SEQ ID NO. 6.
1) Antibody plasmid construction: the light and heavy chain sequences of the antibody hAb-L3H4 (shown as SEQ ID NO.5 and SEQ ID NO. 6) are codon optimized by a human expression system to form the light and heavy chain nucleotide sequence of the hAb-L3H4, then the nucleotide sequence encoding the light and heavy chain signal peptide of the antibody (shown as SEQ ID NO. 7) is added to the 5 'end of the light and heavy chain nucleotide sequences (shown as SEQ ID NO.8 and SEQ ID NO. 9), and the Kozak sequence (sequence ACCACC) is added to the 5' end of the signal peptide sequence. Finally, ecoR I and Not I restriction sites were added to the 5 'and 3' ends of the resulting sequences, respectively. The resulting sequence was synthesized by Nanjing Jinsri Biotechnology Co., ltd and cloned into pcDNA3.1 (+) vector.
2) The antibody was prepared by transiently transfecting HEK293F cells with the pcDNA3.1 (+) vector carrying the light and heavy chains of the hAb-L3H4 antibody described above. One day before transfection, HEK293F cells were shake-cultured overnight at a cell density of (1-2). Times.10 6/mL,37℃,5％CO2, 220 rpm. On the day of transfection, cell viability was measured and transfection was performed at greater than 95%. The plasmids after mixing the light and heavy chains were transiently co-transfected into HEK293F cells at a mass ratio of PEI: dna=4:1 using the transfection reagent PEI (polyethyleneimine hydrochloride, poly sciences) at a mass ratio of 1:1 for antibody expression. After shaking culture at 220rpm in a shaking table at 37℃in 5% CO 2 for 3-4 days, the culture supernatant was collected by centrifugation at 3000rpm for 20 minutes.
3) And (5) purifying the antibody. The antibody with Fc domain was captured from the expression supernatant using HiTrap protein A affinity column (GE 17040301), equilibrated with phosphate buffer at pH 7.2, the supernatant was passed through the affinity column, eluted with elution buffer (100 mM sodium citrate, pH 3.2), and concentrated in PBS for displacement. Subsequently, protein concentration was measured using Nanodrop ND-1000, and the antibody after PBS buffer replacement was frozen in sub-packs at-80 ℃. The purified antibodies were purified by reducing SDS-PAGE to identify purity as shown in FIG. 1. Two bands are shown, around 25kDa and 50kDa, respectively, the band sizes being in agreement with theory. Finally, the hAb-L3H4 antibody is obtained. (the light chain amino acid sequence is shown as SEQ ID NO.5, and the heavy chain amino acid sequence is shown as SEQ ID NO. 6).
Example 2 hAb-preparation of L3H4-vcSN conjugate
1) The toxic small molecule SN38 containing cleavable linker (Mc-VC-PAB) is an active metabolite of topoisomerase I inhibitor irinotecan, named Mc-VC-PAB-SN38, abbreviated VC-SN38, available from MCE company (full MedChemExpress), cat: HY-131057. The molecular structure is shown in figure 3. The small toxic molecules were dissolved in dimethyl sulfoxide (DMSO) to prepare a 5mM stock solution, which was stored in aliquots at-80 ℃.
2) The antibody solution after expression purification was replaced with PBS buffer, 50-fold molar equivalents of TCEP reducing agent was added and incubated overnight at 4 ℃. Then, the solution was replaced with PBS containing 2mM EDTA at 4℃with an ultrafiltration tube to remove residual TCEP in the solution, thereby finally obtaining an antibody having an open interchain disulfide bond. And finally, adding small molecules vc-SN38 into the reaction liquid according to different molar equivalent ratios (1:2, 1:5, 1:10 and 1:15), and reacting for 2 hours at room temperature to enable the maleimide group and the sulfhydryl group to generate Michael addition reaction. After the reaction is finished, the reaction solution is replaced by PBS at 4 ℃ by a centrifugal ultrafiltration tube, residual small molecules in the reaction solution are removed, or the product is directly purified by affinity chromatography again to obtain hAb-L3H4-vcSN, and the obtained coupling product is sterilized by a filter membrane with the aperture of 0.22 mu m and then stored at-80 ℃ for a long time. A schematic diagram of the preparation of the antibody-conjugated drug in this example is shown in FIG. 2.
The products before and after coupling were analyzed by SDS-PAGE, and as shown in FIG. 4, the protein bands after coupling under the reducing condition were significantly shifted upward as compared with the bands before coupling, and the shift ratio was increased as the molar ratio of the charged small molecules was increased. Indicating that the overall molecular weight did increase compared to the naked antibody, the drug-antibody coupling ratio (DAR value) of the conjugate increased. Specifically, from left to right, lane 1 is a molecular weight Marker, lane 2 is a naked antibody, lane 3 represents that the feeding mole ratio of the antibody to the small molecule is 1:2, lane 4 represents that the feeding mole ratio of the antibody to the small molecule is 1:5, lane 5 represents that the feeding mole ratio of the antibody to the small molecule is 1:10, and lane 6 represents that the feeding amount of the antibody to the small molecule is 1:15.
Example 3 detection of the level of binding of antibody-conjugated drugs to cells Using flow cytometry
The change in the level of binding of the hAb-L3H4-vcSN conjugated drug (hereinafter referred to as ADC) to cells before and after the conjugation drug was evaluated by flow cytometry through a cell binding experiment based on the CD24 + DU-145 cell line. Bare anti-hAb-L3H 4 (hereinafter abbreviated as WT) was used as a control.
CD24 + DU-145 cell density was adjusted to 2X 10 6/mL, 100. Mu.l was taken, washed with pre-chilled PBS, incubated in 100. Mu.l of PBS of the above antibody-conjugated drug (ADC) and naked antibody (WT) at an initial concentration of 20. Mu.g/mL, respectively, for 30 minutes at 4℃and after the incubation, the cells were washed twice with chilled PBS, followed by fluorescent labelling in 200. Mu.l of FITC-labeled goat anti-human IgG secondary antibody (Beyotime). After washing, the cells were resuspended in 200. Mu.L of PBS and the Mean Fluorescence Intensity (MFI) of the cell surface was measured by ACEA NovoCyte TM flow cytometry. Shifts in MFI represent changes in antibody affinity levels before and after coupling.
As shown in fig. 5, the above antibody-conjugated drug (ADC) exhibited a binding capacity to HT29 cells comparable to that of naked antibody (WT).
EXAMPLE 4 detection of expression level of CD24 on tumor cell surface by flow cytometry
The number of target antigens on the surface of tumor cells, i.e. the target expression level, is one of the key influencing factors for the therapeutic effect of ADC drugs. The expression level of CD24 on the surface of different tumor cells was examined by flow cytometry. Specifically, prostate cancer cells (22 RV1, DU-145, PC-3), colorectal cancer cells (HT-29, SW480, SW 620), breast cancer cells (MDA-MB-468, SK-BR-3), ovarian cancer cells SKOV3, B lymphoma cells Ramos were cultured in a cell incubator of 5% CO 2 at 37 ℃. Taking all tumor cells in logarithmic growth phase, adjusting the cell density to 2X 10 6/mL, taking 100 mu L, washing and re-suspending by precooled PBS, respectively incubating for 30 minutes at 4 ℃ in 100 mu L of PBS buffer with the concentration of 20 mu g/mL of naked antibody, setting blank control only with cells without antibody incubation, setting cell control only with fluorescent secondary antibody, and setting 3 parallel controls for each sample. After incubation, the cells were centrifuged at 600 Xg for 5min and each tumor cell was washed 2 times with pre-chilled PBS. Followed by fluorescent labeling at 200. Mu.L of FITC-labeled goat anti-human IgG (L+H) secondary antibody (Beyotime). After incubation for 15 min at 4℃and centrifugation at 600 Xg for 5min, the cells were washed 2 times with pre-chilled PBS. Subsequently, each tumor cell was resuspended in 200 μl of pre-chilled PBS. The average fluorescence intensity (MFI) of the cell surface was then measured by ACEA NovoCyte TM flow cytometry. The relative expression level of CD24 on the surface of each tumor cell is represented by the relative average fluorescence intensity (RELATIVE MFI).
Relative mean fluorescence intensity (RELATIVE MFI) =mean fluorescence intensity of experimental group (MFI)/mean fluorescence intensity of blank group (MFI) ×100%. The data was then statistically analyzed and plotted using GRAPHPAD PRISM software.
The expression level of CD24 on the surface of each tumor cell is shown in fig. 6.
Example 5 evaluation of ADC endocytic efficiency Using laser confocal microscope
2X 10 6 MDA-MB-468 tumor cells are taken to the bottom of a confocal dish, and after the cells grow stably, the tumor cells are randomly divided into two groups of control at 4 ℃ and 37 ℃. Wherein different time control groups of 0, 4, 6, 12h, etc. are set under each group. The medium in each group of cell culture dishes was discarded. Complete medium containing 20. Mu.g/mL ADC was added simultaneously and incubated at different temperatures for a fixed period of time. After the incubation, the cells were washed with pre-chilled PBS, followed by treatment of the cells with 200. Mu.l of 4% paraformaldehyde, immunofluorescent permeant (Beyotime) at 4℃for 30min. After washing with pre-chilled PBS, the cells were incubated with 200. Mu.l of 1:1000 diluted primary antibody to human lysosomal LAMP (Abcam) for 30min. After washing twice, immunofluorescent staining was performed by incubation for 45 min in 200 μl of FITC-labeled goat anti-human IgG-Fc secondary antibody and Cy 5-labeled goat anti-rabbit IgG-Fc secondary antibody. After PBS washing is finished, dropwise adding DAPI light-shading stained cell nuclei with PBS diluted 1:1000 for 5min on the cells, and washing with PBS for 3 times; then 200. Mu.l of PBS was gently added dropwise to the cell surface at the bottom of the dish. Endocytosis of the ADC was then assessed by observing co-localization of the ADC with the intracellular lysosome in each experimental group using an Olympus FV3000 (OSR) confocal microscope.
DAPI is a nuclear stain (blue); FITC is a fluorescent label (green) on the secondary antibody, indicating the position of ADC; LAMP-1 is a lysosomal associated membrane protein, indicating the location of lysosomes (red). The experimental results are shown in FIG. 7, and it can be seen that the co-localization of ADC and lysosome LAMP-1 is significantly improved along with the prolonged proportion of incubation time in the environment of 37 ℃, which means that ADC is endocytosed by MDA-MB-468 cells and reaches lysosomes along with the prolonged incubation time, and then dipeptide linker (vc linker) is cut in lysosomes, so that active drugs are released and cells are killed; in the environment of 4℃the co-localization of ADC to lysosomes is still less proportional, even if incubated for a longer period. Indicating that endocytosis of the ADC is an energy dependent process.
Example 6 evaluation of in vitro killing Activity of ADC against tumor cells Using CCK8 assay
Four tumor cells expressing CD24 on their surface were selected: breast cancer cell line MDA-MB-468, colorectal cancer cell line HT29, ovarian cancer cell line SK-OV-3 and prostate cancer cell line DU-145 are used as target cells, and the in vitro killing activity of ADC on tumor cells is detected. According to example 4 of the present invention, the number of CD24 expressed on the surface of four tumor cells was sequentially decreased, and the results are shown in fig. 5. Tumor cells to be used were cultured, and after the cells had grown to the logarithmic phase, the cells were collected by centrifugation and added to 96-well cell culture plates at a cell density of 5X 10 4/mL, with 100. Mu.L of cell suspension added to each well. The 96-well culture plate is placed in a cell culture box containing 5% CO 2 at 37 ℃ for culture overnight until cells adhere to the wall and the growth state is good. After the ADC to be tested is subjected to filtration sterilization by a 0.22 mu m filter membrane, the ADC to be tested is subjected to gradient dilution into a series of antibody solutions by using a corresponding complete culture medium for culturing tumor cells, and then the antibody solutions are added into corresponding cell holes according to the dosage of 100 mu L/hole, three auxiliary holes are arranged in parallel at each concentration, wherein the complete culture medium without adding medicines is used as a negative control, the cells and the medicines are not added, and the complete culture medium is only added as a blank control hole.
After the treated 96-well cell culture plates were placed in a 37℃cell incubator containing 5% CI 2 for 72 hours of incubation, the 96-well plates were removed, the medium in the plates was discarded, and medium containing 10% CCK8 reagent (Beyotime) was added at a volume of 100. Mu.L per well and returned to the 37℃cell incubator for waiting for development. After development to the appropriate time, the 96-well plate was placed in an microplate reader to read the OD450nm value, the reading was recorded and the relative cell viability of each well was calculated.
Relative cell viability (%) = (experimental well-blank well)/(negative well-blank well) ×100%.
The data were then fitted using GRAPHPAD PRISM software and IC50 values of the drug were calculated to evaluate the in vitro killing activity of ADC against tumor cells.
The in vitro killing activity of the antibody conjugated drug hAb-L3H4-vsSN to tumor cells is shown in FIG. 8. It can be seen that the ADC has good killing effect on 4 kinds of CD24 positive tumor cells MDA-MB-468, HT29, SK-OV-3 and DU-145, and the IC50 values are 4.823nM,5.078nM,0.919nM and 0.599nM respectively.
Example 7 evaluation of in vivo anti-tumor Activity of ADC and naked anti-WT Using colorectal cancer isotype mouse model
MC38-hCD24 cells were cultured at 37℃in 5% CO 2 in DMEM containing 10% fetal bovine serum and 2. Mu.g/mL purmycin. MC38-hCD24 tumor cells in the logarithmic growth phase were collected, resuspended in pre-chilled PBS, and counted. The cell concentration was adjusted to 1X 10 7/mL. The cell suspension was inoculated subcutaneously into the right armpit of 9C 57BL/6 cells of 6 to 8 weeks of age using a 1mL syringe, each inoculated with 100. Mu.L, about 1X 10 6 tumor cells. When the average tumor volume reached about 80mm 3, animals were randomly divided into 3 groups of 3 animals per group according to tumor volume. PBS blank, naked antibody (hAb-L3H 4) and ADC (hAb-L3H 4-vsSN) were each 10mg/mL. Adjusting the concentration of the drug, and respectively carrying out intravenous injection on the tail of the mice in the naked antibody group and the ADC group according to the dosage of the drug of 10 mg/kg; PBS control mice were injected tail-intravenously with 100uL PBS solution. Experimental time the vaccinated tumor was set to D0. Once every two days, twice in total. The body weight and tumor size of the mice were measured every 2 to 3 days. When the average tumor volume of the PBS group reached 1500mm 3, the control group stopped observation, and the rest of the administration groups continued observation. The specific calculation formula of the tumor volume is as follows: v= (l×w 2)/2. Where V is tumor volume (mm 3), L is measured tumor length (mm), and W is measured tumor width (mm). The tumor volume of the PBS group mice at D30 exceeded the preset value of 1500mm 3, so D0-D30 data were taken for mapping and analysis.
As shown in FIG. 9A, the naked antibody (hAb-L3H 4) and the antibody conjugated drug (hAb-L3H 4-vcSN) show better tumor growth inhibition capability in a MC38-hCD24 cell tumor model of a C57BL/6 mouse allograft model. Specifically, at the administration dose of 10mg/kg, complete clearance of 2 tumors occurred in 3 mice in the administration group of the antibody-conjugated drug hAb-L3H4-vcSN, and no recurrence occurred; the trend of tumor volume increase was significantly inhibited in the remaining 1 mouse, as shown in fig. 9C. In the case of the administration dose of 10mg/kg, 1 tumor in 3 mice in the nude anti-hAb-L3H 4 administration group was completely cleared, and no recurrence occurred; the remaining 2 mice showed a significant trend of increase in tumor volume after D20 after temporary tumor growth inhibition, as shown in fig. 9D. Fig. 9B is data of the weights of mice in each group during the course of the experiment, and it was observed that there was no significant decrease in the weights of mice in each group during the whole experimental period, and the overall weights showed a slight increase, indicating that neither ADC nor WT group had significant side effects on mice during the experimental period.
The above in vivo activity results show that the antibody-conjugated drug exhibits superior in vivo antitumor activity compared to the naked antibody. The ADC medicine designed by the application has the activity brought by naked anti-blocking CD24-Siglec 10 'eating me' signal, and the toxicity small molecule carried by the ADC medicine can clear the rest tumor cells after monoclonal antibody treatment, especially some tumor stem cells, thereby reducing the possibility of tumor recurrence and achieving better treatment effect.

Claims (10)

1. An antibody-conjugated drug, having a conjugate of general formula Ab- (L-U) n, characterized in that Ab represents an anti-CD 24 humanized monoclonal antibody or a functional fragment thereof, comprising a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown in SEQ ID No.5 and the amino acid sequence of the heavy chain is shown in SEQ ID No. 6;
l represents a coupling linker;
U represents a therapeutic agent selected from a cytotoxic drug, an immunopotentiator or a radioisotope, and n is an integer of 1 to 8.
2. The antibody conjugated drug of claim 1, wherein the therapeutic agent is a cytotoxic drug; the cytotoxic drug is topoisomerase I inhibitor irinotecan or its derivative.
3. The antibody conjugated drug of claim 2, wherein the therapeutic agent is SN38; the SN38 is coupled to the cysteine residue position of the anti-CD 24 humanized monoclonal antibody or functional fragment thereof after the interchain disulfide bond is opened via a coupling linker.
4. The antibody conjugated drug of claim 3, wherein the conjugated linker is Mc-VC-PAB or Mc; when the coupling linker is Mc-VC-PAB, wherein one end of the PAB is coupled to SN38, one end of the Mc is coupled to the humanized monoclonal antibody against CD24 or a functional fragment thereof.
5. The method for preparing the antibody-conjugated drug according to any one of claims 1 to 4, comprising the steps of:
(1) Preparing and obtaining a humanized monoclonal antibody of the CD 24;
(2) Treating the humanized monoclonal antibody against CD24 with a reducing agent to obtain an inter-chain disulfide bond opened humanized monoclonal antibody against CD 24;
(3) And (3) carrying out Michael addition reaction on the SN38 with the coupling joint of Mc-VC-PAB and the humanized monoclonal antibody of the anti-CD 24 with the disulfide bond opened between chains in the step (2) to obtain the antibody coupling drug.
6. The method of claim 5, wherein in step (2), the reducing agent is trichloroethyl phosphate.
7. The method of claim 6, wherein the molar ratio of humanized monoclonal antibody against CD24 to reducing agent is 1:50.
8. The method of claim 5, wherein in the step (3), the molar ratio of SN38 having a coupling linker of Mc-VC-PAB to the humanized monoclonal antibody against CD24 having an open interchain disulfide bond is 10-15:1.
9. Use of an antibody-conjugated drug according to any one of claims 1 to 4 for the preparation of an anti-tumor drug.
10. The use of claim 9, wherein the tumor type is selected from the group consisting of CD24 positive solid tumors, which CD24 positive solid tumors are breast, colon, ovarian, prostate, lung, pancreatic, renal cell, cervical, endometrial, bile duct, gastric adenocarcinoma, or glioblastoma.
CN202310685347.3A 2023-06-09 2023-06-09 Antibody coupling drug and preparation method and application thereof Pending CN118105511A (en)

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