Application of engineered antibody coupling medicine chemically modified by sugar in preparation of HER2 positive cancer treatment product

Abstract

The invention relates to the field of biological medicines, in particular to application of a sugar chemical modified engineered antibody coupling medicine in preparation of an HER2 positive cancer treatment product, and the engineered antibody coupling medicine is a medicine obtained by modifying an anti-HER2 antibody in an original antibody conjugate to reduce combination of an Fc segment of the anti-HER2 antibody and an Fc receptor on macrophages. The probability of interstitial pneumonia of the HER2-positive cancer treatment product is far lower than that of an original antibody conjugate. When the engineered antibody coupled drug chemically modified by sugar is used for treatment, endocytosis of the engineered antibody can be reduced by macrophages, so that the incidence rate of interstitial pneumonia is reduced or the severity of interstitial pneumonia is relieved.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine, and in particular to the use of sugar chemically modified engineered antibody-drug conjugates in the preparation of HER2-positive cancer treatment products. Background Art

[0002] Antibody-drug Conjugate (ADC) is a drug that connects biologically active cytotoxic drugs to monoclonal antibodies (mAb) through chemical bonds, and uses monoclonal antibodies as carriers to target and transport cytotoxic drugs to target cells to exert their effects. This type of drug combines the powerful lethality of cytotoxic drugs with the high targeting, stability and excellent pharmacokinetic characteristics of monoclonal antibodies, and is mainly manifested in: strong therapeutic efficacy, high specificity, weak immunogenicity (not easy to develop drug resistance), long circulation time in serum (slightly shorter than monoclonal antibodies), and low toxicity to non-target sites.

[0003] T-DXd is an antibody-drug conjugate (ADC) targeting human epidermal growth factor receptor 2 (HER2). In relevant clinical studies, although the overall efficacy was significantly improved, drug-induced interstitial pneumonia (ILD) has also attracted attention. The prescribing information of T-DXd contains a warning of ILD, which is usually low-grade (≤Grade 2). Although it can be effectively treated, 2%-3% of patients may develop fatal events, so it is necessary to conduct research to reduce the probability of interstitial pneumonia. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a use of a sugar-chemically modified engineered antibody-drug conjugate in the preparation of a HER2-positive cancer treatment product, so as to solve the problems in the prior art.

[0005] To achieve the above-mentioned object and other related objects, the present invention provides the use of an engineered antibody-drug conjugate in the preparation of a HER2-positive cancer treatment product, wherein the engineered antibody-drug conjugate is a drug in which the anti-HER2 antibody in the original antibody conjugate is modified to reduce the binding of its Fc segment to the Fc receptor on macrophages, and the probability of interstitial pneumonia in the HER2-positive cancer treatment product is lower than that of the original antibody conjugate.

[0006] Preferably, the method for modifying the anti-HER2 antibody is to remove N-glycosidic residues from the anti-HER2 antibody to obtain the N-glycosidic residues of the antibody.

[0007] Preferably, the method for modifying the anti-HER2 antibody is to perform N297A mutation on the anti-HER2 antibody having an amino acid sequence as shown in SEQ ID NO.1.

[0008] Preferably, the method for modifying the anti-HER2 antibody is to mutate the following sites of the anti-HER2 antibody whose amino acid sequence is shown in SEQ ID NO.1: L234A, L235A and P329G.

[0009] As described above, the use of the glycochemically modified engineered antibody-drug conjugate of the present invention in the preparation of HER2-positive cancer treatment products has the following beneficial effects: for the clinical problem of interstitial pneumonia caused by ADC, a solution is proposed to reduce the incidence of interstitial pneumonia by engineering the antibody part of ADC, designing and synthesizing ADC of engineered antibodies. The experimental results show that macrophages can reduce the endocytosis of engineered antibodies, thereby reducing the incidence of interstitial pneumonia or reducing its severity. The research results provide a strong theoretical basis for the development of subsequent animal experiments and clinical trials, and the engineered antibody ADC shows great potential for clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shown is a schematic diagram of the antibody structure.

[0011] Figure 2 Shown are the characterization results after enzyme digestion of the full-length antibody.

[0012] Figure 3 Shown are antibody-Cy3 protein gel electrophoresis and fluorescence imaging.

[0013] Figure 4 The results are shown for THP-1 cells induced by PMA.

[0014] Figure 5 Three macrophage endocytosis antibodies - Cy3 are shown. The obtained data were analyzed and processed by GraphPad and the results are shown in the figure (*p<0.05; **p<0.01; ***p<0.001; no significant difference ns)

[0015] Figure 6 The tumor cell binding antibody-Cy3. The obtained data were analyzed and processed by GraphPad and the results are shown in the figure (*p<0.05; **p<0.01; ***p<0.001; no significant difference ns)

[0016] Figure 7 Shown are the N87 endocytosis confocal and endocytosis curves.

[0017] Figure 8 Shown is a schematic diagram of the ADC structure and characterization.

[0018] Fig. 9 The CCK-8 method is used to detect and compare the killing effects of four ADC drugs on cell lines, and the IC50 values ​​are calculated.

[0019] Fig.10 The figure shows the flow chart of the antibody-Cy3 distribution experiment in mice. The lung organs and tumors of mice were photographed using IVIS, and the obtained data were analyzed and processed using GraphPad. The results are shown in the figure (*p<0.05; **p<0.01; ***p<0.001; no significant difference ns). The lung organs were frozen and sectioned for immunofluorescence experiments.

[0020] Fig.11 The figure shows the schematic diagram of subcutaneous tumor modeling and treatment process. The figure shows the solid photos of the in vitro tumor and the changes in the weight and tumor volume of each group of mice recorded at different time points. The ADC group showed significant differences compared with the PBS group, and the weight of mice in each group did not show significant changes during the treatment (*p<0.05; **p<0.01; ***p<0.001; no significant difference ns). DETAILED DESCRIPTION

[0021] Nonspecific uptake of ADC by normal cells may occur through receptor mediation (such as FcγR, mannose receptor) and pinocytosis. Antibodies exert their effector function by binding to FcγR through their Fc fragment (crystallizable fragment). Typical monoclonal antibodies have N-glycosylation modification at Asn297 in the CH2 region of the Fc fragment.

[0022] The present invention finds that the binding of the Fc segment of ADC to the Fc receptor on macrophages is the cause of interstitial pneumonia when ADC is used.

[0023] This study used antibody modification to reduce the nonspecific endocytosis of T-DXd by macrophages, and used engineered antibodies to design antibody-drug conjugates to reduce the severity of ILD or reduce its incidence. Specifically, this study selected two methods, PNGase F and N297A mutation, to remove N sugars, which would lead to reduced binding of Fc receptors and C1q. At the same time, LALAPG mutations were also selected, including three point mutations, L234A, L235A, and P329G. The LALAPG variant inhibits binding to FcγR and C1q, but does not affect FcRn binding and Fc stability. All three methods can reduce the interaction between the antibody Fc segment and the macrophage FcγR receptor, and reduce the nonspecific absorption of ADC in normal lung tissue. Therefore, this study proposed an engineered antibody solution to the clinical problem of interstitial pneumonia caused by macrophage endocytosis of ADC, in order to reduce the incidence of pneumonia and reduce drug side effects.

[0024] Based on the above content, the present invention provides the use of an engineered antibody-drug conjugate in the preparation of a HER2-positive cancer treatment product, wherein the engineered antibody-drug conjugate is a drug in which the anti-HER2 antibody in the original antibody conjugate is modified to reduce the binding of its Fc segment to the Fc receptor on macrophages, and the probability of interstitial pneumonia in the HER2-positive cancer treatment product is lower than that of the original antibody conjugate.

[0025] The original antibody conjugate is selected from T-DXd, T-DM1, Trastuzumab Duocarmazine, RC48, Tisotumab vedotin-tftv, Loncastuximab tesirine-lpyl, Belantamab mafodotin-blmf, Sacituzumab govitecan, Enfortumab vedotin, Polatuzumab vedotin-piiq, Moxetumomabpasudotox, Inotuzumab ozogamicin, Brentuximab vedotin or Gemtuzumab ozogamicin.

[0026] Trastuzumab deruxtecan (T-DXd) is composed of an anti-HER2 humanized monoclonal antibody (MAAL-9001) and an exetilcan derivative MAAA-1181a (DXd). Part of the amino acid sequence of the antibody is identical to that of trastuzumab.

[0027] In certain embodiments of the present invention, the method for modifying the anti-HER2 antibody is to remove N-glycosylation from the anti-HER2 antibody to obtain the N-glycosylated antibody.

[0028] The method of removing N-glycosylation is an existing technology, for example, using peptide: N-glycanase (abbreviated as PNGase, also known as N-glycosidase) to hydrolyze the oligosaccharides linked to asparagine (Asn) on the protein and release the complete oligosaccharide chain.

[0029] In certain embodiments of the present invention, the PNGase is selected from PNGase F. The cleavage site of PNGase F is the amide bond between the N-acetylglucosamine (GlcNAc) and asparagine residues inside the glycoprotein, and the aspartyl residue on the enzymatically hydrolyzed protein is converted into aspartic acid.

[0030] In certain embodiments of the present invention, full-length Trastuzumab is treated with PNGase F to obtain de-N-glycosylated Trastuzumab (Trastuzumab-0705). PNGase F can remove all N-linked oligosaccharides in anti-HER2 antibodies.

[0031] In certain embodiments of the present invention, based on the total volume of the N-deglycosylation treatment, the concentration ratio of the anti-HER2 antibody to PNGase F is: 25 μg / μL: (10,000-20,000) units / μL.

[0032] In certain embodiments of the present invention, the method for modifying the anti-HER2 antibody is to replace the 297th alanine (A) of the anti-HER2 antibody (amino acid sequence as shown in SEQ ID NO.1) with aspartic acid (N), i.e., perform N297A mutation. This mutation removes the glycosylation of the Fc segment of the anti-HER2 antibody.

[0033] Amino acid sequence of anti-HER2 antibody:

[0034] Anti-HER2 Heavy chain(SEQ ID NO.1)

[0035] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0036] Anti-HER2 Light chain(SEQ ID NO.2)

[0037] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0038] In certain embodiments of the present invention, the method for modifying the anti-HER2 antibody is to mutate the following sites of the anti-HER2 antibody (amino acid sequence as shown in SEQ ID NO.1): L234A, L235A and P329G.

[0039] In certain embodiments of the invention, the macrophages are alveolar macrophages.

[0040] In certain embodiments of the invention, the Fc receptor is an FcγR.

[0041] The HER2-positive cancer is a cancer that can be treated with T-DXd.

[0042] In certain embodiments of the present invention, the HER2-positive cancer is selected from breast cancer, non-small cell lung cancer, esophageal cancer, gastric cancer or colorectal cancer.

[0043] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0044] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0045] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0046] The following examples address the clinical problem of interstitial pneumonia caused by ADCs, and propose a solution to reduce the incidence of interstitial pneumonia by engineering the antibody portion of ADCs, designing and synthesizing engineered antibody ADCs. The experimental results show that macrophages can reduce the endocytosis of engineered antibodies, thereby reducing the incidence of interstitial pneumonia or reducing its severity. The research results provide a strong theoretical basis for the development of subsequent animal experiments and clinical trials, and engineered antibody ADCs show great potential for clinical transformation.

[0047] Materials and Experimental Methods Used in Examples

[0048] Experimental reagents:

[0049] Antibodies: trastuzumab for injection (440 mg, Roche), DXd (MCE), 1× PBS buffer (pH 7.2-7.4, pH 8.5, Solarbio), South American fetal bovine serum (ExCell Bio); double antibody (penicillin / streptomycin, Biosharp); New Zealand fetal bovine serum (Gbico); 1640 complete medium (Gbico); bovine serum albumin (BSA, Sigma-Aldrich); Hoechst33342 staining solution (Biosharp); 4% paraformaldehyde / universal tissue fixative (Biosharp); Cell Counting Kit-8 cell counting reagent (CCK8) (Unionway); 0.25% trypsin + 0.02% EDTA solution (Cienry); BL203B serum-free cell freezing solution (Biosharp); phorbol ester (PMA, Sigma)

[0050] Cell culture:

[0051] Human esophageal cancer cell line OE19, human gastric cancer cell line NCI-N87, and mouse colon cancer cell line CT26 (transfected with human HER2); mouse alveolar macrophage cell line MH-S, mouse mononuclear macrophage leukemia cell line RAW264.7, and human mononuclear leukemia cell line THP-1. All cells were cultured in 1640 medium with 10% South American fetal bovine serum and 1% double antibody, and THP-1 cells were cultured with 0.05mM β-mercaptoethanol. All cells were placed in a humidified incubator containing 95% air and 5% CO2, and the constant temperature was adjusted to 37°C.

[0052] method

[0053] 1. Preparation of engineered antibodies and antibody-fluorescent dye conjugation

[0054] 1. PNGase F treatment of full-length Trastuzumab to obtain de-N-sugared Trastuzumab (Trastuzumab-0705):

[0055] Take 500μg of Trastuzumab, add 3μL 10×GlycoBuffer, 1μL PNGase F, add pure water to the total volume of the reaction system to 20μL, and mix well. Incubate at 37°C for 4-24 hours. Use protein gel electrophoresis and matrix-assisted laser desorption tandem time-of-flight mass spectrometry for structural characterization. The results showed that the molecular weight of the antibody decreased from 148kDa to 145kDa, indicating that the N-sugar on the antibody has been completely hydrolyzed. The desugared antibody and free N-sugar are separated by a desalting column to obtain the desugared antibody (Trastuzumab-0705).

[0056] 2. Synthesis of Trastuzumab (297A mutation) and Trastuzumab (LALAPG mutation):

[0057] N297A mutation: In immunoglobulins, alanine (A) is replaced by aspartic acid (N) at position 297. This mutation removes glycosylation in the Fc region and reduces binding to Fc receptors and C1q.

[0058] LALAPG mutations: including three point mutations: L234A, L235A and P329G. The LALAPG variant inhibits binding to FcγR and C1q, but does not affect FcRn binding and Fc stability.

[0059] The amino acid sequence of the antibody was derived from previous literature and DrugBank. After antibody synthesis, SDS-PAGE and HPLC analysis confirmed that the purity was >95%.

[0060] 3. Antibody-fluorescent dye coupling:

[0061] Fluorescent dyes are labeled on antibodies to achieve immunofluorescence staining of antigens in cells or animals. Three modified Herceptin and full-length Herceptin were coupled with Cy3 fluorescent dye. Take 1 mg of antibody and react at a ratio of antibody: Cy3 = 1:5, add PBS with pH = 8.5, the total reaction volume is 500 μL, and react at room temperature for 4 hours. Use a desalting column to separate antibody-CY3 and free CY3 to obtain labeled antibody-CY3. The ratio of protein concentration to fluorescence intensity is determined to confirm that the number of CY3s connected to the four antibodies is consistent. Subsequently, protein gel electrophoresis is performed on the four antibodies and antibody-CY3, and CY3 is located on the gel using a fluorescent imager, and protein localization is performed using Coomassie Brilliant Blue staining.

[0062] 2. Macrophage endocytosis antibody-Cy3

[0063] In order to compare the internalization capacity of different engineered Herceptin by macrophages, nonspecific internalization experiments were performed. Flow cytometry and confocal microscopy were used for quantitative and qualitative analysis.

[0064] Flow cytometry was used to detect the internalization capacity of macrophages to different modified Herceptin. 1×10 6 Cells / well, different modified antibody-CY3 (LUO-CY3, 0705-CY3, 297A-CY3 and LALAPG-CY3) were added to each well and incubated in an incubator for 4 hours. Next, the cells in each well were collected and rinsed three times in PBS and resuspended in PBS. The mean fluorescence intensity (MFI) of the cells in each sample was collected using a flow cytometer. From the experimental results, it can be concluded that antibody modification will reduce the endocytosis of macrophages.

[0065] Confocal immunofluorescence staining: 1×10 6 Cells were seeded in 35 mm glass bottom dishes, 2 mL of cell culture medium was added, and cultured at 37 ° C overnight. After good adhesion, the culture medium was removed, and the cells were rinsed twice in PBS. The cells were endocytosed with different modified antibodies-CY3 (LUO-CY3, 0705-CY3, 297A-CY3 and LALAPG-CY3) in the incubator for 4 hours, and then rinsed three times in PBS after the endocytosis. The cell nucleus was stained with Hoechst 33342 for 10 minutes. Rinse three times with PBS. After rinsing, the samples were stored in PBS and photographed using a single-photon confocal microscope (A1 HD25, NIKON) to visually show the behavior of cell endocytosis of antibodies.

[0066] 3. Tumor cell binding / endocytosis antibody-Cy3

[0067] The binding ability and internalization phenomenon of different modified Herceptin were detected by flow cytometry and confocal microscopy.

[0068] Flow cytometry: HER2-positive tumor cell lines were selected: human esophageal cancer cell OE19 and human gastric cancer cell NCI-N87. First, 5×10 6 After ice bath, the non-specific antigen epitopes of the cells were blocked with PBS containing 1% bovine serum albumin (BSA) for 30 minutes, and then the cells were resuspended with 1 mL of blocking solution and divided into five groups. 2 μL PBS, LUO-CY3, 0705-CY3, 297A-CY3 and LALAPG-CY3 were added to each group, and incubated in a light-proof shaking table for 45 minutes. After washing twice, the mean fluorescence intensity (MFI) of the cells was detected using a flow cytometer (CytoFLEX LX, Beckman Coulter) to compare the specific binding ability of different modified antibodies to cells.

[0069] Confocal immunofluorescence staining: 1×10 6 Cells were seeded in 35 mm glass bottom dishes, 2 mL of cell culture medium was added, and incubated at 37 ° C overnight. After incubation, the cells were rinsed twice with PBS and blocked with 1% BSA in an ice bath for 30 minutes. After blocking, the cells were co-stained with different modified antibody-CY3 (LUO-CY3, 0705-CY3, 297A-CY3 and LALAPG-CY3) at room temperature for 1 hour, and then rinsed three times with PBS. The nuclei were stained with Hoechst 33342 for 10 minutes and then rinsed three times with PBS. The samples were stored in PBS and photographed using a confocal microscope to observe the cell membrane surface binding of different modified antibodies.

[0070] Confocal microscopy to verify endocytosis: 1 × 10 6 For each group, add 1 mL of culture medium and incubate overnight at 37°C. After attachment, remove the culture medium and rinse once with PBS. Under ice bath conditions, add 2 μL of PBS, LUO-CY3, 0705-CY3, 297A-CY3 and LALAPG-CY3 respectively and stain on ice for 45 minutes. After aspirating PBS, rinse once with pre-cooled PBS, add 1 mL of PBS, and place at 37°C for 0, 15, 30, 60, 120 and 240 minutes of endocytosis experiments. After each group reaches the time, add paraformaldehyde to fix the cells for 20 minutes, wash with PBS, add 1 mL of Hoechst dye, stain at 37°C for 20-30 minutes, and then wash 1-2 times with PBS. Finally, add 1 mL of PBS to keep the cells moist and use a confocal microscope to observe the endocytosis phenomenon.

[0071] ADC Preparation

[0072] The expressed and purified antibody was diluted to 2 mg / mL with PBS, and 10 times the molar amount of tris(2-carbonylethyl)phosphine hydrochloride (TECP) was added and reduced at 37°C for 2 hours to obtain an antibody with open interchain disulfide bonds. Then, 5 times the molar amount of Deruxtecan was reacted at room temperature for 4 hours. Deruxtecan is a conjugate of a derivative (Dxd) of the toxic drug DX-8951 and a maleimide-GGFG peptide linker. After the reaction, ADC and small molecule drugs were separated by a desalting column to obtain four ADCs (LUO-Dxd, 0705-Dxd, 297A-Dxd, LALAPG-Dxd). Mass spectrometry was subsequently used to determine the molecular weight and the number of small molecule drugs connected to each antibody to ensure that the number of Dxd connected to the four antibodies was the same.

[0073] ADC in vitro cytotoxicity assay: 1×10 4 / 100μL suspension inoculated cells, OE19 cells and N87 cells were inoculated in 96-well plates, respectively, and pre-cultured in an incubator for 24h. The drugs (LUO-Dxd, 0705-Dxd, 297A-Dxd, LALAPG-Dxd) were diluted with culture medium in a gradient: 102-10-5μg / mL, the culture medium in the 96-well plate was aspirated, and 100μL of drug solution was added to each column according to different concentration gradients (6 replicates for each concentration), and the plates were placed in an incubator for 72h. The original culture medium was aspirated and 100μL of CCK-8 working solution was added to each well. The culture plate was incubated in an incubator for 1-4 hours (the duration depended on the condition of each cell, and the OD value of the control group was optimal in the range of 0.8-1.2). The absorbance at a wavelength of 450 nm was measured by a microplate reader (SPARK, TECAN), and the obtained OD value was processed by EXCEL to obtain the cell growth rate corresponding to each drug concentration. Finally, the obtained data was processed by the 4-parameter nonlinear regression model of GraphPad, and the inhibition rate curve was fitted to obtain the IC50 value of the drug for the cell line.

[0074] 5. In vivo drug molecule organ distribution experiment

[0075] The experiment used female BALB / c mice aged 3-5 weeks. CT26-HER2 cell tumors from the same strain of mice were implanted subcutaneously in the groin to construct a subcutaneous tumor model. The tumor volume reached 100-200mm. 3Afterwards, the mice were randomly divided into five groups, 5 in each group. Each group of mice was intravenously injected with PBS, LUO-CY3, 0705-CY3, 297A-CY3 and LALAPG-CY3 at a dose of 5 mg / kg. The mice were killed 48 hours after injection, and the organs were removed. The small animal in vivo imaging system (IVIS Lumina SeriesIII, PerkinElmer) was used to image each organ at a wavelength of 520-570nm, and the mean fluorescence intensity (ROI) was quantitatively analyzed. The results of lung quantification and data analysis showed that antibody modification would reduce the deposition of antibody-CY3 in the lungs.

[0076] At the end of the experiment, mouse tumor and lung tissue sections were taken for immunofluorescence staining of CD68 (lung macrophage marker) and F4 / 80 (tumor macrophage marker) to observe the co-localization of related markers and HER2-Cy3.

[0077] Then, mouse tumor and lung tissue sections were taken for immunofluorescence staining of F4 / 80 (macrophage marker) to observe the co-localization of the marker and antibody-CY3. The results of immunofluorescence staining showed that antibody-CY3 and F4 / 80 were co-localized, which indicates that antibody-CY3 is internalized by macrophages.

[0078] VI. In vivo efficacy verification

[0079] Similarly, female BALB / c mice of 3-5 weeks old were used to subcutaneously implant CT26-HER2 cell tumors from the same strain of mice in their groin to construct a subcutaneous tumor model. The tumors grew to a volume of approximately 50-100 mm. 3 The mice were then randomly divided into 5 groups, 5 mice in each group, and given PBS and the other four ADC drugs (LUO-Dxd, 0705-Dxd, 297A-Dxd, LALAPG-Dxd) respectively. The same amount of drugs was injected into the tail vein every 3 days at a dose of 5 mg / kg for a total of 4 injections. The tumor volume and mouse weight were measured every other day, and the endpoint was reached two weeks after the start of treatment. When the tumor volume of the control group mice reached 1500mm 3 , the experiment was stopped, the animals were killed, and each tumor mass was taken out for weighing and photographing. After the experiment, the subcutaneous tumor was removed and the size of the mass was measured. At the same time, tumor tissue samples were retained for subsequent RNA-seq and WB experiments.

[0080] VII. Pneumonia Induction Experiment

[0081] The same strain of mice as mentioned above was also used, with 50 mice in total and 10 mice in each group. PBS and ADC modified with different antibodies (LUO-Dxd, 0705-Dxd, 297A-Dxd, LALAPG-Dxd) were injected through the tail vein. The ADC dose was 5 mg / mL and the drug was administered twice a week. The experimental period was 2-3 months. In the second half of the experiment, orbital blood was collected every half a month for the detection of inflammatory factors. At the end of the experiment, we took mouse tumor and lung tissue sections for HE staining and immunohistochemical analysis to observe the lung tissue structure and the expression changes of inflammatory indicators. At the same time, lung tissue samples were retained for subsequent RNA-seq and WB experiments.

[0082] Example 1. Preparation of engineered antibodies and antibody-fluorescent dye coupling

[0083] Figure 1 Schematic diagram of the antibody structure involved. Figure 2 The results showed that the molecular weight of the antibody decreased from 148 kDa to 145 kDa, indicating that the N-sugar on the antibody had been completely hydrolyzed. Figure 3 The results showed that the molecular weight of the antibody coincided with the fluorescence position, and four antibody-CY3 (LUO-CY3, 0705-CY3, 297A-CY3 and LALAPG-CY3) conjugates were obtained.

[0084] Example 2 Macrophage endocytosis antibody-Cy3

[0085] Figure 4 After THP-1 was induced by PMA, the expression of CD68 increased, and the cell morphology changed significantly, the cells changed from a suspended state to an adherent state, and the organelles became more obvious. Based on the WB results and cell morphology, human macrophages were successfully induced. Figure 5 The flow cytometry and confocal results of three types of macrophage endocytosis show that the engineered antibody reduces the endocytosis of antibody-Cy3 by macrophages.

[0086] Example 3 Tumor cell binding / endocytosis antibody-Cy3

[0087] Figure 6 The flow cytometry and confocal results of three tumor cell binding antibodies-Cy3. The results show that the MFI of the engineered antibody and the full-length antibody are almost the same, with no significant difference, and the confocal results also show the same trend. That is, the engineered antibody will not affect the binding of the antibody Fab segment to the tumor cell antigen. Figure 7 This is the confocal result of tumor cell internalization of antibody-Cy3, and the image was analyzed by fluorescence quantitative analysis using Image J. The results showed that the internalization rate of antibody-Cy3 gradually increased over time, reaching about 60% after 4 hours, and the internalization rates of the four antibody-Cy3 were almost the same.

[0088] Example 4 ADC preparation

[0089] Figure 8 The structure of the synthesized ADC is shown in Figure 2. The structure of the synthesized ADC is characterized after synthesis. From the mass spectrometry results, it can be seen that the number of small molecules connected to the four synthesized ADCs is the same, and about 8 small molecule drugs can be connected. Fig. 9 It is a method that uses CCK8 at the cellular level, and gradient administration inhibition is used to calculate the IC50 of various drugs for different cell lines. The results show that different ADCs have the same cell-killing effect.

[0090] Example 5 In vivo drug molecule organ distribution experiment

[0091] Antibody-Cy3 was injected into the tail vein, and the lungs were dissected out 48 hours later and the fluorescence intensity of the lung organs and tumors was recorded using IVIS. The average fluorescence intensity of each part was calculated using two-way ANOVA, and it can be seen intuitively that the fluorescence intensity of each group of tumor tissues is similar, but the deposition of engineered antibodies in the lungs is significantly reduced. At the end of the experiment, mouse tumor and lung tissue sections were taken for immunofluorescence staining of CD68 (lung macrophage marker) and F4 / 80 (tumor macrophage marker) to observe the co-localization of related markers and antibody-Cy3. The immunofluorescence results are shown in the figure. Fig.10 The results showed that antibody-Cy3 co-localized with fluorescent antibodies of CD68 and F4 / 80, which proved that antibody-Cy3 was significantly aggregated in macrophages in the lungs and tumors. The engineered antibody can effectively reduce its endocytosis by lung macrophages, but has no significant effect on the endocytosis of tumor macrophages. Example 6 In vivo efficacy verification

[0092] After the subcutaneous tumor model was successfully established, after 10 days of drug inhibition, the tumor volume and body weight of each experimental group were recorded at different time points. At the end of the experiment, the dissected in vitro tumors were shown in the figure. The data of each drug group were analyzed by two-way ANOVA. Fig.11 The results showed that the efficacy of each ADC was similar, and there was a significant difference between the PBS blank control group and the tumor growth was inhibited. The body weight of mice in each group did not change significantly during the entire treatment process. After measuring the weight of the in vitro tumor and analyzing it, the tumor weight of the ADC treatment group was also significantly lower than that of the PBS group.

[0093] discuss:

[0094] Based on T-DXd, we engineered the antibody part of the drug. The original T-DXd antibody part is trastuzumab targeting HER2, and we modified it in three ways: the first method is to use the enzyme PNGase F for N-glycan removal; the second method is to introduce the 297A site mutation; the third method is to introduce the LALAPG mutation. First, we de-N-glycanized trastuzumab and structurally characterized the treated antibody and N-glycans by SDS-PAGE and mass spectrometry. Then, the full-length antibody and the engineered antibody were labeled with Cy3 to obtain antibody-Cy3 for subsequent experiments. Next, we selected HER2-positive tumor cells (human esophageal cancer cell line OE19, human gastric cancer cell line NCI-N87, mouse colon cancer cell line CT26 (transfected with HER2)), macrophages (mouse alveolar macrophage cell line MH-S, mouse mononuclear macrophage leukemia cell line RAW264.7, and human macrophage cell line THP-1) for experiments. We compared the binding ability of HER2-positive tumor cells to different engineered antibodies, observed the specific binding behavior of tumor cells to antibodies using confocal microscopy, and performed quantitative analysis using flow cytometry. Subsequently, the endocytosis behavior of MH-S, RAW264.7, and THP-1 (PMA-induced) was observed after incubation with different antibodies-Cy3 for 4 hours at 37°C, and the amount of endocytosis was determined by flow cytometry.

[0095] Based on the above, we used 3-5 week old female BALB / c mice, implanted CT26-HER2 cell tumors in the groin, constructed subcutaneous tumor models, and injected different antibodies-Cy3 into the tail vein of the mice. We used IVIS to observe the organ distribution of fluorescent antibodies in the mice and performed quantitative fluorescence analysis on each organ. At the end of the experiment, we took sections of mouse tumors and lung tissues for immunofluorescence staining of CD68 (lung macrophage marker) and F4 / 80 (tumor macrophage marker) to observe the co-localization of related markers with antibody-Cy3.

[0096] Finally, we synthesized engineered antibody ADCs and performed in vitro killing experiments on tumor cells. The same mice were used to construct a subcutaneous tumor model, and the synthesized ADCs were injected through the tail vein to verify whether the engineering modification of the antibody changed the inhibitory effect of T-DXd on tumors. Pneumonia induction experiments were performed on mice, and T-DXd with different engineered antibodies was injected into the tail vein in groups. Lung tissue sections were taken for H&E and IHC staining, and pathological analysis was performed to observe whether the engineering modification of the antibody reduced the incidence of pneumonia induced by T-DXd.

[0097] Results: SDS-PAGE and mass spectrometry analysis showed that the molecular weight of the antibody dropped from 148 kDa to 145 kDa, indicating that PNGase F successfully removed the N-sugar. This means that we obtained intact N-sugar chains and N-sugar-free antibodies.

[0098] The results of flow cytometry endocytosis experiments showed that the internalization rate of engineered HER2 antibodies by MHS, RAW264.7 and induced THP-1 was 30%-50% lower than that of full-length HER2 antibodies. Images taken with confocal microscopy also showed the same results. This verifies that the nonspecific internalization of engineered HER2 antibodies by macrophages is reduced.

[0099] The results of the antigen-antibody binding experiment by flow cytometry showed that OE19, N87 and CT26-HER2 had basically the same binding ability with the engineered antibody-Cy3, and the results of confocal microscopy also supported this result. This indicates that antibody engineering will not affect the specific binding of antibody-antigen, that is, it will not affect the Fab segment of the antibody.

[0100] The organ distribution data of the animal model showed that the engineered antibody had reduced deposition in the mouse lungs. Immunofluorescence results showed that antibody-Cy3 co-localized with fluorescent antibodies for CD68 and F4 / 80, proving that antibody-Cy3 accumulated in macrophages in the lungs and tumor sites.

[0101] The results of the efficacy validation experiment showed that the efficacy of the ADC drug synthesized from the engineered antibody did not change.

[0102] Conclusion: Overall, our study showed that engineered antibodies can reduce the level of ADC internalization by macrophages, thereby reducing the occurrence of interstitial pneumonia. This finding provides useful clues for further optimizing ADC treatment strategies.

[0103] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present 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 the present invention.

Claims

1. Use of an engineered antibody-drug conjugate in the preparation of a HER2-positive cancer treatment product, wherein the engineered antibody-drug conjugate is a drug in which the anti-HER2 antibody in the original antibody conjugate is modified to reduce the binding of its Fc segment to the Fc receptor on macrophages, and the probability of interstitial pneumonia in the HER2-positive cancer treatment product is lower than that of the original antibody conjugate.

2. The use according to claim 1, characterized in that The original antibody conjugate is selected from T-DXd, T-DM1, Trastuzumab Duocarmazine, RC48, Tisotumab vedotin-tftv, Loncastuximab tesirine-lpyl, Belantamab mafodotin-blmf, Sacituzumab govitecan, Enfortumab vedotin, Polatuzumab vedotin-piiq, Moxetumomab pasudotox, Inotuzumab ozogamicin, Brentuximab vedotin or Gemtuzumab ozogamicin.

3. The use according to claim 1, characterized in that The method for modifying the anti-HER2 antibody is to remove N-sugar from the anti-HER2 antibody to obtain the N-sugar-removed antibody.

4. The use according to claim 3, characterized in that The step of N-glycosidase treatment comprises the following steps: using N-glycosidase to treat the anti-HER2 antibody to remove N-glycosidase to obtain the N-glycosidase antibody.

5. The use according to claim 4, characterized in that The N-glycosidase is PNGase F.

6. The use according to claim 1, characterized in that: The method for modifying the anti-HER2 antibody is to perform N297A mutation on the anti-HER2 antibody with the amino acid sequence as shown in SEQ ID NO.

1.

7. The use according to claim 1, characterized in that The method for modifying the anti-HER2 antibody is to mutate the following sites of the anti-HER2 antibody whose amino acid sequence is shown in SEQ ID NO.1: L234A, L235A and P329G.

8. The use according to claim 1, characterized in that The macrophages are alveolar macrophages.

9. The use according to claim 1, characterized in that The HER2-positive cancer is a cancer that can be treated with the original antibody conjugate.

10. The use according to claim 1, characterized in that The HER2-positive cancer is selected from breast cancer, non-small cell lung cancer, esophageal cancer, gastric cancer or colorectal cancer.

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