A design method for reducing side effects of antibody drug conjugates

Abstract

The present application relates to a kind of design methods for reducing antibody drug conjugate side effects.The design method includes: analyzing the binding site of antibody and macrophage surface receptor in antibody drug conjugate, carrying out site-directed modification (such as amino acid substitution and chemical modification) on the binding site on antibody, or, screening the competitive blocker of the binding site on receptor, the antibody drug conjugate is combined with the competitive blocker.The present application deeply analyzes the mechanism of adverse reaction induced by ADC drug, first determines the key molecular interface of the accumulation of ADC in macrophage and inflammatory response, and designs corresponding blocking strategy, which can effectively reduce the non-specific uptake of ADC in macrophage and inflammatory response, while not affecting its therapeutic activity, so as to realize the safety optimization of ADC drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a design method for reducing the side effects of antibody-drug conjugates. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a class of targeted drugs that chemically link monoclonal antibodies to highly effective cytotoxic drugs. As a significant breakthrough in cancer treatment in recent years, ADCs leverage the specific recognition capabilities of antibodies to precisely deliver highly effective cytotoxic drugs to tumor cells, achieving the dual advantages of "highly effective killing + reduced systemic toxicity." Trastuzumab deruxtecan (T-DXd, also known as DS-8201) is a HER2-targeting ADC that has demonstrated significant anti-tumor activity in multiple clinical trials and was approved by the FDA in 2019 for the treatment of metastatic HER2-positive breast cancer. In 2024, T-DXd further became the first ADC approved for a "tissue-independent indication," for the treatment of tumors with HER2 activating mutations, regardless of their tissue origin.

[0003] However, accompanying clinical application is the prevalent and severe toxicity caused by T-DXd and other ADCs, especially adverse events related to inflammatory responses, such as drug-induced interstitial lung disease / pneumonia (ILD), fever and cytokine-like reactions, gastrointestinal inflammation (mucositis, diarrhea), bone marrow suppression followed by infection or inflammation, liver inflammation, and skin inflammation. These adverse events not only significantly affect patients' quality of life but may also be life-threatening, severely limiting the safe application and efficacy of ADCs.

[0004] Existing treatments include dose reduction, treatment interruption or discontinuation, corticosteroids, and supportive care, but these measures cannot fundamentally address the problem of abnormal accumulation and uptake of ADCs in non-target cells (such as macrophages). Furthermore, traditional ADC drug design methods generally include three basic units: antibody design, drug carrier design, and conjugate design. Although ADCs are typical biopharmaceutical drugs, consisting of nanoparticles built on a protein backbone and possessing "nanoparticulate" properties, classical pharmaceutical research has not adequately considered this aspect, which is one of the important reasons for off-target effects and side effects (such as interstitial pneumonia or pulmonary fibrosis) of ADC drugs.

[0005] In summary, there is an urgent need for an innovative approach that, starting from the molecular binding mechanism, reduces the non-specific uptake of ADCs in macrophages, thereby lowering the risk of lung inflammation and ILD. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a design method for reducing the side effects of antibody-drug conjugates, with the aim of developing low-side-effect ADC drugs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a design method for reducing the side effects of antibody-drug conjugates, the design method comprising: Analyze the binding sites of antibodies and macrophage surface receptors in antibody-drug conjugates, perform site-specific modifications on the binding sites of antibodies (such as amino acid substitution and chemical modification), or screen for competitive inhibitors of the binding sites on the surface receptors, and use the antibody-drug conjugates in combination with the competitive inhibitors.

[0008] This invention deeply analyzes the mechanism of adverse reactions induced by antibody-drug conjugates (such as T-DXd), and finds that adverse reactions are related to the uptake of antibody-drug conjugates by macrophages. Two types of solutions are designed in a targeted manner: (1) Molecular engineering modification strategy, which analyzes the binding sites on the antibody and performs site-specific modifications (such as amino acid substitution and chemical modification); (2) Competitive blocking agent strategy, which uses competitive blocking agents to block the binding of the two, which can effectively reduce the non-specific uptake and inflammatory response of antibody-drug conjugates in macrophages, while not affecting their therapeutic activity, thereby optimizing the safety of ADC drugs.

[0009] Optionally, the antibody in the antibody-drug conjugate includes trastuzumab.

[0010] Optionally, the macrophage surface receptors include scavenger receptors.

[0011] Optionally, the scavenger receptor may include the MSR1 receptor.

[0012] Optionally, the binding sites on the trastuzumab include T167 (heavy chain), D215 (heavy chain), V128 (light chain), Q112 (light chain), L115 (light chain), G8 (light chain), Y33 (heavy chain), Y52 (heavy chain), H91 (light chain), and T31 (light chain), and the binding sites on the MSR1 receptor include Q208, R124, A90, N221, A224, E225, R362, R351, Q407, and W371.

[0013] Optionally, the competitive blocker includes at least one of fucoidan, dextran sulfate, heparin, lipopolysaccharide, polynucleotide, nucleic acid mimic, oxidized lipoprotein, modified lipoprotein, or transferrin.

[0014] In this invention, the process of analyzing the binding sites of antibodies to scavenger receptors on the surface of macrophages in antibody-drug conjugates and screening competitive inhibitors for binding sites on scavenger receptors can be carried out based on methods such as immunoprecipitation, ELISA, molecular docking, and point mutation verification.

[0015] In a second aspect, the present invention provides a site-modified version of trastuzumab, wherein the amino acid sequence of the site-modified version of trastuzumab includes a sequence after site-modification based on the sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2; the site-modified site includes the antibody binding site as described in claim 1.

[0016] SEQ ID NO.1 (heavy chain): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0017] SEQ ID NO.2 (light chain): DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0018] The positions of disulfide bridges in the antibody are: H22-H96, H147-H203, H264-H324, H370-H428, H229-H'229, H232-H'232, L23-L88, L134-L194, and H223-L214.

[0019] Optionally, the site for targeted modification includes at least one of Y33 (heavy chain), Y52 (heavy chain), H91 (light chain), or T31 (light chain).

[0020] Optionally, the site-directed modification (amino acid substitution mutation) includes at least one of T167A, T167V, D215A, D215K, Q112A, Q112E, V128A, V128D, L115A, L115D, G8A, Y33A, Y33F, Y33H, Y33W, Y33R, Y33K, Y52A, Y52R, Y52F, Y52H, Y52W, Y52S, H91A, H91Q, H91N, H91F, H91K, H91R, T31A, T31S, T31V, T31L, T31W, T31D, or T31E.

[0021] This invention identifies and verifies that MSR1-R362 and Trastuzumab-Y33 are key binding sites for non-specific uptake of ADCs; this interaction is the molecular basis for ADC side effects such as inflammation and pulmonary toxicity. By site-directed mutation or engineering of Trastuzumab-Y33 residues, the non-specific binding with MSR1-R362 can be reduced; this can significantly reduce the inflammatory response caused by uptake by non-tumor cells while maintaining targeting activity.

[0022] Thirdly, the present invention provides an antibody-drug conjugate comprising the trastuzumab site-directed modifier described in the second aspect and the drug conjugated thereto.

[0023] Fourthly, the present invention provides a combination drug composition comprising the antibody-drug conjugate and a competitive inhibitor from the design method for reducing the side effects of antibody-drug conjugates in the first aspect.

[0024] Optionally, the combined pharmaceutical composition may further include pharmaceutically acceptable excipients.

[0025] Optionally, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations; Optionally, the pharmaceutical excipients include any one or a combination of at least two of the following: delivery carrier, filler, binder, wetting agent, disintegrant, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0026] Fifthly, the present invention provides the use of the trastuzumab site-specific modifier described in the second aspect, the antibody-drug conjugate described in the third aspect, or the combination drug composition described in the fourth aspect in the preparation of antitumor drugs.

[0027] Optionally, the tumor includes HER2-positive tumors, such as breast cancer.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides an in-depth analysis of the mechanism by which ADC drugs induce adverse reactions. For the first time, it clarifies the key molecular interface between ADC accumulation and inflammatory response in macrophages and designs a multi-level intervention and application strategy that can effectively reduce side effects while maintaining the efficacy of ADC, thereby optimizing the safety of ADC drugs. Attached Figure Description

[0029] Figure 1 This figure shows the distribution of T-DXd in the major organs of mice after tail vein injection.

[0030] Figure 2 The image shows the results of immunofluorescence staining characterizing the distribution of lung cells after tail vein injection of T-DXd. Figure A shows the distribution of T-DXd cells compared to F4 / 80. + Macrophage colocalization detection results: Figure B shows the colocalization detection results of T-DXd and alveolar epithelial cells, and Figure C shows the colocalization detection results of T-DXd and mesenchymal fibroblasts. The figure below is a magnified view of the area corresponding to the dashed line, with a scale bar of 20 μm.

[0031] Figure 3 To characterize the MSR1 in the lungs after tail vein injection of T-DXd using immunofluorescence staining + Macrophage distribution results, scale bar 10 μm.

[0032] Figure 4 Figure showing the results of T-DXd uptake by macrophages after MSR1 knockdown.

[0033] Figure 5 Figure showing the T-DXd inflammation induction results in macrophages after MSR1 knockdown.

[0034] Figure 6 This is a schematic diagram illustrating the experimental principle of immunoprecipitation characterizing the binding of T-DXd and MSR1.

[0035] Figure 7 The image shows the results of immunoprecipitation characterization of the interaction between T-DXd and MSR1.

[0036] Figure 8 The figure shows the ELISA results characterizing the binding of T-DXd and MSR1.

[0037] Figure 9 The figure shows the ELISA results characterizing the binding of Trastuzumab to MSR1.

[0038] Figure 10 This diagram illustrates the interface between Trastuzumab and MSR1 for molecular docking characterization.

[0039] Figure 11 The diagram shows the main sites of binding of Trastuzumab to MSR1 in point mutation experiments.

[0040] Figure 12 This diagram illustrates the main modes of operation between Trastuzumab Y33 and MSR1 R362.

[0041] Figure 13 The figure shows the results of ELISA experiments characterizing the effect of the Y33A mutation on the binding of Trastuzumab to HER2.

[0042] Figure 14 The figure shows the results of ELISA experiments characterizing the effects of different blocking agents on the binding of Trastuzumab to MSR1.

[0043] Figure 15 A schematic diagram illustrating the mechanism by which Fuco blocks the binding of Trastuzumab to MSR1 through molecular docking characterization.

[0044] Figure 16 The figure shows the results of ELISA experiments characterizing the effect of Fuco blocker on the binding of T-DXd to HER2.

[0045] Figure 17 This is a confocal microscope image of a three-dimensional lung organoid model, with a scale bar of 100 μm.

[0046] Figure 18 This is a graph showing the IL-1β level results in a three-dimensional lung organoid model.

[0047] Figure 19 This figure shows the toxicity results induced by T-DXd in a three-dimensional lung organoid model.

[0048] Figure 20 Figure showing the results of Fuco alleviating inflammation induced by different HER2-targeting ADCs. Detailed Implementation

[0049] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0050] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0051] This invention addresses the common problem of non-specific uptake of HER2-targeting ADCs by lung macrophages during clinical use, leading to adverse reactions such as inflammatory responses and drug-induced interstitial lung disease. It delves into the mechanisms by which ADCs cause adverse reactions, discovering that ADC drugs can be recognized and uptaken by the macrophage scavenger receptor MSR1 (CD204) on the surface of macrophages. This triggers ceramide-dependent lysosomal instability, resulting in the activation and release of cathepsin B, inducing inflammation-related adverse reactions. Further analysis of the interaction mechanism between ADCs and MSR1 reveals a direct binding between the key MSR1 residue R362 and Y33 on the ADC antibody Trastuzumab. This is the molecular basis for the non-specific uptake of HER2-targeting ADCs by lung macrophages; that is, the binding of MSR1-R362 and ADC-Y33 is a crucial molecular event leading to related side effects. Specifically, this invention designs a solution that significantly reduces the non-targeted uptake of ADCs by macrophages by performing Y33 point mutations (such as Y33F, Y33R, Y33A, etc.) or by blocking the binding of R362 to Y33 (by screening and designing competitive blocking agents, such as fucoidan, dextran sulfate, heparin, etc.). This reduces the inflammatory response and toxicity induced by ADCs while maintaining the specific binding of ADCs to HER2 and their anti-tumor activity.

[0052] It is understood that, in addition to directly mutating the antibody, the hydrogen bonds or electrostatic interactions at the interface can also be disrupted by mutating or conservatively substituting key residues on the receptor side. For example, MSR1-R362 can be mutated to alanine (R362A), lysine (R362K), etc., and neighboring residues such as E360 and K363 can also regulate binding ability in a similar manner. The above mutations, as well as Trastuzumab-Y52A and Trastuzumab-T31A mutations, can also produce binding inhibition effects similar to those produced by the Y33A mutation, thereby effectively reducing the non-targeted uptake of ADCs mediated by MSR1. In addition, besides MSR1, key binding residues of other scavenger receptors or macrophage surface receptors can also achieve the function presented in this invention through similar mutation strategies, namely, reducing non-specific uptake of ADCs without affecting antigen-targeted binding.

[0053] Competitive inhibitors, such as small molecule inhibitors, aptamers, cyclic peptides, or direct antibody inhibitors, specifically bind to the interaction interface between MSR1 and trastuzumab. These inhibitors prevent non-specific recognition of MSR1 by the ADC through competitive binding or steric hindrance, thereby reducing macrophage uptake. This strategy can be used independently or in combination with residue mutations or ADC structural optimization to provide multiple approaches to reduce non-specific uptake.

[0054] In ADC molecular design, the affinity of antibody fragments or Fc regions for binding interfaces such as R362-Y33 can be reduced by adjusting these fragments. Modifications or point mutations of residues near Y33 (e.g., R31, F34) in the Trastuzumab Fab region weaken the binding stability with R362 while maintaining high affinity for HER2. Deglycosylation or key site mutations (e.g., N297A) in the Fc region reduce non-specific binding to the scavenger receptor family, indirectly weakening the R362-Y33-dependent uptake pathway. Optimization of drug linkers reduces hydrophobic exposed regions, decreasing the likelihood of the ADC being recognized by MSR1.

[0055] The above strategies can be used in combination. Combined strategies include, but are not limited to: Mutation + Blocker: Based on mutations of key residues in the ADC or receptor, combined use of blockers targeting interfaces such as R362-Y33 can further reduce macrophage uptake; Mutation + ADC Structure Modification: Based on mutations of key residues, optimizing the linker or Fc region structure enhances control over non-specific uptake; Blocker + ADC Structure Modification: Using blockers to inhibit binding, while simultaneously reducing incidental contact with MSR1 through ADC molecule optimization, achieving dual protection; Triple combination strategy: Combining mutations, blockers, and ADC structure modification provides the most comprehensive non-specific uptake inhibition regimen, minimizing the risk of side effects. This combined strategy is flexible, allowing for the selection of single or multiple strategies based on different ADC types, receptor characteristics, or clinical application needs.

[0056] In addition to directly intervening in the structure of the Y33-R362 interface, the purpose of this invention can also be indirectly achieved by regulating the expression or function of MSR1: downregulating the expression of MSR1 using siRNA, shRNA, or CRISPR technology to reduce the binding opportunities of R362-Y33; temporarily blocking the R362-Y33 binding interface by delivering specific short peptides or blocking agents through nanocarriers to reduce the phagocytosis rate of ADCs mediated by scavenger receptors.

[0057] In the specific embodiments of this invention, T-DXd (trastuzumab, catalog number HY-138298A) was purchased from MedChemExpress, USA; fucoidan (catalog number HY-132179) was purchased from MedChemExpress, USA; dextran sulfatedase (catalog number D110733) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; lipopolysaccharide (catalog number HYD1056) was purchased from MedChemExpress, USA; heparin (catalog number HY-17567) was purchased from MedChemExpress, USA; and mannose (YS-D182401) was purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. Example 1 This embodiment verifies MSR1-mediated nonspecific uptake by the ADC.

[0058] In vivo drug administration experiments were conducted using 6-8 week old male C57BL / 6 human HER2 transgenic mice (B-hHER2, mouse strain number 110812, BioMice). All animal experimental procedures followed ethical guidelines for laboratory animals and were approved by the Laboratory Animal Ethics Committee of the National Center for Nanoscience and Technology (ethics approval number: NCNST21-2407-0415). Four mice were randomly divided into two groups. In the experimental group, T-DXd was injected intravenously (iv) at a dose of 10 mg / kg. In the control group, only an equal volume of physiological saline was injected. Subsequently, 6 hours after injection, major organ tissues, including lung, liver, heart, kidney, and bone marrow, were collected and homogenized. The content of T-DXd in each tissue was quantitatively detected using a sandwich ELISA method with a monoclonal antibody against the cytotoxic load DXd of T-DXd. The total protein content of the tissues was determined by the BCA method, and the T-DXd level was expressed as ng / mg total protein. The lungs of mice treated with physiological saline served as the control group. Biological distribution results as follows Figure 1 As shown, T-DXd accumulates most significantly in the lungs, followed by significant distribution in the kidneys, bone marrow, and other tissues.

[0059] Simultaneously, another group of T-DXd-treated mice (n = 4) had their lung tissues fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Immunofluorescence staining was performed using antibodies targeting the epithelial cell marker EpCAM, the macrophage marker F4 / 80, and the fibroblast marker Vimentin, in combination with T-DXd detection antibodies. Co-localization analysis of T-DXd signals with each cell type marker signal was used to determine the uptake and distribution characteristics of T-DXd in different lung cell populations. Immunofluorescence (IF) imaging revealed that red-labeled T-DXd and some green-labeled F4 / 80... + Macrophage colocalization ( Figure 2 Figure A in the middle suggests that only a specific population of macrophages is involved in T-DXd uptake. In contrast, alveolar epithelial cells (EpCAM) + , Figure 2 (Figure B) and mesenchymal fibroblasts (vimentin) + , Figure 2 (Figure C) No significant T-DXd uptake was observed.

[0060] In addition, MSR1 was simultaneously labeled using multicolor IF staining technology. + Macrophages (green) and T-DXd (red), results show that T-DXd is involved in MSR1 + Major accumulation within macrophages provides direct visual evidence of non-targeted uptake of T-DXd by macrophages expressing MSR1. Figure 3 ).

[0061] Example 2 This embodiment conducts an experiment on the lack of MSR1 functionality.

[0062] To further elucidate the interaction between T-DXd and the macrophage surface scavenger receptor MSR1, in vitro experiments were conducted using THP-1-derived macrophages, and the effect of MSR1 knockdown on T-DXd uptake was compared.

[0063] The experimental procedure included: 1 × 10⁻⁶ wild-type or MSR1-knockdown THP-1 macrophages 5 Cells (100 cells / well) were seeded in 8-well slides and co-cultured in complete culture medium for 12 h. Then, T-DXd (100 nM) was added and incubated for another 24 h. Immunofluorescence staining with anti-DXd monoclonal antibody was performed, and the uptake of T-DXd in different cells was observed using confocal microscopy. The uptake amount was determined by quantitative fluorescence method. Simultaneously, the release level of IL-1β in the culture supernatant was detected by ELISA.

[0064] Experimental results showed that wild-type macrophages (Mø WT ) exhibited significant T-DXd endocytic capacity, while MSR1 knockdown macrophages (Mø MSR1- / - T-DXd uptake decreased significantly. Quantitative analysis further confirmed this. MSR1- / - T-DXd accumulation in cells was significantly reduced ( Figure 4 (P = 0.0045), suggesting that MSR1 is a key mediator of macrophage uptake of T-DXd. Notably, compared with the control group that only added culture medium, T-DXd treatment induced significant IL-1β secretion, while MSR1 knockdown significantly reduced IL-1β secretion (P = 0.0045). Figure 5 (P = 0.0056), suggesting that MSR1 plays a key role in ADC-induced inflammasome activation.

[0065] Example 3 This embodiment analyzes the MSR1-Trastuzumab binding mechanism.

[0066] The physical binding of MSR1 to T-DXd was verified by co-immunoprecipitation (Co-IP). THP-1 macrophages were treated with T-DXd (40 μg / mL), trastuzumab, or isotype control IgG for 2 h, and cell lysates were collected. Anti-human IgG pre-coated with Protein A / G magnetic beads bound to the Fc region of T-DXd was used for enrichment, followed by Western blotting detection using antibodies against MSR1 and cytotoxic DXd-loaded cells. Figure 6To assess the specificity of the interaction between the two, unconjugated DXd-containing Trastuzumab, isotype control IgG, and untreated lysate were used as controls. Experimental results are as follows: Figure 7 As shown, T-DXd treatment led to the co-precipitation of MSR1 and T-DXd, clearly demonstrating the physical interaction between the ADC and the receptor. Trastuzumab itself could also co-precipitate MSR1, but not T-DXd, suggesting that MSR1 binding is independent of chemically conjugated drug loading.

[0067] Furthermore, the binding affinity between T-DXd and MSR1 was quantitatively analyzed using ELISA. T-DXd or trastuzumab was coated into the wells of an ELISA plate, and after blocking, different concentrations of recombinant MSR1 protein were added and incubated at room temperature for 2 h. Subsequently, HRP-labeled secondary antibody and TMB chromogenic substrate were added, and absorbance was measured at 450 nm. EC50 was calculated using a four-parameter logistic regression model. 50 Value. Experimental results show that the EC of T-DXd 50 Approximately 120.5 nM ( Figure 8 Trastuzumab is 141.6 nM ( Figure 9 This indicates that DXd coupling has a limited impact on MSR1 identification.

[0068] To gain a structural understanding, this embodiment employs computer simulation analysis. Based on experimental results showing that drug loading has little impact on MSR1 binding, this embodiment constructs a computational model of the MSR1-Trastuzumab Fab fragment (excluding DXd) complex. Specific methods: The human MSR1 protein structure predicted by AlphaFold (UniProt ID: P21757) is used as the receptor model. The trastuzumab Fab structure is derived from the resolved crystal structure (PDB ID: 6MH2). Protein-protein docking is performed to obtain a potential MSR1-trastuzumab complex model. Key interaction interface residues are analyzed, visualized, and residue-level interaction mapping is performed. The results show that the scavenger receptor cysteine-rich domain (SRCR) of MSR1 is the main binding interface for Trastuzumab. Predicted contact residues include MSR1 R362, R351, Q407, W371 (marked in red) and Trastuzumab Y33 (heavy chain), Y52 (heavy chain), H91 (light chain), T31 (light chain) (marked in black). Figure 10 ).

[0069] To verify the calculation results, this embodiment used single-point amino acid substitution (Alanine scanning) to mutate candidate Fab residues (Y33A, Y52A, H91A, T31A) in the Trastuzumab antibody, and determined their effect on MSR1 binding by ELISA. The results are as follows: Figure 11 As shown, each mutation can reduce affinity to some extent, with the Y33A mutation causing a significant decrease in MSR1 binding affinity (EC). 50 (From 100 nM to about 400 nM), confirming that Y33 is one of the important residues for stabilizing the MSR1-Trastuzumab interaction.

[0070] Further molecular structure analysis revealed that Y33 is located in the complementarity-determining region 1 (CDR-H1) of the Trastuzumab heavy chain and is in close contact with MSR1 R362 through a network of hydrogen bonds and electrostatic interactions. Figure 12 This constitutes the key molecular basis for the non-specific uptake of ADCs in macrophages.

[0071] To further analyze the effect of mutations on the binding of Trastuzumab to HER2, the binding affinity between the mutant and HER2 was quantitatively analyzed using ELISA. Specifically, HER2 was coated into the wells of an ELISA plate, and after blocking, different concentrations of Trastuzumab and its mutants were added and incubated at room temperature for 2 h. Subsequently, HRP-labeled secondary antibody and TMB chromogenic substrate were added, and absorbance was measured at 450 nm. EC50 was calculated using a four-parameter logistic regression model. 50 Values. The results showed that the Y33A mutation in Trastuzumab Fab had almost no effect on HER2 targeting binding (EC). 50 Approximately 0.046 nM, wild-type EC 50 Approximately 0.057 nM), which is comparable to the T31A mutation negative control antibody ( Figure 13 This indicates that the Y33 residue is specific for binding to MSR1, but does not interfere with the classic HER2 recognition of Trastuzumab.

[0072] Based on the above results, the binding ability of Trastuzumab antibody to MSR1 can be reduced by mutating it, thereby reducing adverse reactions.

[0073] Example 4 This embodiment involves the screening of competitive inhibitors and analysis of their mechanisms of action.

[0074] Reported MSR1 ligands were screened, including dextran sulfate (DS), heparin, fucoidan (Fuco), and lipopolysaccharide (LPS) (Figure 11). Mannose was selected as a CD206 ligand. + Macrophage-targeting ligand design and control group use.

[0075] To identify ligands that can effectively block the MSR1-Trastuzumab interaction, a competitive ELISA assay was designed in this embodiment. Recombinant MSR1 was immobilized on a 96-well plate, blocked, and then the candidate ligand was added in the presence of Trastuzumab, followed by incubation at room temperature for 2 h. Subsequently, HRP-labeled secondary antibody and TMB chromogenic substrate were added, and absorbance was measured at 450 nm. EC50 was calculated using a four-parameter logistic regression model. 50 Value. Experimental results are as follows: Figure 14 As shown, among all tested ligands, Fuco exhibited the strongest competitive effect, leading to a decrease in the EC50 of Trastuzumab. 50 The concentration was increased from approximately 100 nM to approximately 2.2 μM. Other ligands may also produce similar effects under suitable conditions.

[0076] Furthermore, molecular docking simulations yielded the following results: Figure 15 As shown, adjacent O-sulfate groups on a single fucosylate ring of Fuco, as well as 2-O-sulfate groups on the adjacent ring, form a synergistic binding relationship with the arginine-rich region of the MSR1 SRCR domain, particularly R362, via electrostatic interactions. Furthermore, the branched structure and variable sulfation degree of Fuco may achieve simultaneous occupancy or steric hindrance of the SRCR domain surrounding R362. Based on this, this invention uses Fuco as a regioselective MSR1 binding inhibitor for further research.

[0077] To rule out the potential impact of Fuco on the binding of T-DXd to HER2, the binding of T-DXd to HER2 in the presence of Fuco was further analyzed and tested. The experimental procedure included immobilizing HER2 protein on a 96-well plate, blocking it, adding T-DXd in the presence of Fuco, and incubating at room temperature for 2 h. Subsequently, HRP-labeled secondary antibody and TMB chromogenic substrate were added, and absorbance was measured at 450 nm. EC50 was calculated using a four-parameter logistic regression model. 50 Value. Result as follows Figure 16 As shown, Fuco blockade has almost no effect on the targeted binding of T-DXd to HER2 (ECG). 50(Approximately 0.052 nM for the wild type and approximately 0.063 nM for the wild type), indicating that Fuco is specific for MSR1 blockade without interfering with the classic HER2 recognition of T-DXd.

[0078] Example 5 This embodiment includes tests on inflammatory response and toxicity relief.

[0079] To partially simulate the lung microenvironment, THP-1 macrophages, BEAS-2B cells, and HELF cells differentiated from phorbol PMA (10 ng / mL, 12 h) were mixed at a ratio of 1:8:1 and seeded in ultra-low adsorption 96-well round-bottom culture plates, and three-dimensional lung organoids were formed using the hanging drop method. In this embodiment, inflammatory responses and toxicity mitigation were tested in the three-dimensional lung organoid model. The experimental procedure included pre-incubation of the three-dimensional lung organoids with Fuco (1 μg / mL) for 6 h, followed by stimulation with T-DXd (100 nM). The activation level of caspase-1 in cells was assessed using the FAM FLICA caspase-1 detection kit (using the fluorescent inhibitor probe FAM-YVAD-FMK to specifically label active caspase-1 in live cells). A treatment with only culture medium was used as a control group. The experimental results showed that Fuco significantly alleviated the T-DXd-induced inflammatory response in the three-dimensional lung organoid model.

[0080] Furthermore, confocal microscopy results showed that Fuco could inhibit the activation of Caspase-1 (…). Figure 17 (See the figure above, P=0.0056). Furthermore, the secretion level of IL-1β in the culture supernatant was detected by ELISA, and the results showed that Fuco reduced IL-1β levels. Figure 18 The image below shows... P =0.0020), and the cell protective effect of Fuco was analyzed using Cell Counting Kit-8 (CCK-8) reagent. The results showed that Fuco reduced cytotoxicity ( Figure 19 , P <0.0001).

[0081] Example 6 This embodiment verifies the broad-spectrum protective effect.

[0082] To verify the protective effect of Fuco blockade against other Trastuzumab-related ADCs, this embodiment further analyzed ILD-related ADCs, including Trastuzumab duocarmazine (SYD985, approved for metastatic breast cancer) and Trastuzumab emtansine (T-DM1, a DM1-linked cytotoxic drug). The specific procedure was as follows: THP-1 cells were pre-incubated with Fuco (1 μg / mL) for 6 h, followed by stimulation with SYD985 and T-DM1 (100 nM). The secretion level of IL-1β in the culture supernatant was further detected by ELISA. The treatment with only culture medium was used as a blank control.

[0083] The experimental results showed that in macrophages, treatment with either SYD985 or T-DM1 (100 nM) induced significant IL-1β secretion, suggesting innate immune activation. Notably, Fuco pretreatment (1 μg / mL, 6 h) significantly attenuated this inflammatory response. Figure 20 This further supports its interference with the MSR1-Trastuzumab interaction.

[0084] In summary, this invention provides an in-depth analysis of the mechanisms by which ADC drugs induce adverse reactions, and for the first time clarifies the key molecular interfaces of ADC accumulation and inflammatory response in macrophages. Two feasible technical pathways are designed: (1) eliminating the binding ability of Trastuzumab to MSR1 through point mutation or molecular modification without affecting HER2 binding; (2) using competitive inhibitors (such as fucoidan) and other anionic polysaccharides to cover MSR1, selectively blocking the interaction between Trastuzumab and MSR1. Both methods can effectively reduce the non-specific uptake and inflammatory response of HER2-targeting ADCs in macrophages without affecting their anti-HER2 activity, thereby optimizing the safety of ADC drugs. Furthermore, these methods can not only be applied to the safety optimization of Trastuzumab series ADCs, but also extended to other antibody-drug conjugates, providing an operable solution for the molecular screening, mechanism research, and clinical application of ADCs, demonstrating significant scientific research value and clinical translational potential.

[0085] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A design method for reducing the side effects of antibody-drug conjugates, characterized in that, The design method includes: Analyze the binding sites of antibodies and macrophage surface receptors in antibody-drug conjugates, perform site-specific modifications on the binding sites of antibodies, or screen for competitive inhibitors of the binding sites on receptors, and combine the antibody-drug conjugates with the competitive inhibitors.

2. The design method for reducing the side effects of antibody-drug conjugates according to claim 1, characterized in that, The antibody-drug conjugate includes trastuzumab; Optionally, the macrophage surface receptors include scavenger receptors; Optionally, the scavenger receptor includes the MSR1 receptor.

3. The design method for reducing the side effects of antibody-drug conjugates according to claim 2, characterized in that, The binding sites on the trastuzumab include T167, D215, V128, Q112, L115, G8, Y33, Y52, H91, and T31, and the binding sites on the MSR1 receptor include Q208, R124, A90, N221, A224, E225, R362, R351, Q407, and W371.

4. The design method for reducing the side effects of antibody-drug conjugates according to any one of claims 1-3, characterized in that, The competitive blocker includes at least one of fucoidan, dextran sulfate, heparin, lipopolysaccharide, polynucleotide, nucleic acid mimic, oxidized lipoprotein, modified lipoprotein, or transferrin.

5. A site-specific modifier of trastuzumab, characterized in that, The amino acid sequence of the trastuzumab site-modified variant includes the sequence after site-modification based on the sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2; The site for targeted modification includes the antibody binding site described in claim 1.

6. The trastuzumab site-modified form according to claim 5, characterized in that, The site for targeted modification includes at least one of T167, D215, V128, Q112, L115, G8, Y33, Y52, H91, or T31; Optionally, the fixed-point modification includes at least one of T167A, T167V, D215A, D215K, Q112A, Q112E, V128A, V128D, L115A, L115D, G8A, Y33A, Y33F, Y33H, Y33W, Y33R, Y33K, Y52A, Y52R, Y52F, Y52H, Y52W, Y52S, H91A, H91Q, H91N, H91F, H91K, H91R, T31A, T31S, T31V, T31L, T31W, T31D, or T31E.

7. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate includes the trastuzumab site-directed modifier as described in claim 5 or 6 and the drug conjugated thereto.

8. A combination drug composition, characterized in that, The combination drug composition includes the antibody-drug conjugate and competitive blocker as described in claim 1, which are designed to reduce the side effects of antibody-drug conjugates.

9. The combination drug composition according to claim 8, characterized in that, The combined pharmaceutical composition further includes pharmaceutically acceptable excipients; Optionally, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations; Optionally, the pharmaceutical excipients include any one or a combination of at least two of the following: delivery carrier, filler, binder, wetting agent, disintegrant, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

10. The use of the trastuzumab site-directed modifier of claim 5 or 6, the antibody-drug conjugate of claim 7, or the combination drug composition of claim 8 or 9 in the preparation of antitumor drugs.

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