An antibody conjugate based on humanized anti-CD147 antibody F(ab')2 fragment and application thereof

Abstract

The application belongs to the technical field of biology and particularly relates to an antibody conjugate drug based on a humanized anti-CD147 antibody F(ab')2 fragment and application thereof. The antibody conjugate drug is formed by coupling a humanized anti-CD147 antibody F(ab')2 fragment and a maytansine derivative through a non-cleavable linker; the light chain amino acid sequence of the F(ab')2 fragment is shown as SEQ ID NO. 1, and the heavy chain amino acid sequence is shown as SEQ ID NO. 2. Compared with the existing HcHAb18-DM1, the drug provided in the application has a smaller molecular weight, better solid tumor penetration, lower non-specific binding and lower effective dose, thereby improving the anti-tumor efficacy and reducing the systemic toxicity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antibody-drug conjugate based on the humanized anti-CD147 antibody F(ab')2 fragment and its application. Background Technology

[0002] CD147 (also known as HAb18G / EMMPRIN) is a transmembrane glycoprotein highly expressed on the surface of various malignant tumor cells, involved in tumor invasion, metastasis, and angiogenesis. CD147 is significantly highly expressed in non-small cell lung cancer (NSCLC) tissues and is associated with poor patient prognosis; therefore, CD147 has become a potential target for anti-NSCLC drug development.

[0003] HcHAb18 is a humanized, modified anti-CD147 chimeric antibody. Previous studies have conjugated it with the microtubule inhibitor maytansine derivative (DM1) via a non-cleavable linker (SMCC) to obtain the ADC drug HcHAb18-DM1 (metuzumab-matansine derivative conjugate, see DOI: 10.1016 / j.bbrc.2019.04.046). This drug specifically binds to CD147 molecules and is internalized by tumor cells, releasing DM1 to exert its anti-tumor effect.

[0004] However, HcHAb18-DM1 still has the following shortcomings: (1) Large molecular weight: Full-length antibody (about 150 kDa) has limited penetration ability in solid tumor tissue and is difficult to reach deep areas of tumor tissue.

[0005] (2) Long half-life: The complete Fc segment makes the drug circulate in the blood for a longer time. On the one hand, it increases the risk of non-specific binding of the antibody Fc segment to normal tissues. On the other hand, it may cause the toxin-laden cells to be released prematurely in the circulating blood, resulting in systemic toxic side effects.

[0006] (3) Limited therapeutic window: Due to the above defects, higher doses are required in clinical practice to achieve effective intratumoral concentration, but the toxicity also increases accordingly.

[0007] To address these issues, some researchers have attempted to use antibody fragments (such as F(ab′)2) to replace full-length antibodies. However, their application in the construction of ADC drugs still faces technical challenges, including complex conjugation processes, unstable drug-antibody ratio (DAR), and decreased affinity. Summary of the Invention

[0008] The purpose of this invention is to provide an improved ADC drug for the treatment of non-small cell lung cancer, namely, a metuzumab F(ab′)2 fragment-maytansin derivative conjugate (HcHAb18 F(ab′)2-DM1), its preparation method, and its application. Compared with the existing HcHAb18-DM1, the drug provided by this invention has a smaller molecular weight, better penetration into solid tumors, lower nonspecific binding, and a lower effective dose, thereby improving antitumor efficacy and reducing systemic toxicity.

[0009] The technical solution provided by this invention is as follows: This invention provides an antibody-drug conjugate based on the humanized anti-CD147 antibody F(ab')2 fragment, which is formed by conjugating the metuzumab F(ab')2 fragment with a maytansine derivative through a non-breakable linker; the light chain amino acid sequence of the metuzumab F(ab')2 fragment is shown in SEQ ID NO.1, and the heavy chain amino acid sequence is shown in SEQ ID NO.2.

[0010] Preferably, the linker is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester.

[0011] The present invention also provides a method for preparing the antibody-drug conjugate, comprising the following steps: Using maytansine derivatives and unbreakable linkers as raw materials, a Michael addition reaction was carried out under the action of triethylamine to obtain linker-matansine derivatives; The antibody-drug conjugate is obtained by conjugating the linker-matansine derivative with the metuzumab F(ab′)2 fragment.

[0012] Preferably, the linker is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester, and the mass ratio of the maytansin derivative to succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester is 1.475:1; The molar ratio of the linker-matansin derivative to the metotuzumab F(ab´)2 fragment is 9.5:1.

[0013] Preferably, the Michael addition reaction is carried out at room temperature under nitrogen protection for 6 hours; The conjugation of the linker-matansine to the metutuzumab F(ab′)2 fragment was carried out under nitrogen protection, at 25°C and a rotation speed of 120 rpm for 6 hours.

[0014] Preferably, the metutuzumab F(ab′)2 fragment is obtained by purifying metutuzumab by digestion with pepsin. The digestion conditions are: the mass ratio of pepsin to metutuzumab is 1:2~10, the digestion temperature is 37℃, and the digestion time is 3~3.5 hours.

[0015] The present invention also provides the use of the antibody-drug conjugate described above in the preparation of a medicament for treating non-small cell lung cancer.

[0016] Preferably, the non-small cell lung cancer is non-small cell lung cancer expressing CD147 molecules.

[0017] The present invention also provides a pharmaceutical formulation for treating non-small cell lung cancer, comprising the antibody-drug conjugate and a pharmaceutically acceptable carrier.

[0018] Preferably, the pharmaceutical preparation is a lyophilized powder. This lyophilized powder is prepared using conventional pharmaceutical methods, such as mixing HcHAb18 F(ab′)2-DM1 with an appropriate amount of lyophilization protectant, dispensing, pre-freezing, primary drying, secondary drying, and then sealing with a stopper.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: Compared with HcHAb18-DM1 (metuzumab-maytansin derivative conjugate), the novel ADC drug based on the metuzumab F(ab´)2 fragment provided by this invention can reduce the molecular weight of the antibody drug, thereby shortening the drug's half-life, reducing the probability of premature release of the drug load in the blood, avoiding non-specific binding of the Fc fragment of the full-length antibody to normal tissues, and effectively improving the penetration of the ADC drug in solid tumors. Attached Figure Description

[0020] Figure 1 It is the DAR value of HcHAb18 F(ab´)2-DM1 detected by ultraviolet light.

[0021] Figure 2 The DAR value of HcHAb18 F(ab´)2-DM1 was detected by mass spectrometry.

[0022] Figure 3 This is a molecular sieve chromatography assay to determine the purity of HcHAb18 F(ab´)2-DM1.

[0023] Figure 4 SPR is used to detect the affinity of HcHAb18 F(ab´)2 for HAb18G / CD147 antigen.

[0024] Figure 5The SPR assay detects the affinity of HcHAb18 F(ab´)2-DM1 for the HAb18G / CD147 antigen.

[0025] Figures 6-7 The ADC and its corresponding antibody are used as single drugs to inhibit the proliferation of various cells.

[0026] Figure 8 This is the tumor volume-time curve after HcHAb18 F(ab´)2-DM1 administration.

[0027] Figure 9 This represents the changes in body weight of mice in each group.

[0028] Figure 10 The results show the changes in tumor weight at the endpoint in mice under different drug dosages.

[0029] Figure 11 The tumor inhibition rate-dose curve of HcHAb18-DM1 against tumor volume is shown in Figure (A) and the tumor inhibition rate-dose curve of HcHAb18 F(ab´)2-DM1 against tumor volume is shown in Figure (B).

[0030] Figure 12 This represents the survival time of mice in each group in the NCI-H520 cell diffusion model. Detailed Implementation

[0031] This invention aims to overcome the shortcomings of existing anti-CD147 ADC drugs (HcHAb18-DM1), such as large molecular weight, poor penetration into solid tumors, excessively long half-life leading to non-specific binding and premature release of toxins, and to provide a novel ADC drug based on the metuzumab F(ab′)2 fragment, its preparation method and application.

[0032] Specifically, the present invention aims to solve the following technical problems: (1) To provide an ADC drug targeting CD147 molecules with a significantly reduced molecular weight to enhance its penetration depth in non-small cell lung cancer tissue; (2) The Fc fragment of the antibody is removed by pepsin digestion to avoid non-specific binding of the antibody to normal tissues (such as Fc receptor positive cells), while shortening the drug half-life and reducing the premature release of toxins in circulation. (3) Establish a stable and scalable one-step conjugation process to obtain antibody fragment-drug conjugates with appropriate DAR values ​​(3±0.5) and maintaining antigen affinity; (4) Verify whether this novel antibody-drug conjugate (ADC) is superior to the original HcHAb18-DM1 in terms of in vitro cell proliferation inhibition, in vivo tumor-bearing model tumor inhibition rate, and survival prolongation, and determine the lower effective dose (ED). 50 ).

[0033] CD147: A tumor-associated antigen highly expressed on the surface of tumor cells; HcHAb18 (metuzumab): A humanized modified anti-CD147 chimeric antibody HcHAb18, see patent document with application number 201410320878; HcHAb18-DM1 (Metuzumab-Maytansine Derivative Conjugate): A conjugate of humanized anti-CD147 chimeric antibody HcHAb18 and maytansine derivative. HcHAb18 F(ab´)2-DM1 (Metuzumab F(ab´)2 fragment-Mertansine derivative conjugate): The F(ab)2 fragment of a humanized modified anti-CD147 chimeric antibody HcHAb18, conjugated with Mertansine (DM1); it can be effectively internalized by cancer cells and shows effective anti-tumor effects in non-small cell lung cancer cells.

[0034] The light chain sequence of the F(ab)2 fragment of HcHAb18 is shown in SEQ ID NO.1: MGWSCIILFLVATATGSIVMTQTPTFLVVSAGDRVTITCKASOSVINDVAWYQQKPGQSPKLLIFYASNRNTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSPPFTFG SGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The heavy chain sequence of the F(ab)2 fragment of HcHAb18 is shown in SEQ ID NO.2: MGWSCIILFLVATATGEVKLEESGGGLVQPGGSMKLSCVASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHAPYYTESVKGRFTISRDDSKSIIYLQMNNLRAEDTGIYYCTRDSTATHW GQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP For HcHAb18-DM1 (metutuzumab-matansine derivative conjugate), please refer to the article published by our team (DOI: 10.1016 / j.bbrc.2019.04.046). The HcHAb18 F(ab´)2-DM1 (metutuzumab F(ab´)2 fragment-matansine derivative conjugate) provided by this invention is based on HcHAb18-DM1 (metutuzumab-matansine derivative conjugate), and the antibody has been optimized by removing the Fc fragment, which allows the entire ADC drug to function better.

[0035] Example 1 An antibody-drug conjugate based on the humanized anti-CD147 antibody F(ab')2 fragment (labeled as HcHAb18 F(ab')2-DM1) was prepared according to the following steps: 1) The F(ab´)2 fragment of metotuzumab was prepared by pepsin digestion. The digestion conditions were: a pepsin to metotuzumab mass ratio of 1:5, a digestion temperature of 37℃, and a digestion time of 3 hours. (The digestion conditions can be varied within the following range: a pepsin to metotuzumab mass ratio of 1:2~10, a digestion temperature of 37℃, and a digestion time of 3~3.5 hours).

[0036] 2) Purification using a GE purification column: This process was performed on an AKTA Basic protein purification system, using Capto L gel (Cytiva) as the column separation medium. The F(ab)2 and Fc fragments were separated using the affinity principle. Purification conditions: 1×PBS equilibration buffer (pH 7.2-7.4) and 0.05M glycine-hydrochloric acid were used as the isocratic elution buffer, with a constant flow rate of 3.0 mL / min. After filtering through a 0.22 μm filter, the sample was manually loaded using a syringe. The elution curve was monitored in real-time at 280 nm using an online UV detector, and the eluted fractions of the antibody fragments were collected to obtain high-purity metuzrexate F(ab')2 fragments.

[0037] The purified solution was collected and then concentrated using 50 ml / 30 kDa ultrafiltration centrifuge tubes from Millipore at 4°C and 4000 rpm until the concentration of the methotrexate F(ab´)2 fragment was 20 mg / ml, in order to control the total volume of the coupling reaction system.

[0038] 3) The maytansine derivative (DM1) and the linker SMCC were mixed and subjected to a Michael addition reaction in the presence of triethylamine to obtain the linker-matansine derivative (SMCC-DM1). The specific synthetic process is as follows:

[0039] First, weigh 90.0 mg of maytansine derivative (DM1) powder, add 8 ml of distilled anhydrous tetrahydrofuran (C4H8O) solution at room temperature, and simultaneously introduce nitrogen (N2) as a protective gas. Shake and stir to obtain maytansine derivative solution.

[0040] Next, weigh 61.0 mg of SMCC powder, add 3 ml of anhydrous tetrahydrofuran (THF), and shake well. Mix the resulting solution with a tetrahydrofuran (THF) solution of maytansine derivative (DM1), and then add 2-3 drops of triethylamine (C6H2O). 15 N) is used to catalyze the connection reaction between the two, and the reaction lasts for 6 hours and is carried out under the condition of nitrogen (N2) as a protective gas.

[0041] Finally, the reaction product was purified using a silica gel column: the column was first packed with dichloromethane (CH2Cl2), and then eluted with a mixture of dichloromethane (CH2Cl2) and methanol (CH3OH) at a volume ratio of 30:1. The reaction product was evaporated to dryness using a rotary evaporator, yielding 97.1 mg of a white powdery solid product, with a yield of 74.3%. The purity of the product was determined to be 96.5% by liquid chromatography-mass spectrometry, meeting the expected requirements; the mass-to-charge ratio (m / z) was 1072.40, confirming that the powder was a linker-maytansin derivative (SMCC-DM1).

[0042] 4) Dissolve the linker-matansine derivative (SMCC-DM1) powder in N,N-dimethylacetamide (DMA) organic solvent, and then add it dropwise to a buffer solution containing metosulfonamide F(ab´)2 (HcHAb18 F(ab´)2), wherein the molar ratio of SMCC-DM1 to HcHAb18 F(ab´)2 is 9.5:1. Shake and stir well, and purge with nitrogen (N2) protective gas. Wrap with aluminum foil to protect from light, place in a constant temperature shaker, set the crosslinking temperature to 25℃, the speed to 120 rpm, and the crosslinking reaction time to 6 hours.

[0043] 5) The reaction solution from step 4) was concentrated using a 50ml / 30kDa ultrafiltration centrifuge tube from Millipore at 4°C and 4000 rpm until the monoclonal antibody concentration was 20mg / ml, which would facilitate subsequent purification operations.

[0044] 6) Purify the above reaction solution using a GE Superdex-200 molecular sieve chromatography column. Concentrate the purified solution using Millipore 50ml / 30kDa ultrafiltration centrifuge tubes at 4°C and 4000 rpm until the monoclonal antibody concentration reaches 10mg / ml. Filter and dispense the solution using a sterile filter and store it in a -80°C freezer.

[0045] The above preparation process utilizes the indestructible linker succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (SMCC) and employs a one-step cross-linking process to cross-link maytansine derivative (DM1) with metotuzumab fragment (HcHAb18 F(ab')2) to prepare HcHAb18 F(ab')2-DM1, i.e., the ADC drug. The preparation process route is as follows: .

[0046] Scale-up preparation reaction: After three scale-up reactions, different amounts of HcHAb18F(ab´)2-DM1 were prepared, as shown in Table 1.

[0047] Table 1. Batch list of HcHAb18 F(ab´)2-DM1 preparation. 1. Preliminary quality analysis (1) Detection of drug-antibody conjugate ratio (DAR) of HcHAb18 F(ab´)2-DM1 by ultraviolet light method 1) HcHAb18 F(ab´)2-DM1 was used as the test sample, and its solvent solution PBS was used as the control.

[0048] 2) Dilute the sample to 10 mg / ml with 1X PBS buffer and set aside.

[0049] 3) Use a UV spectrophotometer to measure the absorption curves of the metutuzumab-matansine derivative conjugate or the metutuzumab F(ab´)2 fragment-matansine derivative conjugate, and record the absorbance values ​​at 252nm and 280nm.

[0050] 4) Calculate the DAR value according to the following formula for calculating the antibody-drug ratio (DAR).

[0051] in The calculated DAR value of HcHAb18 F(ab´)2-DM1 is 3.28. Figure 1 As shown.

[0052] (2) Mass spectrometry detection of DAR value of HcHAb18 F(ab´)2-DM1 1) Removal of the sugar chains from the sample: Take 300 μg of the metuzin F(ab´)2 fragment-maytansin derivative conjugate from the sample and dilute it to 5 mg / ml or 3 mg / ml with 1X PBS. Adjust the pH to 7.5, add 5 μl of PNGase F enzyme, and react at 37°C for 20 hours. Afterward, centrifuge the reaction system at 12,000 rpm for 15 minutes at room temperature and collect the supernatant for mass spectrometry analysis.

[0053] 2) Liquid Chromatography (HPLC) system settings: Column pressure ≤3000 psi, column temperature 50℃, flow rate 0.2 ml / min, single injection volume 16 μg, mobile phase A is a deionized aqueous solution containing 0.1% formic acid (HCOOH), and mobile phase B is an acetonitrile (C2H3N) solution containing 0.1% formic acid (HCOOH). Gradient conditions were set as follows: A:B = 95%:5% for minutes 0-3; A:B = 5%:95% for minutes 3-6; and A:B = 95%:5% for minutes 6-8.

[0054] 3) Mass spectrometer settings: Declustering voltage (DP) set to 120~140 V; electrospray voltage set to 4000 V; source temperature set to 350℃.

[0055] 4) Calculate the average DAR value according to the percentage content of each DAR value in the mass spectrum using the following formula.

[0056] Where n represents the degree of crosslinking, and Dn% represents the percentage of couplings with a degree of crosslinking of n.

[0057] The calculated DAR value of HcHAb18 F(ab´)2-DM1 is 3.01, as shown in Table 2. Figure 2 As shown.

[0058] Table 2. DAR values ​​of HcHAb18 F(ab´)2-DM1 detected by mass spectrometry (3) Molecular sieve chromatography was used to determine the purity of HcHAb18F(ab´)2-DM1. 1) Considering analytical efficiency, the sample to be tested is first concentrated to less than 1 ml.

[0059] 2) Equilibrate the GE Superdex-75 (16 / 60) molecular sieve column using buffer solution.

[0060] 3) Use a 1ml syringe to directly load the sample.

[0061] 4) Set the flow rate of the chromatograph to 0.5 ml per minute, and the eluent to the succinic acid buffer solution with a pH of 5.0 prepared above.

[0062] 5) Collect and analyze signals when the absorbance at a wavelength of 280 nm in the ultraviolet light is greater than 100 mAU.

[0063] Molecular sieve chromatography showed that the purity of HcHAb18 F(ab´)2-DM1 was 97.70%, with an elution time of 13.5 minutes. The polymer, accounting for 2.30%, eluted before the main peak, with an elution time of 11.4 minutes, as shown in Table 3. Figure 3 As shown.

[0064] Table 3 Purity of HcHAb18 F(ab´)2-DM1 as determined by molecular sieve chromatography (4) SPR detection of antigen affinity of HcHAb18 F(ab´)2-DM1 1) The metutuzumab F(ab´)2 fragment-maytansin derivative conjugate and the metutuzumab F(ab´)2 fragment were respectively coupled to the CM5 chip (purchased from Cytiva), and the mobile phase flow rate was set to 25ul per minute.

[0065] 2) Use a blocking solution, namely hydrochloric acid-ethanolamine, to block the vacancy sites on the chip surface, and set the flow rate of the mobile phase to 25 μL per minute.

[0066] 3) Set the concentration gradient of the antigen protein CD147 from high to low as 1.6 nmol / L, 0.8 nmol / L, 0.4 nmol / L, 0.2 nmol / L, 0.1 nmol / L and 0 nmol / L. These 6 concentrations were flowed vertically over the chip surface coupled with different analytes. The mobile phase flow rate was set to 50 μL per minute, the binding time was 180 seconds and the dissociation time was 720 seconds. The process curve and equilibrium dissociation constant (KD value) were recorded to evaluate the affinity.

[0067] The equilibrium dissociation constant (KD) between HcHAb18 F(ab´)2 and HAb18G / CD147 antigen is 9.08 × 10⁻⁶. -10 The dissociation constant KD between HcHAb18F(ab´)2-DM1 and the HAb18G / CD147 antigen is 5.92 × 10⁻⁶. -10The fact that both are on the same order of magnitude indicates that the cross-linking reaction did not change the affinity between the antibody and the antigen. Figures 4-5 As shown.

[0068] In summary, the above testing items were collected, and the test results were used as a preliminary standard and a reference for the preparation of subsequent samples.

[0069] 2. Efficacy and mechanism of action of metotuzumab F(ab´)2 fragment-matansine derivative conjugate against non-small cell lung cancer The cell lines used in the experiments were three human non-small cell lung cancer cell lines (A549, NCI-H226, and NCI-H520) and one normal human embryonic lung cell line (MRC-5). All cells were routinely cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum (incubated at 37°C with 5% carbon dioxide gas), and all were adherent cell lines. To ensure the optimal condition of the cell models used, all cells were directly obtained from the cell bank, revived, and passaged, with the passage number controlled to within 15 generations to prevent cell aging.

[0070] (1) Drug activity detection 1) Dissolve the WST-1 powder with the electron coupling solution provided in the kit, mix well by blowing, then aliquot and freeze at -20°C for later use.

[0071] 2) Select cell lines in the logarithmic growth phase as the cell model to be tested. After confirming good growth under a microscope, digest the cells moderately with trypsin, collect them in centrifuge tubes, and mix well to form a single-cell suspension. Observe under a hemocytometer to count the cells, finding that each suspension contains 1×10⁻⁶ cells per milliliter. 4 Each cell.

[0072] 3) The A549, NCI-H226, and NCI-H520 human lung cancer cell lines were seeded at 2000 cells per well, and the MRC-5 normal human embryonic lung cell line was seeded at 5000 cells per well. The cell suspension was seeded into 96-well plates and cultured overnight until the cells adhered and entered a normal proliferation state.

[0073] 4) Discard the upper layer of culture medium and add 200 μl of fresh culture medium containing different drug interventions to each well. The specific drug concentration gradients were designed as follows: HcHAb18-DM1, HcHAb18 F(ab´)2-DM1, and the two corresponding naked antibodies HcHAb18 (unconjugated maytansine derivative) and HcHAb18 F(ab´)2 (unconjugated maytansine derivative) were all used at a maximum concentration of 1000 μg / ml, with each adjacent gradient being a 10-fold downdilution, for a total of 8 concentration gradients. The two small molecule drugs, paclitaxel and S-methyl DM1, had a maximum concentration of 10 μg / ml, with each adjacent gradient being a 10-fold downdilution, for a total of 8 concentration gradients. Three wells were designed for each concentration, with an intervention time of 72 hours. Fresh culture medium was added to the blank control.

[0074] 5) After the intervention time is up, add 10 μl of WST-1 solution directly to each well and incubate at 37°C for 1 hour.

[0075] 6) Take out the 96-well plate, place it on a shaker and shake it slowly for 5 minutes.

[0076] 7) Use a full-wavelength microplate reader to scan and read the absorbance of the 96-well plate, with the wavelength set to 450 nm.

[0077] The results showed that both ADC drugs, HcHAb18-DM1 and HcHAb18 F(ab´)2-DM1, inhibited the proliferation of NCI-H520, A549, and NCI-H226 tumor cells, with HcHAb18 F(ab´)2-DM1 showing a more significant effect. HcHAb18 and HcHAb18 F(ab´)2, however, showed almost no inhibitory effect. Considering the instability of DM1, S-methyl DM1 was selected as the small molecule research object (i.e., the reference standard) and compared with paclitaxel, a first-line chemotherapy drug for lung cancer. Both drugs inhibited the proliferation of NCI-H520, A549, and NCI-H226 tumor cells, with HcHAb18 F(ab´)2-DM1 showing better efficacy, as shown in Table 4. Figures 6-7 As shown.

[0078] Table 4. Inhibition of tumor cell proliferation by two ADC drugs and maytansine derivatives alone, and paclitaxel. (2) Pharmacodynamics of HcHAb18 F(ab´)2-DM1 -A549 cell tumor-bearing model 1) Establishing a tumor-bearing animal model Non-small cell lung cancer cells (A549) in the logarithmic growth phase were digested with trypsin, washed three times with RPMI-1640 incomplete medium, and resuspended in RPMI-1640 incomplete medium containing 20% ​​Matrigel. The cell concentration was adjusted to 3 × 10⁻⁶ cells / year. 7 The tumor was injected subcutaneously into the left axilla of nude mice within 12 hours, with a volume of 200 μl per mouse, to establish a subcutaneous tumor-bearing nude mouse model.

[0079] The average volume of the observed tumor nodules reached 100 mm. 3 At that time, the selection was based on the size of the tumor nodules and the animal's weight. Animals that had not formed tumors, had excessively large tumors, had ulcerated tumor surfaces, or were underweight or overweight were not selected. After the selection, the model was established.

[0080] 2) Establish a cell diffusion animal model Non-small cell lung cancer (NSCLC) cells (NCI-H520) in the logarithmic growth phase were digested and washed as described above. The cell pellet was collected and resuspended in RPMI-1640 incomplete culture medium, and the cell concentration was adjusted to 3 × 10⁻⁶ cells / mL. 6 Cells / ml. Then, 100 μl of the above cell suspension was injected into the tail vein of nude mice. After 10 days, the diffusion model was established. After randomization into groups, the mortality or survival events of each group were recorded, and the survival time was calculated.

[0081] 0) Administration method After randomization, the animals were marked using the ear-tag method. Ear tags were inserted into the right ear of each mouse, and the group and ear tag number were recorded. The saline group and each dose group of HcHAb18 F(ab´)2-DM1 were administered once a week for a total of four times; the administration method was tail vein injection. The grouping details are shown in Table 5.

[0082] Table 5. Pharmacodynamic grouping and dosage of HcHAb18 F(ab´)2-DM1 4) Experimental observation and data processing Throughout the experiment, the health status of the models was closely monitored, with model weight and tumor nodule volume data measured and recorded twice weekly. The minor and major axes of the tumor nodules were measured using calipers to calculate tumor volume and compare differences in tumor growth curves between groups. Tumor volume was calculated using the following formula: V = (major axis × minor axis) 2The relative tumor volume (RTV) is calculated using the following formula: RTV = Daily tumor volume / Initial tumor volume (before drug administration). The relative tumor proliferation rate (T / C%) is calculated using the following formula: T / C% = Average RTV of the drug treatment group / Average RTV of the control group × 100%. For result determination, when the relative tumor proliferation rate (T / C%) is greater than 40%, the drug treatment is considered ineffective; when the relative tumor proliferation rate (T / C%) is less than or equal to 40% and the statistical analysis P ≤ 0.05, the drug treatment is considered effective. The tumor volume inhibition rate (IRTV) can be calculated using the formula IRTV(%) = 1 - T / C%.

[0083] from Figure 8 It was found that the tumor volume growth was slower in all HcHAb18 F(ab´)2-DM1 dose groups compared to the saline group, and the tumor volume was reduced in the two high-dose groups of 8 mg / kg and 16 mg / kg compared to before administration.

[0084] Table 6 shows that, compared to the control group (saline), the relative tumor volume inhibition rates of the HcHAb18 F(ab´)2-DM1 8 mg / kg and 16 mg / kg groups were 76.96% and 84.16%, respectively, both higher than 60%. The relative tumor volume inhibition rates of the HcHAb18 F(ab´)2-DM1 2 mg / kg and 4 mg / kg groups were 30.79% and 50.22%, respectively, both lower than 60%, but the endpoint tumor volume was significantly lower than that of the saline group. The two low-dose groups of HcHAb18 F(ab´)2-DM1 (0.5 mg / kg and 1 mg / kg) showed no significant tumor-inhibiting effect.

[0085] Table 6 Tumor volume inhibition rate from Figure 9 It can be observed that the body weight of each model group remained basically unchanged or increased slightly, and there was no significant difference in body weight among the groups, suggesting that in the pharmacodynamic experiment of HcHAb18 F(ab´)2-DM1, the body weight of the model was not significantly different from that of the saline group within the dose range of 0 to 16 mg / kg.

[0086] according to Figure 10 As a result, the tumor weight at the end of the model decreased with increasing dosage, and the tumor weight at the end of the 2, 4, 8 and 16 mg / kg groups was significantly lower than that of the saline group.

[0087] The results in Table 7 show that, compared with the saline group, the relative tumor weight inhibition rates (IRTW) of the 2, 4, 8, and 16 mg / kg HcHAb18 F(ab´)2-DM1 groups were 32.25%, 44.43%, 70.67%, and 74.44%, respectively, all of which were significantly different.

[0088] Table 7. Relative tumor weight inhibition rate of HcHAb18 F(ab´)2-DM1 pharmacodynamics from Figure 11 The curves show that by fitting the relative tumor volume inhibition rate-dose curve and the relative tumor weight inhibition rate-dose curve respectively, it can be found that after taking the logarithm, the curves exhibit an "S" shape. The ED calculated from the HcHAb18-DM1 tumor volume inhibition rate-dose curve... 50 The value was 8.03 mg / kg; the ED was calculated based on the relative tumor weight inhibition rate-dose curve. 50 The value was 8.19 mg / kg, which suggests a single effective dose of 38 mg to 40 mg for a 60 kg adult. The ED was calculated using the dose-response curve of HcHAb18 F(ab´)2-DM1 against tumor volume. 50 The value was 3.97 mg / kg; the ED was calculated based on the relative tumor weight inhibition rate-dose curve. 50 The value is 4.46 mg / kg. This suggests an effective single dose of 19 mg to 22 mg for a 60 kg adult. For the same efficacy, HcHAb18 F(ab´)2-DM1 requires a lower dose.

[0089] In the NCI-H520 cell diffusion model, compared with the saline control group, the HcHAb18-DM1, HcHAb18 F(ab´)2-DM1, and HcHAb18 monoclonal antibody groups, when administered with cisplatin in combination with gemcitabine, all significantly prolonged the survival of mice injected with NCI-H520 cells via tail vein, with survival times of 40 days, 53 days, 60 days, and 32.5 days, respectively (see Table 8). Figure 12 .

[0090] Table 8. Duration of ADC efficacy The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An antibody-drug conjugate based on the humanized anti-CD147 antibody F(ab')2 fragment, characterized in that, It is formed by coupling the metuzumab F(ab′)2 fragment with a maytansine derivative through a non-breakable linker; the light chain amino acid sequence of the metuzumab F(ab′)2 fragment is shown in SEQ ID NO.1, and the heavy chain amino acid sequence is shown in SEQ ID NO.

2.

2. The antibody-drug conjugate according to claim 1, characterized in that, The linker is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester.

3. A method for preparing the antibody-drug conjugate according to claim 1, characterized in that, Includes the following steps: Using maytansine derivatives and unbreakable linkers as raw materials, a Michael addition reaction was carried out under the action of triethylamine to obtain linker-matansine derivatives; The antibody-drug conjugate is obtained by conjugating the linker-matansine derivative with the metuzumab F(ab′)2 fragment.

4. The preparation method according to claim 3, characterized in that, The linker is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester, and the mass ratio of the maytansin derivative to succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester is 1.475:1; The molar ratio of the linker-matansin derivative to the metotuzumab F(ab´)2 fragment is 9.5:

1.

5. The preparation method according to claim 3, characterized in that, The Michael addition reaction was carried out at room temperature under nitrogen protection for 6 hours. The coupling of the linker-matansin derivative with the metutuzumab F(ab′)2 fragment was carried out under nitrogen protection, at 25°C and a rotation speed of 120 rpm for 6 hours.

6. The preparation method according to claim 3, characterized in that, The metutuzumab F(ab′)2 fragment was obtained by purifying metutuzumab by digestion with pepsin. The digestion conditions were as follows: the mass ratio of pepsin to metutuzumab was 1:2~10, the digestion temperature was 37℃, and the digestion time was 3~3.5 hours.

7. The use of the antibody-drug conjugate according to any one of claims 1 to 2 in the preparation of a medicament for treating non-small cell lung cancer.

8. The application according to claim 7, characterized in that, The non-small cell lung cancer mentioned refers to non-small cell lung cancer that expresses the CD147 molecule.

9. A pharmaceutical formulation for treating non-small cell lung cancer, characterized in that, It includes the antibody-drug conjugate as described in any one of claims 1 to 2 and a pharmaceutically acceptable carrier.

10. The pharmaceutical preparation according to claim 9, characterized in that, The pharmaceutical preparation is a lyophilized powder.

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