Preparation method and application of an antibody conjugate containing sLex tetrasaccharide structure

Abstract

The application provides a preparation method of an antibody conjugate containing an sLex tetrasaccharide structure, and comprises the following steps: S1, Endo S2 enzyme and alpha-L-fucosidase are sequentially added into a target antibody, glycosylation is analyzed respectively, and modification site conflict or sugar chain structure interference is avoided; S2, the antibody accurately characterized in S1, B4GalT1 and UDP-Gal are added into a Tris-HCl system containing Mn 2+ , incubation is performed, and reaction solution I is obtained; S3, reaction solution I, ST3Gal3 and CMP-Neu5Ac are mixed in a Tris-HCl system containing Mg 2+ , incubation is performed, CMP-N-acylneuraminic acid phosphodiesterase reaction is further added, Protein A purification is performed, and purified antibody I is obtained; S4, purified antibody I, alpha1, 3 FucT-2HR and GDP-Fuc are mixed in a Tris-HCl system containing Mg 2+ , incubation is performed, and Protein A purification is performed, so that the target product is obtained. The modified antibody obtained by the application can enhance the targeting property, further enhance the immune blocking activity of the antibody by enhancing the binding affinity of the antibody and the tumor cell surface antigen, realize the double enhancement of the "targeting property" and the "activity", and has the advantages of high activity, high specificity, high stability, high safety, high efficiency, high accuracy, high selectivity, high sensitivity, high reliability, high accuracy, high selectivity, high sensitivity, high reliability, high accuracy, high selectability, high sensitivity, high reliability, high accuracy, high selectivity, high sensitivity, high reliability and high accuracy.

Description

Technical Field

[0001] This invention belongs to the field of antibody-drug conjugate preparation technology, specifically relating to a method for preparing antibody-drug conjugates containing an sLeX tetrasaccharide structure and its application. Background Technology

[0002] Malignant tumors are a major disease posing a serious threat to human life and health worldwide. According to the World Health Organization's (WHO) "Global Cancer Report 2020," there were over 19.3 million new cancer cases and over 10 million cancer deaths globally that year, with both incidence and mortality rates continuing to rise. In the field of cancer treatment, traditional methods such as surgery, chemotherapy, and radiotherapy, while able to control tumor progression to some extent, have limitations such as poor targeting, strong side effects, and a high risk of drug resistance, making it difficult to meet the clinical demand for highly effective and low-toxicity cancer treatment regimens. In recent years, tumor immunotherapy, with its unique mechanism of "activating the body's own immune system to attack tumor cells," has become a revolutionary breakthrough in cancer treatment. Among these, tumor checkpoint antibody therapy is one of the core technologies of immunotherapy. It works by blocking the binding of "immune checkpoint molecules" (such as PD-1, PD-L1, CTLA-4, etc.) on the surface of tumor cells to receptors on the surface of immune cells, thereby relieving the "immunosuppression" of the immune system by the tumor and restoring the killing activity of immune cells against tumor cells. As of 2024, more than 30 tumor checkpoint antibody drugs have been approved for marketing worldwide (such as pembrolizumab and nivolumab), and the related technologies have been widely used in the treatment of various solid tumors and hematological malignancies, such as melanoma, non-small cell lung cancer, and gastric cancer.

[0003] However, current tumor checkpoint antibody therapy still faces key bottlenecks: on the one hand, some patients experience a treatment response rate of only 20%-40% due to insufficient immune cell infiltration in the tumor microenvironment and low antibody targeting efficiency in tumor tissue; on the other hand, antibody drugs are easily cleared from the body, requiring multiple high-dose administrations, which not only increases the economic burden on patients but may also cause immune-related adverse reactions (such as pneumonia and colitis), limiting their clinical application. Therefore, how to improve the targeting, tumor tissue enrichment capacity, and anti-tumor activity of tumor checkpoint antibodies through technological improvements has become a core research direction in the field of tumor immunotherapy.

[0004] Currently, the mechanisms of action of marketed and investigational tumor checkpoint antibodies mainly rely on "blocking immune checkpoint signaling pathways." However, the targeted delivery efficiency and local accumulation capacity of antibody molecules in tumor tissues remain insufficient. In existing technologies, to improve anti-tumor efficacy, researchers often employ an "antibody-drug conjugate (ADC)" strategy, linking cytotoxic drugs to antibodies via a linker, and utilizing the antibody's targeting ability to deliver the drug to tumor cells. However, this technology has significant drawbacks: first, cytotoxic drugs are highly toxic, easily causing off-target damage to normal tissues, and may induce severe hematologic toxicity, gastrointestinal toxicity, and other side effects; second, ADC drugs only target "tumor cells expressing specific antigens," failing to address the problem of insufficient immune cell infiltration in the tumor microenvironment, thus having limited therapeutic effects on "cold tumors" (tumors with low immune cell infiltration).

[0005] Sialyl Lewis X (sLeX, chemical name: α-2,3-sialyl-β-1,4-(α-1,3-fucosylation)-N-acetylglucosamine) is a naturally occurring oligosaccharide molecule widely expressed on the surface of leukocytes. Its core biological function is as a ligand for selectins, mediating the specific binding of leukocytes to vascular endothelial cells, thereby enabling the directed migration of leukocytes to sites of inflammation or tumors. In recent years, studies have found that the expression levels of E-selectin and P-selectin on the surface of vascular endothelial cells in tumor tissues are significantly higher than those in normal tissues, and the binding of sLeX to selectins can mediate the targeted enrichment of modified molecules to tumor sites. However, no studies have yet directly coupled sLeX to tumor checkpoint antibodies to achieve a synergistic anti-tumor effect of "antibody targeted enrichment in tumor tissues + immune checkpoint blockade" by utilizing the specific binding of sLeX to tumor vascular endothelial selectins. Summary of the Invention

[0006] Based on the limitations of the existing technology, this invention proposes an "antibody conjugate containing Sialyl Lewis X sugar" scheme, with the core motivation stemming from the following three points: a) Solving the problem of poor targeting of existing tumor checkpoint antibodies: Existing checkpoint antibodies mainly rely on the "passive targeting" of tumor cell surface antigens, which are easily taken up by normal tissues, resulting in insufficient antibody concentration in tumor tissues; while sLeX can specifically bind to tumor vascular endothelial selectin, conjugating it to the antibody, and guide the antibody to the tumor site through the "active targeting" mechanism, thereby increasing the local concentration of the antibody in the tumor tissue.

[0007] b) Achieving the synergistic effect of "targeted delivery + immune activation": The targeting effect of sLeX can increase the accumulation of antibodies in the tumor microenvironment, thereby enhancing the blocking efficiency of antibodies against tumor immune checkpoints. At the same time, it may improve the immunosuppressive state of the tumor microenvironment by mediating the migration of immune cells to the tumor site, forming a synergistic anti-tumor effect with checkpoint blocking, breaking through the limitations of existing technologies in treating "cold tumors".

[0008] c) Avoiding the toxicity risks of existing ADC technologies: Existing ADC technologies rely on cytotoxic drugs to exert their effects, resulting in strong side effects; this approach enhances the targeting and activity of the antibody itself through "sLeX modification," eliminating the need for conjugation with toxic drugs, significantly reducing treatment side effects, and improving the safety of clinical applications.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing an antibody-conjugate containing an sLeX tetrasaccharide structure, characterized by comprising the following steps: S1. Add antibody and Endo S2 enzyme to Tris-HCl, incubate for the first time, and analyze the structure and glycoform of the N-linked glycan chain at Asn297 of the product; add α-L-fucosidase to the above reaction system, incubate for the second time, and analyze the fucose modification on the N-linked glycan chain at Asn297 of the product. S2, Take the antibody, B4GalT1, and UDP-Gal from the Asn297 N-linked glycan precisely characterized in S1, and add them to the Mn-containing... 2+ In the Tris-HCl system, incubation was performed to obtain reaction solution I; S3, take reaction solution I, ST3Gal3, CMP-Neu5Ac, containing Mg 2+ Mix with Tris-HCl and incubate; then add CMP-N-acylneuraminidine phosphodiesterase for reaction. After the reaction is complete, perform Protein A purification to obtain purified antibody I. S4. Take purified antibody I, α1,3 FucT-2HR (HR: heptad repeats), GDP-Fuc, and Mg-containing... 2+ The mixture was incubated with Tris-HCl; after the reaction was completed, Protein A was purified to obtain the target product.

[0010] Furthermore, the antibodies screened in step S1 through two analyses include the PD-1 inhibitor nivolumab, the PD-L1 inhibitor durvalumab, the HER2 inhibitor trastuzumab, and the EGFR inhibitor cetuximab.

[0011] Furthermore, the pH of the entire reaction system in step S1 is 7.5; First incubation conditions: 37℃ for 8-12 minutes; Second incubation conditions: 37℃ for 10-14 hours.

[0012] Furthermore, the pH of the reaction system in step S2 is 7.5; The molar ratio of antibody, B4GalT1, and UDP-Gal in the reaction system was 10:1:100, with B4GalT1 activity ≥10U / mg. Mn 2+ The concentration is 8~12mM; Incubation conditions: Incubate at 37℃ and 130~170rpm for 10~14h.

[0013] Furthermore, the pH of the reaction system in step S3 is 7.5; The initial molar ratio of antibody, ST3Gal3, and CMP-Neu5Ac added to reaction solution I in the reaction system is 10:1:100, wherein the activity of ST3Gal3 is ≥5U / mg; Mg 2+ The concentration is 8~12mM; Incubation conditions: Incubate at 37℃ for 7~9 hours.

[0014] Furthermore, the pH of the reaction system in step S4 is 8.0; The molar ratio of purified antibody I, α1,3 FucT-2HR, and GDP-Fuc in the reaction system was 10:1:100, wherein the activity of α1,3 FucT-2HR was ≥8U / mg; Mg 2+ The concentration is 8~12mM; Incubation conditions: Incubate at 37℃ for 10~14h.

[0015] In some specific embodiments, preferably, the Protein A purification conditions in steps S3 and S4 are the same: equilibration with 5 CV of PBS; loading volume of 0.5 mL / min; elution buffer: 0.1 M glycine-HCl, pH=2.7; neutralization buffer: 1 M Tris-HCl, pH=8.5.

[0016] The application of antibody-drug conjugates containing sLeX tetrasaccharide structures prepared by the above method in the treatment of tumors, inflammation and autoimmune diseases.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) sLeX tetrasaccharide targeted modification technology at the N-glycosylation site at the 297th position of the Fc end of the antibody: Without changing the antigen-binding function of the antibody Fab segment, the sLeX tetrasaccharide structure is precisely modified at the conserved N-glycosylation site at the 297th position of the Fc end of the IgG antibody. By utilizing the specific binding of sLeX with E-selectin / P-selectin, which are highly expressed in the tumor microenvironment, the limitation of single-targeting of existing monoclonal antibodies is broken through, and a dual-targeting mechanism of "antigen recognition + inflammation chemotaxis anchoring" is constructed to solve the technical bottleneck of poor penetration of solid tumors and insufficient local drug concentration in tumors.

[0018] (2) Standardized enzymatic modification and purification system based on GlycoMab platform: The innovative three-step enzymatic reaction of B4GalT1, ST3Gal3 and HpFucT is used to achieve glycosylation modification. Combined with the two-step Protein A column purification process of "first purification after sialylation + second purification after fucosylation", there is no need to perform gene modification of host cells such as CHO cells. It overcomes the defects of poor glycoform consistency, large batch fluctuation and long R&D cycle of traditional cell engineering methods. At the same time, it is compatible with four core target antibodies of PD-1 / PD-L1 / HER2 / EGFR, realizing the universality of modification process and large-scale production.

[0019] (3) Antibody functional optimization design with “targeted killing-immune regulation” synergy: By modifying with sLeX, the antibody is endowed with immune cell recruitment activity while retaining the original target binding and killing function of the antibody. For PD-1 / PD-L1 antibody, it can alleviate T cell exhaustion and drug resistance. For HER2 / EGFR antibody, it can enhance antibody-dependent cytotoxicity (ADCC). This solves the core problem of high drug resistance rate and significant individual heterogeneity of efficacy caused by the single mechanism of action of existing monoclonal antibodies. The results were verified by humanized A549 tumor model. The tumor inhibition rate of the modified antibody was increased by 20% compared with the unmodified antibody. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the reaction in step 1 of Example 1.

[0021] Figure 2 This is a graph showing the analysis results of step 1 in Example 1.

[0022] Figure 3 This is a schematic diagram of the reaction in step 2 of Example 1.

[0023] Figure 4 This is a graph showing the analysis results of step 2 in Example 1.

[0024] Figure 5 This is a schematic diagram of the reaction in step 3 of Example 1.

[0025] Figure 6 This is a graph showing the analysis results of step 3 in Example 1.

[0026] Figure 7 This is a schematic diagram of the reaction in step 4 of Example 1.

[0027] Figure 8 This is a graph showing the analysis results of step 4 in Example 1.

[0028] Figure 9 This is a graph showing the test results of quality control 1 in Example 1.

[0029] Figure 10 This is a graph showing the test results of quality control 2 in Example 1.

[0030] Figure 11 , 12 Figures 1 and 13 show the product detection results at each stage in Example 2.

[0031] Figure 14 , 15 Figures 1 and 16 show the product detection results at each stage in Example 3.

[0032] Figure 17 The results are for evaluating the antitumor effects of the products prepared in Examples 1 and 2.

[0033] Figure 18 The results show the evaluation of the antitumor effect of the product prepared in Example 3. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0035] Example 1 This invention targets nivolumab (a PD-1 antibody, clinically used for melanoma, non-small cell lung cancer, and other solid tumors) as the antibody, and provides a method for the directional construction of an sLeX (Neu5Ac-α2,3-Gal-β1,4-[Fuc-α1,6]-GlcNAc) tetrasaccharide structure on the N-linked glycan chain at Asn297 of the Fc segment of a monoclonal antibody using the GlycoMab platform. The specific steps are as follows: Step 1: Precise characterization of the N-linked glycan of the target antibody 1.1 Technical Objectives The initial glycoform composition (e.g., high-mannose Man5-9, heterozygous Man3GlcNAc2, complex GlcNAc2Man3Gal2, etc.) and the proportion of exposed terminal residues (e.g., the proportion of free GlcNAc and Gal) of the N-linked glycan at Asn297 of the Fc segment of the antibody to be modified were analyzed to exclude abnormal glycan structures (e.g., excessive fucosylation at the terminal or truncated glycans). This provides data support for the "substrate suitability" of subsequent enzymatic modifications and avoids modifications at non-target sites or glycan conflicts.

[0036] 1.2 Key Technical Means Glycan release: Recombinant Endo S2 enzyme (≥1000 U / mL, specifically cleaving the β-1,2 glycosidic bond between the first and second GlcNAc) was used. The antibody and Endo S2 enzyme were incubated at 37°C for 10 min in a Tris-HCl (pH 7.5) system to ensure the release of the N-linked glycan at Asn297 (without affecting the O-linked glycan) (see reaction procedure below). Figure 1 ).

[0037] Glycan analysis: The precise molecular weight of the glycans was determined by high-resolution mass spectrometry (HRMS, resolution 140000 FWHM) (error ≤ 5ppm), and the structure and sugar form of the N-linked glycan at Asn297 were finally confirmed.

[0038] Core fucose analysis: α-L-fucosidase (AFU) was added to the above reaction solution (which specifically cleaves the α-1,6-Fuc glycosidic bond on the core GlcNAc of the antibody Asn297 glycan chain), and the mixture was incubated at 37°C for 24 h in a Tris-HCl (pH 7.5) system to ensure the release of core fucose at the Asn297 position (without affecting the O-linked glycan chain).

[0039] Glycan analysis: The precise molecular weight of the glycans was determined by high-resolution mass spectrometry (HRMS, 140,000 FWHM) (error ≤ 5 ppm), ultimately confirming the presence of core fucose modification in the N-linked glycan at Asn297 (analytical results are shown in [link to results]). Figure 2 ).

[0040] Step 2: β-1,4-galactosylation modification of GlcNAc at the Asn297 glycan terminus and mass spectrometry quality control 2.1 Technical Objectives The sLeX core precursor (GlcNAc-β1,4-Gal) was constructed by selectively linking galactose (Gal) to the C4 hydroxyl group of GlcNAc residues using the glycosidic bond catalysis of galactosyltransferase B4GalT1. The modification efficiency and site specificity were verified by HRMS to ensure that Gal was linked only to the target GlcNAc and that there was no non-specific modification of other glycan residues (such as Man and Fuc).

[0041] 2.2 Key Technical Means Enzymatic modification system: 50 μL system contains 20 μg antibody, B4GalT1 0.1 eq (expressed in CHO cells, specific activity ≥10 U / mg), UDP-Gal 100 eq (≥98%), Tris-HCl (pH 7.5, containing 10 mM Mn). 2+ ); Mn 2+ As an essential cofactor for B4GalT1, it enhances catalytic efficiency by stabilizing the enzyme-substrate ternary complex. Incubation was performed at 37°C in a water bath (150 rpm) for 12 hours (see reaction procedure). Figure 3 ).

[0042] HRMS quality control: After dilution, the reaction solution was detected in ESI positive ion mode (m / z 500-3000). The judgment criterion was: the antibody molecular weight increased by 162 Da compared with that before modification (molecular weight of a single Gal residue, C6H). 12 O6-H2O=162), the abundance of the target modified peak is ≥90%, and the intensity of impurity peaks is <5% (excluding non-specific modifications) (see results). Figure 4 ).

[0043] Step 3: α-2,3-sialylation modification of Gal terminus, mass spectrometry quality control and Protein A purification 3.1 Technical Objectives Site-specific catalysis by sialic acid (Neu5Ac) is achieved by linking the hydroxyl group at the C3 position of the Gal residue to form the "GlcNAc-β1,4-Gal-α2,3-Neu5Ac" unit. Free enzyme (B4GalT1, ST3Gal3), unmodified antibody, and glyconucleotide substrate (UDP-Gal, CMP-Neu5Ac) are removed by Protein A affinity chromatography, thus initially improving the purity of the product.

[0044] 3.2 Key Technical Means Enzymatic modification system: 100 μL system contains the complete reaction solution from step 2, ST3Gal3 0.1 eq (expressed in SF9 insect cells, specific activity ≥5 U / mg), CMP-Neu5Ac 100 eq (≥98%), Tris-HCl (pH 7.5, containing 10 mg / mL). 2+); Mg² + To enhance the binding affinity of the enzyme to CMP-Neu5Ac, incubate at 37°C for 8 hours. Add CMP-N-acylneuraminic acid phosphodiesterase to hydrolyze the byproduct CMP (the product of CMP-sialic acid transfer catalyzed by ST3Gal3), thereby relieving the product inhibition of ST3Gal3 by CMP (see reaction procedure). Figure 5 ).

[0045] Dual quality control and purification: HRMS validation: The antibody molecular weight increased by 291 Da compared to step 2 (molecular weight of a single Neu5Ac, C). 11 H 19 NO9-H2O=291), target peak abundance ≥90%; Protein A purification: 1 mL column (capacity ≥20 mg / mL), equilibrate with PBS (5 CV) → load at 0.5 mL / min → elute with 0.1 M glycine-HCl (pH 2.7) → neutralize with 1 M Tris-HCl (pH 8.5), HRMS validation confirms preliminary purity ≥92% (see results). Figure 6 ).

[0046] Step 4: α-1,6-fucosylation modification of GlcNAc, secondary purification and initial preparation of the final product 4.1 Technical Objectives The sLeX tetrasaccharide structure was fully constructed by substrate-specific catalysis of the fucosyltransferase HpFucT, which links fucose (Fuc) to the C6 hydroxyl group of the GlcNAc residue linked to Gal. The residual HpFucT and GDP-Fuc were further removed by secondary Protein A purification to ensure that the purity of the final product was ≥95%.

[0047] 4.2 Key Technical Means Enzymatic modification system: 200 μL system contains 100 μg of antibody purified in step 3, 0.1 eq of α1-3FucT-2HR (expressed in E. coli, specific activity ≥8 U / mg), 100 eq of GDP-Fuc (≥98%), and Tris-HCl (pH 8.0, containing 10 mg / mL). 2+ ); Mg² + Activate the HpFucT catalytic domain and incubate at 37°C for 12 h (see reaction procedure). Figure 7 ).

[0048] Secondary purification: The Protein A purification process in step 3 was repeated, and HRMS verification showed that the purity of the final product was ≥95%, laying the foundation for subsequent quality control (results see...). Figure 8 ).

[0049] Dual quality control steps for the final product (focusing on molecular weight and SEC testing) Quality Control 1: High-resolution mass spectrometry (HRMS) verification of molecular weight accuracy 1.1 Technical Principles: Based on the precise quality analysis capabilities of HRMS (resolution 140,000 FWHM, quality accuracy ≤ 5 ppm), the actual molecular weight of sLeX-modified antibodies is determined and compared with the "molecular weight of unmodified antibody + theoretical molecular weight of sLeX tetrasaccharide" to verify whether the sLeX tetrasaccharide is completely linked (without missing sugar residues or excessive modification).

[0050] 1.2 Key Detection Details Theoretical molecular weight calculation: The theoretical molecular weight of sLeX tetrasaccharide = Gal (162 Da) + Neu5Ac (291 Da) + Fuc (146 Da) = 599 Da; Taking nivolumab as an example, the molecular weight of the unmodified antibody is about 146223 Da, and the theoretical molecular weight after modification should be 146223 + 599 × 4 = 148619 Da.

[0051] Actual detection and judgment: The modified antibody was diluted (1 μg / μL) and detected in ESI positive ion mode (m / z 500-3000). The error between the actual molecular weight and the theoretical value should be ≤5 ppm. At the same time, the secondary mass spectrometry (MS / MS) should detect sLeX characteristic fragment ions (m / z 512.1: Neu5Ac-Gal-GlcNAc; m / z 657.2: Neu5Ac-Gal-GlcNAc-Fuc) to ensure the integrity of the tetrasaccharide structure (see results). Figure 9 ).

[0052] Quality Control 2: Size Exclusion Chromatography (SEC) for Detecting Key Antibody Indicators 2.1 Technical Principles SEC (Separation by Electron Microscopy) achieves separation based on differences in molecular volume: antibody monomers (smaller in size) can enter the interior of the gel particles as they flow through the chromatographic column (porous gel packing material), resulting in longer retention times; polymers (such as dimers and polymers, which are larger in size) cannot enter the gel interior, resulting in shorter retention times; degradation products (such as fragmented antibodies, which are even smaller in size) have the longest retention times. SEC allows for the quantitative analysis of antibody aggregation, monomer purity, and the degree of degradation, indicators that are directly related to antibody stability, immunogenicity, and clinical efficacy.

[0053] 2.2 Key Detection Parameters and Indicators SEC detection conditions: Column (e.g., TSKgel G3000SWxl, 7.8×300mm), mobile phase (0.1M phosphate buffer - 0.2M NaCl, pH 6.8), flow rate 0.5mL / min, column temperature 30℃, detection wavelength 280nm, injection volume 10μL, run time 30min (results see...). Figure 10 ).

[0054] Core testing indicators: (1) Proportion of antibody monomer peak area: This reflects the proportion of the main active forms of the antibody and is the core indicator of SEC detection. The proportion of monomer peak area should be ≥98%. If it is lower than this value (e.g., <95%), it indicates that the antibody has significant aggregation, which may cause immunogenicity (e.g., induce the production of anti-drug antibodies) or reduce the efficacy in vivo.

[0055] (2) Polymer peak area ratio: including dimers (retention time about 12-13 min) and polymers (retention time about 10-11 min), the total polymer ratio should be ≤2%. Polymers will increase the risk of reaction at the injection site and cannot effectively bind to the target, so they need to be strictly controlled.

[0056] (3) Peak area ratio of degradation products: Including antibody fragments (such as Fab fragments and Fc fragments, with a retention time of about 16-18 min), the proportion of degradation products should be ≤1%. Degradation products do not have complete targeting function and may release immune-active fragments, affecting the safety of medication.

[0057] (4) Consistency of retention time: The coefficient of variation (CV) of the retention time of monomer peaks in the same batch or between different batches should be ≤0.5% to reflect the stability of antibody molecule conformation and avoid functional differences caused by conformational changes.

[0058] Example 2 In this embodiment, the sLeX (Neu5Ac-α2,3-Gal-β1,4-[Fuc-α1,6]-GlcNAc) tetrasaccharide structure was constructed using the PD-L1 antibody durvalumab (targeting PD-L1, clinically used for unresectable stage III non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, and other solid tumors) as the target antibody. The specific steps and procedures are basically the same as in Example 1, except that the target antibody was changed from the PD-1 antibody nivolumab to the PD-L1 antibody durvalumab. The final detection results are shown in the following figures. Figure 11 , 12 13.

[0059] Example 3 This embodiment uses the EGFR antibody cetuximab (targeting EGFR, clinically used for RAS wild-type metastatic colorectal cancer, head and neck squamous cell carcinoma, and other solid tumors) as the target antibody to construct the sLeX (Neu5Ac-α2,3-Gal-β1,4-[Fuc-α1,6]-GlcNAc) tetrasaccharide structure. The specific steps and procedures are basically the same as in Example 1, the difference being that the target antibody is changed from the PD-1 antibody nivolumab to the EGFR antibody cetuximab. The final detection results are shown in the following figures. Figure 14 , 15 16.

[0060] Furthermore, to understand the antitumor effects of the sLeX-modified antibodies prepared in the above embodiments, the following studies were also conducted: Construction of humanized A549 tumor model and evaluation of the anti-tumor effects of sLeX-modified PD1 and PDL1 antibodies I. Experimental Objective A humanized tumor model based on the co-culture of human peripheral blood mononuclear cells (PBMCs) and A549 lung cancer cells was constructed. Animal experiments were used to quantitatively evaluate the difference in antitumor efficacy of PD-1 / PD-L1 antibodies before and after sLeX modification, and to verify the optimization effect of GlycoMab platform glycosylation modification technology on antibody therapy.

[0061] II. Experimental Materials (a) Reagents and Culture Media 1. Core reagents: Lymphocyte separation medium (Yeasen, catalog number 40503ES60), CD3 / CD28 T cell activation reagent (Thermo, catalog number 11131D), mitomycin C (concentration 30μg / mL), recombinant human interleukin-2 (IL-2, final concentration 100IU / mL).

[0062] 2. Culture medium: RPMI 1640 basal medium, supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% glutamine to construct a complete culture medium.

[0063] (II) Cells and Animals 1. Cells: Human non-small cell lung cancer A549 cell line (routine laboratory culture and preservation), fresh human peripheral blood-derived PBMCs (clinical samples obtained in compliance with regulations).

[0064] 2. Animals: Immunodeficient mice (such as NSG mice, 6-8 weeks old, female, SPF-grade environment).

[0065] (III) Instruments and Equipment High-speed refrigerated centrifuge, cell culture incubator (37℃, 5% CO2), cell counting plate, 6-well cell culture plate, sterile syringe, etc.

[0066] III. Experimental Methods (I) PBMC cell isolation and activation culture (Day 0) 1. Sample pretreatment: Take fresh human peripheral blood, dilute it with sterile PBS at a volume ratio of 1:1, mix gently to avoid blood clotting.

[0067] 2. Density gradient centrifugation: Add lymphocyte separation medium to the centrifuge tube, and slowly spread the diluted blood onto the top layer of the separation medium along the tube wall at a volume ratio of 1:2 (maintaining clear stratification). Centrifuge at 400×g for 20 min at room temperature, avoiding braking during centrifugation to prevent stratification.

[0068] 3. Cell collection and washing: After centrifugation, the liquid separates into three layers. The middle layer of white lymphocytes (PBMCs) is aspirated into a new sterile centrifuge tube, 10 mL of sterile PBS is added, and the tube is centrifuged at 200 × g for 10 min at room temperature. The washing is repeated twice to remove residual separation liquid and impurities.

[0069] 4. Activation culture: Resuspend PBMCs in complete culture medium and count the cells, taking 2 × 10⁶ cells. 6 Seed cells into 6-well culture plates, add 25 μL of CD3 / CD28 T cell activation reagent pre-washed with PBS to each well, and incubate in a cell culture incubator to start the T cell activation process (for detailed operation, please refer to the Yeasen official technical manual: https: / / www.yeasen.com / products / detail / 967).

[0070] (II) Co-culture of PBMCs and A549 cells (Day 2) 1. Tumor cell pretreatment: A549 cells in logarithmic growth phase were incubated with 30 μg / mL mitomycin C for 1 h (to inhibit cell proliferation and avoid excessive growth interfering with PBMC activation), followed by washing twice with sterile PBS and counting for later use.

[0071] 2. Construction of co-culture system: A549 cells were added to the PBMC culture system at a cell ratio of A549 cells:PBMC = 1:10, and the cells were co-cultured in an incubator for 2 days. This co-culture step was repeated twice to enhance the antigen recognition ability of PBMCs for A549 cells.

[0072] 3. Cell mixing and inoculation: Collect PBMCs after two co-cultures and mix them with fresh logarithmic growth phase A549 cells at a ratio of 1:4. Gently pipette the mixture until homogeneous, and use a sterile syringe to draw up the mixed cell suspension and inoculate it subcutaneously on the right side of immunodeficient mice (the amount inoculated per mouse is adjusted according to the experimental design to ensure consistent tumor formation).

[0073] (III) Antibody administration and tumor monitoring 1. Group design: After inoculation, mice were randomly divided into 5 groups: PBS group, sLeX-modified PD-1 antibody group, sLeX-modified PD-L1 antibody group, unmodified PD-1 control group, and unmodified PD-L1 antibody control group (each group had at least 5 mice to ensure statistical power).

[0074] 2. Administration regimen: After inoculation, wait until the tumor volume reaches approximately 100 mm. 3 The administration will begin at a certain time and will be administered via intraperitoneal injection. The administration cycle will be 3 weeks, with a total of 6 administrations (the specific dosage will be determined based on the preliminary experiment to ensure that the dosage is consistent across groups).

[0075] 3. Tumor monitoring: The long diameter (L) and short diameter (W) of the tumor were measured twice a week. The tumor volume was calculated using the formula V=(L×W²) / 2, and the tumor growth curves for each group were recorded.

[0076] IV. Experimental Results After 3 weeks and 6 doses of treatment, compared with the unmodified PD-1 / PD-L1 antibody control group, both the sLeX-modified PD-1 antibody group and the PD-L1 antibody group showed more significant tumor inhibition effects. Specifically, the tumor growth rate of the modified antibody group was significantly slowed down, and the tumor inhibition rate of the PD-1 antibody was increased by 18.84% and that of the PD-L1 antibody was increased by 15.01% compared with the control group. Furthermore, no obvious abnormal toxic reactions (such as sudden weight loss or behavioral abnormalities) were observed in the mice during the experiment. This preliminarily verifies the optimizing effect of sLeX glycosylation modification on the anti-tumor activity of PD-1 / PD-L1 antibodies (see results below). Figure 17 ).

[0077] V. Experimental Conclusions This study successfully constructed a humanized A549 tumor model using a method involving PBMC isolation and activation, co-culture with A549 cells, and subcutaneous inoculation into immunodeficient mice. Animal experimental data demonstrate that sLeX glycosylation modification technology based on the GlycoMab platform can effectively enhance the antitumor efficacy of PD-1 / PD-L1 antibodies, providing reliable in vivo experimental evidence for the clinical translation of this modification technology and the development of novel antibody drugs. Further in-depth studies can be conducted on pharmacokinetics and immune microenvironment mechanisms to improve the efficacy evaluation system for modified antibodies.

[0078] A549 tumor model construction and evaluation of the anti-tumor effect of sLeX-modified EGFR antibody. I. Experimental Objective A nude mouse xenograft model of A549 lung cancer cells was constructed. Animal experiments were conducted to quantitatively evaluate the difference in antitumor efficacy of EGFR antibodies (taking cetuximab as an example) before and after sLeX modification, and to verify the optimizing effect of GlycoMab platform glycosylation modification technology on the therapeutic effect of EGFR antibodies.

[0079] II. Experimental Materials (a) Reagents and Culture Media 1. Core reagents: sLeX-modified cetuximab, unmodified cetuximab (purchased from reagent vendor, purity ≥95%), PBS buffer (pH 7.4).

[0080] 2. Culture medium: RPMI 1640 basal medium, with the addition of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin to construct a complete culture medium.

[0081] (II) Cells and Animals 1. Cells: Human non-small cell lung cancer A549 cell line (routinely cultured and preserved in the laboratory, in the logarithmic growth phase).

[0082] 2. Animals: BALB / c nude mice (6-8 weeks old, female, SPF-grade environment, acclimatized to the environment for 1 week before starting experiments).

[0083] (III) Instruments and Equipment Cell culture incubator (37℃, 5% CO2), high-speed centrifuge, cell counting chamber, sterile syringe, vernier caliper, electronic balance.

[0084] III. Experimental Methods (I) Preparation of A549 cells A549 cells in the logarithmic growth phase were collected by digestion with 0.25% trypsin, digestion was terminated by adding complete culture medium, centrifuged at 1000×g for 5 min, the supernatant was discarded, and the cells were resuspended in sterile PBS buffer. The cells were washed twice, centrifuged again, and the cell concentration was adjusted to 5×10⁶ cells / mL with PBS. 6 Quantity / mL, keep on ice for later use.

[0085] (II) Construction of nude mouse xenograft model Subcutaneous inoculation was used. After disinfection of the right back of each nude mouse, 0.2 mL of A549 cell suspension (containing 1×10⁻⁶ cells) was drawn into the cell using a sterile syringe. 6 Inject one cell at a time, gently pressing the injection site for 1-2 minutes to prevent leakage of the cell suspension. Observe the nude mice daily after inoculation for tumor formation at the injection site. When the tumor volume reaches approximately 100 mm³, begin the drug administration phase.

[0086] (III) Group-based drug administration and tumor monitoring 1. Group design: Tumor-forming nude mice were randomly divided into 3 groups of 6 mice each: sLeX-modified cetuximab group, unmodified cetuximab control group, and PBS group. 2. Dosing regimen: The drug was administered via intraperitoneal injection over a period of 3 weeks, for a total of 6 administrations, twice a week. The volume of each administration was adjusted according to the weight of the nude mice (approximately 10 μL / g).

[0087] 3. Tumor monitoring: The long diameter (L) and short diameter (W) of the tumor were measured twice a week. The tumor volume was calculated using the formula V=(L×W²) / 2, and tumor growth curves were plotted for each group. At the same time, the weight of nude mice was measured once a week, and weight changes were recorded to assess drug toxicity.

[0088] IV. Experimental Results After 3 weeks and 6 doses of treatment, compared with the unmodified cetuximab control group, the sLeX-modified cetuximab group showed a more significant tumor inhibition effect: the tumor inhibition rate was 20% higher than that of the control group; and during the experiment, there was no significant decrease in body weight of nude mice in any group, and no toxic reactions such as behavioral abnormalities or skin lesions occurred, indicating that sLeX modification enhanced the anti-tumor activity of EGFR antibodies without increasing drug toxicity (see results). Figure 18 ).

[0089] V. Experimental Conclusions This study successfully established a lung cancer xenograft model in nude mice via subcutaneous inoculation of A549 cells. Animal experimental data confirmed that sLeX glycosylation modification technology based on the GlycoMab platform can effectively enhance the antitumor efficacy of EGFR antibody (cetuximab) with good safety. These results provide reliable in vivo experimental evidence for the further development and clinical translation of sLeX-modified EGFR antibodies, and further investigation into its applicability and mechanism of action in different tumor models is warranted.

[0090] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0091] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an antibody-conjugate containing an sLeX tetrasaccharide structure, characterized in that, Includes the following steps: S1. Add IgG antibody and Endo S2 enzyme to Tris-HCl, perform the first incubation, and analyze the structure and glycoform of the Asn297 N-linked glycan to ensure the release of the Asn297 N-linked glycan. Then add α-L-fucosidase to the above reaction system, perform the second incubation, and analyze the fucose modification on the Asn297 N-linked glycan to ensure the release of the core fucose at Asn297. S2, Take the antibody, galactosyltransferase B4GalT1, and UDP-Gal from S1 that accurately characterize the N-linked glycan chain at Asn297, and add them to the Mn-containing... 2+ In the Tris-HCl system, incubation was performed to ensure that Gal was only linked to the target GlcNAc without non-specific modification of other sugar chain residues, to obtain reaction solution I; S3, Take reaction solution I, sialic acid glycosyltransferase ST3Gal3, CMP-Neu5Ac, and Mg-containing... 2+ The mixture was incubated with Tris-HCl, and sialic acid Neu5Ac was linked to the hydroxyl group at the C3 position of the Gal residue. Then, CMP-N-acylneuraminic acid phosphodiesterase was added to react and the byproduct CMP was hydrolyzed to relieve the inhibition of sialic acid glycosyltransferase ST3Gal3 by CMP. After the reaction was completed, Protein A was used for purification to obtain purified antibody I. S4. Take purified antibody I, fucosyltransferase Hpα1,3-FucT, GDP-Fuc, and Mg-containing... 2+ The mixture was incubated with Tris-HCl; after the reaction was completed, Protein A was purified to obtain the target product.

2. The method according to claim 1, characterized in that, The antibodies in step S1 include the PD-1 inhibitor nivolumab, the PD-L1 inhibitor durvalumab, the HER2 inhibitor trastuzumab, and the EGFR inhibitor cetuximab.

3. The method according to claim 1, characterized in that, In step S1, the pH of the entire reaction system is 7.

5. First incubation conditions: 37℃ for 8-12 minutes; Second incubation conditions: 37℃ for 10-14 hours.

4. The method according to claim 1, characterized in that, The pH of the reaction system in step S2 is 7.

5. The molar ratio of antibody, galactosyltransferase B4GalT1, and UDP-Gal in the reaction system was 10:1:100, and the activity of galactosyltransferase B4GalT1 was ≥10U / mg. Mn 2+ The concentration is 8~12mM; Incubation conditions: Incubate at 37℃ and 130~170rpm for 10~14h.

5. The method according to claim 1, characterized in that, The pH of the reaction system in step S3 is 7.5; The initial molar ratio of antibody, sialyl glycosyltransferase ST3Gal3, and CMP-Neu5Ac added to reaction solution I in the reaction system was 10:1:100, wherein the activity of sialyl glycosyltransferase ST3Gal3 was ≥5U / mg; Mg 2+ The concentration is 8~12mM; Incubation conditions: Incubate at 37℃ for 7~9 hours.

6. The method according to claim 1, characterized in that, The pH of the reaction system in step S4 is 8.

0. The molar ratio of purified antibody I, fucosyltransferase Hpα1,3 FucT, and GDP-Fuc in the reaction system was 10:1:100, wherein the activity of fucosyltransferase Hpα1,3 FucT was ≥8U / mg. Mg 2+ The concentration is 8~12mM; Incubation conditions: Incubate at 37℃ for 10~14h.

7. The method according to claim 1, characterized in that, The purification conditions for Protein A in steps S3 and S4 are the same: equilibration with 5 CV of PBS; Sample loading rate: 0.5 mL / min; Eluent: 0.1 M glycine-HCl, pH=2.7; Neutralization solution: 1 M Tris-HCl, pH=8.

5.

8. The use of the antibody-drug conjugate containing the sLeX tetrasaccharide structure prepared by the method of any one of claims 1-7 in the preparation of a drug for treating tumors, wherein the tumor is a solid tumor.

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