An antibody drug conjugate targeting cd46 and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an antibody conjugated drug targeting CD46 and a preparation method and application thereof. The application is based on a single-domain antibody 31-17-1-A12 with cross binding of human and cynomolgus monkey CD46, and a high-activity cytotoxic load is connected to a cysteine position of a human immunoglobulin G1 crystalline fragment (human IgG1 Fc) connected to a C terminal of the single-domain antibody 31-17-1-A12 through site-specific conjugation technology, so as to finally prepare a novel antibody conjugated drug (Antibody-Drug Conjugate, ADC). The ADC prepared by the application has specificity of resisting human and cynomolgus monkey CD46, a drug-antibody ratio is about 4, and in the detection of killing CD46 high-expression tumor cells in vitro, the killing efficiency is significantly better than that of naked antibody without conjugated cytotoxic load, and good tumor targeted treatment potential is exhibited.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibody-drug conjugate targeting CD46, its preparation method, and its application. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a breakthrough therapy in the field of cancer treatment in recent years. These drugs combine the precise targeting ability of monoclonal antibodies with the killing effect of highly effective cytotoxic drugs to achieve targeted elimination of tumor cells, hence the nickname "biological missiles." ADCs possess advantages such as precise targeting, low systemic toxicity, ability to overcome some drug resistance mechanisms, and good potential for combination therapy, and have broad prospects for research and application. ADCs mainly consist of three core structural parts: first, the antibody, which acts as a navigation structure, specifically recognizing and binding to tumor cell surface antigens to guide the drug to precisely target the lesion; second, the linker, which acts as a bridge between the antibody and the toxic payload, ensuring the drug's stable presence in the bloodstream and allowing it to lyse and release toxins under specific conditions within tumor cells; and third, the cytotoxic payload (toxin), which is the active warhead that exerts its anti-tumor effect, efficiently killing cancer cells after entering the tumor cell.

[0003] In terms of mechanism of action, ADCs represent an optimized combination of targeted therapy and chemotherapy: compared to traditional chemotherapy, they offer higher selectivity and lower systemic toxicity; compared to single-target therapy, their potent toxic payloads deliver stronger killing power and can circumvent some drug resistance issues. The killing process of ADCs is a precise targeted delivery procedure: first, the drug specifically recognizes and binds to the target antigen highly expressed on the surface of tumor cells via antibodies; subsequently, the formed complex enters the cell via endocytosis, forming an endosome; after the endosome matures into a lysosome, under the action of an acidic environment and related enzymes, the linker breaks, releasing a highly active toxin; the toxin diffuses into the cytoplasm or nucleus, inducing tumor cell apoptosis by damaging DNA and inhibiting microtubule polymerization.

[0004] As of 2025, more than 15 ADC drugs have been approved for marketing globally, covering indications for solid tumors such as breast cancer, lung cancer, gastric cancer, and lymphoma, as well as hematological malignancies. However, no ADC drugs have yet been approved for colorectal cancer, and this area remains a key area for future research and development. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody-drug conjugate targeting CD46, its preparation method, and its application. The antibody-drug conjugate targeting CD46 described in this invention has a drug-to-antibody ratio (DAR) > 3.5, and exhibits significantly superior killing activity against CD46-overexpressing tumor cells in vitro compared to naked antibody (31-17-1-A12 purified antibody linked to human IgG1 Fc), and can be used for targeted tumor therapy.

[0006] This invention provides an antibody-drug conjugate targeting CD46, comprising a single-domain antibody 31-17-1-A12, a human IgG1 Fc, and a cytotoxic payload, wherein the human IgG1 Fc is linked to the C-terminus of the single-domain antibody 31-17-1-A12, and the cytotoxic payload is conjugated to the single-domain antibody 31-17-1-A12 via a cysteine ​​residue on the human IgG1 Fc; the amino acid sequence of the single-domain antibody 31-17-1-A12 is shown in SEQ ID NO:1. The amino acid sequence of the human IgG1 Fc is shown in SEQ ID NO:2; The cytotoxic load conjugated with human IgG1 Fc cysteine ​​residues are located at positions 6 and 9, starting from the N-terminus of the amino acid sequence shown in SEQ ID NO:2; The cytotoxic loads include Deruxtecan (Dxd) or Monomethyl auristatin E (MMAE).

[0007] Preferably, the DAR in the antibody-drug conjugate targeting CD46 is >3.5.

[0008] The present invention also provides a method for preparing the antibody-drug conjugate targeting CD46 as described in the above technical solution, comprising the following steps: conjugating a cytotoxic load with a purified 31-17-1-A12 antibody via a Michael addition reaction to obtain an antibody-drug conjugate targeting CD46; wherein the purified 31-17-1-A12 antibody is formed by linking human IgG1 Fc to the C-terminus of the single-domain antibody 31-17-1-A12, and the cytotoxic load is conjugated with a cysteine ​​residue on the human IgG1 Fc.

[0009] Preferably, the method includes the following steps: mixing the purified 31-17-1-A12 antibody with tris(2-carboxyethyl)phosphine hydrochloride and ethylenediaminetetraacetic acid to carry out a reduction reaction to obtain a reduction reaction system; A cytotoxic load was added to the reduction reaction system to carry out a coupling reaction, resulting in an antibody-drug conjugate targeting CD46.

[0010] Preferably, each 1 mg of the antibody-drug conjugate contains 0.07 mg of cytotoxic load.

[0011] Preferably, the coupling reaction is carried out at a rotation speed of 200 rpm, a temperature of 25°C, and a time of 5 h.

[0012] Preferably, the mass ratio of the 31-17-1-A12 purified antibody to tris(2-carboxyethyl)phosphine hydrochloride is 1 mg: 22 μg; and the volume ratio of tris(2-carboxyethyl)phosphine hydrochloride to ethylenediaminetetraacetic acid is 4:25.

[0013] Preferably, the oscillation speed of the reduction reaction is 200 rpm; the temperature of the reduction reaction is 25°C; and the time of the reduction reaction is 17 h.

[0014] Preferably, the 31-17-1-A12 purified antibody is derived from in vivo expression or in vitro synthesis; the biological materials used for in vivo expression include expression cassettes encoding nucleotides of the 31-17-1-A12 purified antibody, recombinant vectors, recombinant bacteria, or recombinant cells.

[0015] The present invention also provides the application of the antibody-drug conjugate described in the above technical solution or the antibody-drug conjugate prepared by the preparation method described in the above technical solution in the preparation of drugs for the treatment of colorectal cancer.

[0016] Beneficial effects: This invention provides an antibody-drug conjugate (ADC) targeting CD46, its preparation method, and its application. Based on the single-domain antibody 31-17-1-A12, which cross-binds to human and cynomolgus monkey CD46, this invention utilizes site-directed conjugation technology to attach a highly active cytotoxic payload to the cysteine ​​residue of human IgG1 Fc linked to the C-terminus of the single-domain antibody 31-17-1-A12, ultimately creating a novel ADC. The ADC prepared by this invention exhibits specificity against human and cynomolgus monkey CD46, with a DAR > 3.5. In in vitro detection of CD46-overexpressing tumor cells, its killing efficacy is significantly superior to that of the naked antibody without conjugated toxin, demonstrating good potential for targeted tumor therapy. Particularly noteworthy is the natural cross-species reactivity of the single-domain antibody used in this invention. This allows the ADC constructed based on it to be directly applied to preclinical efficacy and safety evaluation models in cynomolgus monkeys, etc., without the need to develop additional species-alternative antibodies, thus significantly simplifying the translation process from candidate drug to clinical research. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 This is a graph showing the protein purity of the purified antibody (naked antibody) 31-17-1-A12 in Example 1; Figure 2 This is a graph showing the HIC-UPLC detection results of the antibody-drug conjugate in Example 2; Figure 3 This is a graph showing the LC-MS detection results of the antibody-drug conjugate in Example 2; Figure 4 This is a diagram showing the killing effect of the antibody-drug conjugate on CD46-positive tumor cells in Example 3. Detailed Implementation

[0019] This invention provides an antibody-drug conjugate targeting CD46, comprising a single-domain antibody 31-17-1-A12, a human IgG1 Fc, and a cytotoxic payload, wherein the human IgG1 Fc is linked to the C-terminus of the single-domain antibody 31-17-1-A12, and the cytotoxic payload is conjugated to the single-domain antibody 31-17-1-A12 via a cysteine ​​residue on the human IgG1 Fc; the amino acid sequence of the single-domain antibody 31-17-1-A12 is shown in SEQ ID NO:1.

[0020] The amino acid sequence shown in SEQ ID NO:1 of this invention is: QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYNIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASSDSCGYYYTAGLNYWGQGTQVTVSS.

[0021] The amino acid sequence of the human IgG1 Fc described in this invention is shown in SEQ ID NO:2, specifically: DKTHT C PP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The cysteine ​​residues of the cytotoxic load conjugated to human IgG1 Fc in this invention are located at positions 6 and 9 from the N-terminus of the amino acid sequence shown in SEQ ID NO:2, i.e., the positions of the bolded italicized amino acids in the amino acid sequence shown in SEQ ID NO:2.

[0022] The cytotoxic load described in this invention can be a DNA damaging agent or a tubulin inhibitor, further comprising a topoisomerase I inhibitor or an olistatin, and further comprising Dxd or MMAE. As one embodiment, the DAR in the CD46-targeting antibody-drug conjugate described in this invention is >3.5.

[0023] The present invention also provides a method for preparing the antibody-drug conjugate targeting CD46 as described in the above technical solution, comprising the following steps: conjugating a cytotoxic load with a purified 31-17-1-A12 antibody via a Michael addition reaction to obtain an antibody-drug conjugate targeting CD46; wherein the purified 31-17-1-A12 antibody is formed by linking human IgG1 Fc to the C-terminus of the single-domain antibody 31-17-1-A12, and the cytotoxic load is conjugated with a cysteine ​​residue on the human IgG1 Fc.

[0024] In one embodiment, the present invention involves a reduction reaction of a mixture of purified 31-17-1-A12 antibody with tris(2-carboxyethyl)phosphine hydrochloride and ethylenediaminetetraacetic acid (EDTA) to obtain a reduction reaction system. In another embodiment, the mass ratio of the purified 31-17-1-A12 antibody to tris(2-carboxyethyl)phosphine hydrochloride is 1 mg:22 μg; the volume ratio of tris(2-carboxyethyl)phosphine hydrochloride to EDTA is 4:25. The reduction reaction is carried out in a constant-temperature metal bath at a shaking speed of 200 rpm; the temperature is 25°C; and the reaction time is 17 h. In another embodiment, the purified 31-17-1-A12 antibody can be derived from in vivo expression or in vitro synthesis; the biological material used for in vivo expression can be an expression cassette encoding the nucleotides of the purified 31-17-1-A12 antibody, a recombinant vector, recombinant bacteria, or recombinant cells; in yet another embodiment, the initial vector of the recombinant vector can be a eukaryotic expression vector, and further, a plasmid vector. The present invention does not impose any particular limitation on the construction method of the biomaterials, and conventional genetic engineering methods in the field can be used.

[0025] After the reduction reaction is completed, the present invention adds a cytotoxic load to the reduction reaction system to carry out a coupling reaction, thereby obtaining an antibody-drug conjugate targeting CD46. As one embodiment, each 1 mg of antibody-drug conjugate contains 0.07 mg of cytotoxic load; the oscillation speed of the coupling reaction is 200 rpm; the temperature of the reduction reaction is 25°C; and the reduction reaction time is 5 h.

[0026] This invention employs a site-specific coupling method using cysteine ​​thiol groups to covalently link antibodies to cytotoxic payloads. Human IgG1 Fc naturally contains multiple pairs of conserved cysteine ​​residues linked by interchain disulfide bonds, maintaining the antibody's spatial conformation. Under mild reducing conditions, the reducing agent selectively cleaves the interchain disulfide bonds without disrupting the internal folding structure of the antibody molecule, exposing highly active free thiol groups (-SH) to the paired cysteine ​​residues. The small molecule prodrugs used in this invention all possess maleimide (MC) active functional groups. These maleimide groups can undergo a specific Michael addition reaction with the exposed free thiol groups of cysteine ​​under neutral and mild aqueous conditions, forming stable, non-hydrolyzable thioether covalent bonds. This coupling method features mild reaction conditions, few side reactions, and does not impair antibody-antigen binding activity. Furthermore, the controlled reduction process precisely regulates the amount of free thiol groups generated, thereby stably controlling DAR (Drug Adsorption-Reduction) and ultimately producing antibody-drug conjugates with high uniformity, stable structure, and excellent reproducibility.

[0027] This invention also provides the application of the antibody-drug conjugates described in the above-described technical solutions or the antibody-drug conjugates prepared by the preparation methods described in the above-described technical solutions in the preparation of one or more products for tumor diagnosis, tumor treatment, tumor clinical research, and drug development; the tumor includes tumors with CD46-positive cell surfaces. As one embodiment, the CD46-positive cell surface tumors of this invention can be colorectal cancer. This invention uses enzyme-linked immunosorbent assays (ELISA) to detect the specific binding ability of the above-described antibody-drug conjugates to CD46-positive tumor cells (such as HCT116, RKO, SW480, and SW620), confirming that the antibody-drug conjugates of this invention can specifically recognize and kill CD46-positive tumor cells. Therefore, the antibody-drug conjugates of this invention can be applied to the immunotherapy of malignant tumors, especially CD46-positive tumors.

[0028] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1 Preparation of purified antibody 31-17-1-A12 Based on the eukaryotic expression vector (pcDNA3.4-Fc antibody expression vector) formed by the single-domain antibody 31-17-1-A12, which targets human and cynomolgus monkey CD46 and was previously developed in CN202511107289.1, after transient transfection of 293F cells, the culture supernatant was collected, and the recombinant antibody protein was isolated and purified using AmMag™ Protein A Magnetic Beads. The purity of the recombinant protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific steps are as follows: 1) Remove the LVTransm transfection reagent (iCarEab, Cat#LVTran100) and the 31-17-1-A12 antibody eukaryotic expression vector from the refrigerator, thaw at room temperature, and then mix thoroughly by pipetting up and down.

[0030] 2) Warm the phosphate buffer solution (PBS; Gbico, Cat#14190-250) to room temperature, take 2 mL of PBS into one well of a 6-well plate, add 100 μg of antibody expression vector, mix thoroughly by pipetting, add 300 μL of LLVTransm, mix again by pipetting, and let stand at room temperature for 10 minutes to obtain the DNA / LVTransm complex.

[0031] 3) Add the above DNA / LVTransm complex to 100 mL of 293F cells (from the ATCC cell bank), gently shake to mix thoroughly, and transfer the cells to a 37°C, 5% CO2 incubator for further culture at 130 rpm.

[0032] 4) After continuous culture for 5 days, the supernatant of the culture medium was collected by centrifugation, and the filtrate was transferred to a sterile centrifuge tube (Corning, Cat#430052). The antibody was purified using AmMag™ Protein A Magnetic Beads (Nanjing Genscript Biotech Co., Ltd., Cat#L00695).

[0033] 5) The purified protein was concentrated and buffer replaced using 10 kDa ultrafiltration tubes (Beyotime, Cat#FUF051-5pcs), with the buffer replaced by PBS.

[0034] 6) After determining the concentration of the purified antibody using Nanodrop 2000, perform reducing SDS-PAGE detection.

[0035] SDS-PAGE experimental results are as follows: Figure 1 As shown, the protein purity of the purified antibody 31-17-1-A12 is >95%, and it can be used for ADC conjugation.

[0036] The purified antibody 31-17-1-A12 is conjugated with human IgG1 Fc, and its amino acid sequence is as follows: QVQLVESGGGLVQPG GSLRLSCAASGFTLDYYNIGWFRQAPGKEREGVSCISSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCASSDSCGYYYTAGLNYWGQGTQVTVSS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:3), wherein the italicized and underlined amino acid portion is the amino acid sequence of the single-domain antibody 31-17-1-A12, and the upright amino acid portion represents the amino acid sequence of human IgG1 Fc.

[0037] Example 2 ADC Coupling Preparation The antibody purified from 31-17-1-A12 was conjugated to Dxd or MMAE toxin at the cysteine ​​position of the Fc region via Michael addition reaction. The optimal conjugation conditions were determined by optimizing the buffer pH, antibody-toxin ratio, conjugation temperature, and conjugation time. Single-domain antibody-toxin conjugates with an average DAR > 3.5 were prepared, and the conjugates were analyzed by HIC-UPLC and LC-MS, as follows: 1) Take 10 mg of purified 31-17-1-A12 antibody (10 mL, 133 nmol) and add it to a 15 mL centrifuge tube. The antibody is stored in the following solution: 1 mg / ml 50 mM L-histidine (MCE, Cat#HY-N0832), 15 g / L trehalose (Bailingwei Technology, Cat#99-20-7), 100 mM sodium chloride (Beijing Solarbio Science & Technology Co., Ltd., Cat#7647-14-5), pH 7.0; 2) Add 80 μL (6 eq, 769 nmol) of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP; Aladdin, Cat#16600082, TCEP is a 10 mM aqueous solution) and 500 μL (final concentration 5 mM) of ethylenediaminetetraacetic acid (EDTA; MCE, Cat#HY0Y0682, prepared with aqueous solution to a concentration of 100 mM, pH 7.0). Place the reaction in a constant temperature metal bath and reduce at 200 rpm and 25℃ for 17 h.

[0038] 3) Add 200 μL (15 eq, 2 μmol) MC-GGFG-Dxd (Shanghai Xiyao Pharmaceutical, Cat#1599440-13-7, prepared with dimethyl sulfoxide to a concentration of 10 mM, where dimethyl sulfoxide (DMSO) was purchased from MCE, Cat# HY-Y0320C) or 200 μL (15 eq, 2 μmol) MC-VC-PAB-MMAE (MCE, Cat#HY-15575, prepared with DMSO to a concentration of 10 mM), place the reaction solution in a constant temperature metal bath, and couple at 200 rpm and 25℃ for 5 h.

[0039] 4) After coupling, HIC-UPLC was used for analysis. The chromatographic column used was a BioCore HIC-Butyl high-performance hydrophobic column (NanoChrom, Cat#B713-050100-04610S). Combined with LC-MS, the peak time of the coupling product and the coupling efficiency were determined.

[0040] 5) The conjugated product was purified by affinity chromatography using AmMag™ Protein A Magnetic Beads to remove unconjugated small molecules.

[0041] HIC-UPLC results are as follows Figure 2 As shown, A is the HIC-UPLC detection result of the naked antibody; B is the HIC-UPLC detection result of 1-A12-Fc-Dxd; and C is the HIC-UPLC detection result of 1-A12-Fc-MMAE.

[0042] Depend on Figure 2 It can be concluded that, using the naked antibody as a control group, both newly synthesized ADCs exhibited novel chromatographic peaks with different hydrophobicities, confirming that the cytotoxic load was successfully covalently attached to the purified antibody, and that the two ADCs were successfully conjugated. The naked antibody showed a sharp, symmetrical main peak, indicating good homogeneity and separation. In addition to the main peak, both ADC samples exhibited a series of other peaks, indicating that the ADC products are a mixture of DAR, a common phenomenon in ADC synthesis.

[0043] The synthesized ADC was further analyzed by LC-MS, and the results are as follows: Figure 3 As shown, A is the LC-MS detection result of the bare antibody; B is the LC-MS detection result of 1-A12-Fc-Dxd; and C is the LC-MS detection result of 1-A12-Fc-MMAE.

[0044] Since the molecular weight of small molecules is fixed, according to Figure 3The molecular weights of the detected antibodies and conjugates were used to calculate the DAR of the two ADCs to be approximately 4 (as shown in Table 1), which is within the currently accepted appropriate range for DAR (2~8).

[0045] Table 1. Results of complete molecular weight determination

[0046] Example 3 ADC in vitro killing of target cells detection HCT116, RKO, SW480, and SW620 cells were prepared and adjusted to the logarithmic growth phase. The ADC prepared in Example 2 was serially diluted (starting at 1000 nM, 3-fold diluted for 10 sites) and co-cultured with target cells (cell confluence 80-90%) for 120 h. Cell viability was assessed, and IC50 was calculated. 50 Value. The naked antibody without cytotoxic load was used as a negative control (experimental groups: naked antibody, 1-A12-Fc-Dxd, 1-A12-Fc-MMAE), and the specific steps are as follows: 1) Resuscitate HCT116, RKO, SW480, and SW620 cells (ATCC cell bank) and adjust them to the logarithmic growth phase using the corresponding complete culture medium.

[0047] The HCT116 cells were derived from poorly differentiated colorectal adenocarcinoma, retaining wild-type p53 and mutant KRAS genes. The complete culture medium used was McCoy's 5A medium (Gibco, Cat#16600082) + 10% serum (FBS; VivaCell, Cat#C04001-500). RKO cells were derived from poorly differentiated colorectal cancer, retaining wild-type p53 and KRAS genes. The complete culture medium used was MEM medium (Gibco, Cat#12492013) + 10% serum (FBS; VivaCell, Cat#C04001-500). SW480 cells were derived from in situ rectal adenocarcinoma and p53 and KRAS gene mutations. The complete culture medium used was DMEM high glucose medium (Gibco, Cat#41965-062) + 10% serum (FBS; VivaCell, Cat#C04001-500). SW620 cells were derived from lymph node metastases and p53 and KRAS gene mutations. The complete culture medium used was Leibovitz's L-15 medium (Gibco, Cat#11415064) + 10% serum (FBS; VivaCell, Cat#C04001-500).

[0048] 2) Discard the culture medium, take 3 mL of DuPont phosphate buffer solution (DPBS; Shanghai Yuanpei Biotechnology Co., Ltd., Cat#B210KJ) and add it along the inner wall of the culture dish, then shake horizontally 10 times.

[0049] 3) Discard the DPBS, add 3 mL of Trypsin-EDTA 0.25% trypsin (Gibco, Cat#25200072) along the inner wall of the culture dish, and shake horizontally 3 times.

[0050] 4) Discard the trypsin and immediately place the culture dish under an inverted microscope for observation. When the cells are observed to shrink back and no longer connect to form sheets, and when some cells are observed to drift down with the tilting direction after tilting the culture dish, immediately add 4 mL of complete culture medium along the inner wall of the culture dish. Use a pipette to pipette the digested cells into a cell suspension, and then transfer it to a 15 mL centrifuge tube. Count the cells and calculate the cell viability.

[0051] 5) Centrifuge the cell suspension at 300 rcf for 3 min, discard the supernatant, resuspend the pellet in 4 mL of complete culture medium, wash 2-3 times with DPBS, and prepare a cell suspension (3 × 10⁻⁶). 4 (units / mL).

[0052] 6) Add 200 μL of sterile water to the wells around the perimeter of the 96-well plate; add 100 μL of the corresponding cell suspension to the remaining wells, gently tap the perimeter of the 96-well plate to promote uniform distribution of cells throughout the culture wells, and incubate at 37℃ in a 5% CO2 incubator for 24 h.

[0053] 7) Prepare ADC dilutions with an initial concentration of 1000 nM using the corresponding complete culture medium, and dilute them in a 3-fold gradient, resulting in a total of nine concentration gradients.

[0054] 8) Add 100 μL of ADC dilution to the corresponding cell wells, and make 3 replicates for each concentration gradient of each ADC dilution (the final concentration of ADC in the first well is 1000 nM). Incubate at 37℃ and 5% CO2.

[0055] 9) After culturing for 120 h, when the confluence of cells in the drug-free control wells is greater than 80%, cell viability is detected using a cell viability assay kit (iCarEab, Cat#ICA-CVL-100A). The culture medium in the well plate is completely discarded, 100 μL of cell viability assay reagent is added, and the plate is incubated in the dark for 5 min. 200 μL is then transferred from each well to a white, opaque 96-well plate, and the luminescence value is read using a full-wavelength multi-mode microplate reader.

[0056] The results of in vitro lethality testing are shown in Table 2 and Figure 4As shown, A represents the killing effect of ADC on HCT116 cells; B represents the killing effect of ADC on RKO cells; C represents the killing effect of ADC on SW480 cells; and D represents the killing effect of ADC on SW620 cells.

[0057] Table 2 Results of ADC in vitro killing of target cells

[0058] Depend on Figure 4 The results in Table 2 show that 1-A12-Fc-Dxd and 1-A12-Fc-MMAE have killing effects on all target cells (HCT116, RKO, SW480, SW620), with 1-A12-Fc-MMAE showing stronger killing activity on all target cells.

[0059] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antibody-drug conjugate targeting CD46, characterized in that, The device includes a single-domain antibody 31-17-1-A12, a human IgG1 Fc, and a cytotoxic payload. The human IgG1 Fc is linked to the C-terminus of the single-domain antibody 31-17-1-A12, and the cytotoxic payload is coupled to the single-domain antibody 31-17-1-A12 via a cysteine ​​residue on the human IgG1 Fc. The amino acid sequence of the single-domain antibody 31-17-1-A12 is shown in SEQ ID NO:

1. The amino acid sequence of the human IgG1 Fc is shown in SEQ ID NO:2; The cytotoxic load conjugated with human IgG1 Fc cysteine ​​is located at positions 6 and 9, starting from the N-terminus of the amino acid sequence shown in SEQ ID NO:2; The cytotoxic loads include Deruxtecan or Monomethyl auristatin E.

2. The antibody-drug conjugate targeting CD46 according to claim 1, characterized in that, The drug-antibody ratio in the antibody-drug conjugate targeting CD46 is >3.

5.

3. The method for preparing the antibody-drug conjugate targeting CD46 according to claim 1 or 2, characterized in that, The process includes the following steps: conjugating a cytotoxic payload with a purified 31-17-1-A12 antibody via a Michael addition reaction to obtain an antibody-drug conjugate targeting CD46; the purified 31-17-1-A12 antibody is formed by linking human IgG1 Fc to the C-terminus of the single-domain antibody 31-17-1-A12, and the cytotoxic payload is conjugated with a cysteine ​​residue on the human IgG1 Fc.

4. The preparation method according to claim 3, characterized in that, The steps include the following: The purified antibody 31-17-1-A12 was mixed with tris(2-carboxyethyl)phosphine hydrochloride and ethylenediaminetetraacetic acid for a reduction reaction to obtain the reduction reaction system. A cytotoxic load was added to the reduction reaction system to carry out a coupling reaction, resulting in an antibody-drug conjugate targeting CD46.

5. The preparation method according to claim 4, characterized in that, Each 1 mg of the antibody-drug conjugate contains 0.07 mg of cytotoxic load.

6. The preparation method according to claim 4, characterized in that, The coupling reaction was performed at a rotation speed of 200 rpm, a temperature of 25°C, and a time of 5 h.

7. The preparation method according to claim 4, characterized in that, The mass ratio of the purified 31-17-1-A12 antibody to tris(2-carboxyethyl)phosphine hydrochloride is 1 mg: 22 μg; the volume ratio of tris(2-carboxyethyl)phosphine hydrochloride to ethylenediaminetetraacetic acid is 4:

25.

8. The preparation method according to claim 4, characterized in that, The reduction reaction was carried out at a shaking speed of 200 rpm; the reduction reaction was carried out at a temperature of 25°C; and the reduction reaction was carried out for 17 h.

9. The preparation method according to claim 4, characterized in that, The 31-17-1-A12 purified antibody is derived from in vivo expression or in vitro synthesis; the biological materials used for in vivo expression include expression cassettes, recombinant vectors, recombinant bacteria or recombinant cells encoding nucleotides of the 31-17-1-A12 purified antibody.

10. The use of the antibody-drug conjugate according to claim 1 or 2 or the antibody-drug conjugate prepared by the preparation method according to any one of claims 3 to 9 in the preparation of a drug for treating colorectal cancer.

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