Glycoengineered antibody conjugate drugs and methods of making the same

Abstract

The application belongs to the technical field of biological medicine, and discloses a glycoengineered antibody conjugated drug and a preparation method thereof, wherein the preparation method comprises the following steps: (1) preparing an antibody; (2) engineering the Fc segment N-glycan of the antibody, so that the antibody has enhanced sialylation modification, and a glycoengineered antibody is obtained; (3) coupling the glycoengineered antibody obtained in the step (2) with a linker and a cytotoxic payload, so that a glycoengineered antibody conjugated drug is obtained. By improving the sialylation level of the Fc segment N-glycan of the antibody, the distribution of the obtained ADC in glycogen aggregates is reduced relative to the corresponding unmodified control ADC, and the lysosomal processing, payload release and antitumor activity are improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to a glycoengineered antibody-drug conjugate and its preparation method. The preparation method enhances the lysosomal processing efficiency of the antibody-drug conjugate (ADC) by engineering the N-glycan chain of the Fc segment of the antibody. The glycoengineered ADC and related drug compositions can be further prepared through this method, thereby obtaining a candidate antibody screening method. Background Technology

[0002] Antibody-drug conjugates (ADCs) achieve targeted killing of tumor cells by linking highly specific antibodies to cytotoxic payloads via linkers. For ADCs employing cleavable peptide linkers, such as trastuzumab deruxtecan (T-DXd) and datopotamab deruxtecan (Dato-DXd), their antitumor effects depend on the efficient processing and cleavage of the ADC by proteases in lysosomes after entering tumor cells, thereby releasing the cytotoxic payload.

[0003] However, a significant proportion of patients in clinical practice still exhibit primary or acquired resistance to ADC therapy. Previous research on ADC resistance has mainly focused on target antigen downregulation, receptor-mediated endocytosis impairment, drug efflux pump overexpression, and changes in payload metabolism or sensitivity. These mechanisms can explain resistance to some specific ADCs, but for ADCs targeting different targets that rely on lysosomal protein hydrolysis, there is still a lack of directly addressable preparation strategies to address their common lysosomal processing limitations.

[0004] Existing studies suggest that metabolic reprogramming of tumor cells can alter the lysosomal environment; however, current technologies have not clarified whether abnormal glycogen structures can directly restrict the processing and payload release of ADCs using cleavable peptide linkers in lysosomes, nor have they proposed technical solutions to reduce the interaction between ADCs and glycogen condensates by altering the N-glycan structure of the antibody Fc fragment.

[0005] The N-glycans of the antibody Fc fragment are important post-translational modifications of antibodies. Glycosylation modes, including fucosylation, galactosylation, and sialylation, can affect antibody effector function, half-life, tissue distribution, Fc receptor binding, and complement activation. However, current techniques have not clarified whether Fc glycosylation affects the partitioning behavior of ADCs in lysosomal glycogen condensates, nor have they proposed a preparation strategy to improve the lysosomal processing efficiency of ADCs by regulating the sialylation of the Fc N-glycans. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a glycoengineered antibody-drug conjugate and its preparation method. By increasing the sialylation level of the N-glycan chain of the antibody Fc segment, the resulting ADC exhibits reduced distribution in glycogen condensates relative to the corresponding unmodified control ADC, thereby enhancing lysosomal processing, payload release, and antitumor activity.

[0007] To achieve the above objectives, according to one aspect of the present invention, a method for preparing a glycoengineered antibody-drug conjugate is provided, comprising the following steps: (1) Prepare antibodies; (2) The Fc segment N-glycan chain of the antibody is engineered to give the antibody enhanced sialylation modification, thereby obtaining a glycoengineered antibody; (3) The glycoengineered antibody obtained in step (2) is coupled with a linker and a cytotoxic payload to obtain a glycoengineered antibody-conjugated drug.

[0008] As a further preferred embodiment of the present invention, in step (2), the Fc segment N-glycan chain of the antibody is engineered, specifically including: (i) Overexpression of sialyltransferase; And optionally (ii) further overexpression of galactosyltransferase and / or knockout of fucosyltransferase; This increases the sialylation level of the Fc segment N-glycan chain of the antibody; The enhanced sialylation modification includes increased levels of α2,6-linked sialic acid and / or α2,3-linked sialic acid. The enhancement refers to an increase in the corresponding sialylation level compared to the control antibody, which has the same amino acid sequence and other preparation conditions as the control antibody except for the one that did not undergo the corresponding glycoengineering modification in step (2).

[0009] As a further preferred embodiment of the present invention, the antibody expression host cell is a Chinese hamster ovary (CHO) cell; The sialyl transferase includes ST6GAL1 and / or ST3GAL4; The galactosyltransferase includes B4GALT1; The fucotransferase includes FUT8.

[0010] As a further preferred embodiment of the present invention, in step (1), the antibody is capable of targeting HER2 or TROP2; preferably, the antibody is trastuzumab or datopotamab; In step (3), the linker is a cleavable peptide linker; preferably, the linker is a GGFG linker. The cytotoxic payload is deruxtecan (DXd) or a derivative thereof.

[0011] According to another aspect of the present invention, a glycoengineered antibody-drug conjugate prepared by the above-described preparation method is provided.

[0012] According to another aspect of the invention, the use of the above-described glycoengineered antibody-drug conjugate in the preparation of a medicament for treating tumors is provided.

[0013] As a further preferred embodiment of the present invention, the tumor is HER2-positive breast cancer or TROP2-positive breast cancer.

[0014] According to another aspect of the present invention, a method for screening glycoengineered antibody-drug conjugate candidates with improved lysosomal processing efficiency is provided, comprising the following steps: S1: Provides candidate glycoengineered antibodies and their corresponding unmodified control antibodies; S2: Under the same conditions, detect the partition coefficients of the candidate glycogen engineered antibody and the unmodified control antibody in glycogen aggregates; S3: Select candidate glycoengineered antibodies with a partition coefficient lower than that of the unmodified control antibody as pre-selected antibodies; S4: The preselected antibody and the unmodified control antibody are conjugated with the same cleavable peptide linker and cytotoxic payload, respectively, to obtain the candidate antibody-drug conjugate and the corresponding unmodified control antibody-drug conjugate.

[0015] As a further preferred embodiment of the present invention, in step S2, equal amounts of fluorescently labeled candidate glycogen engineered antibody and unmodified control antibody are mixed with glycogen under the same in vitro phase separation conditions, the fluorescence intensity of each antibody in the aggregate phase and the dilution phase is quantified, and the partition coefficient of each antibody is calculated using the following formula. Distribution coefficient = fluorescence intensity of aggregate phase / fluorescence intensity of dilute phase.

[0016] The enhanced sialylation modification described in this invention can be detected by Sambucus nigra lectin and / or by Maackia amurensis lectin II, and can be further confirmed by N-glycan chromatography.

[0017] The technical solution conceived in this invention can reduce ADC capture by glycogen condensates and enhance lysosomal processing and payload release by optimizing the N-glycan chain of the antibody Fc fragment without changing the ADC target antigen, linker type, or payload type. This invention is particularly applicable to cleavable peptide linker ADCs that rely on lysosomal protein hydrolysis, such as HER2-targeting ADCs and TROP2-targeting ADCs.

[0018] By systematically comparing complete deglycosylation, single-gene glycoengineering, and multiple-glycoengineering strategies, the inventors discovered that increasing the sialylation level of the N-glycan chain in the Fc fragment of an antibody can reduce the partitioning of ADCs in glycogen condensates. Specifically, ST6GAL1-mediated sialylation showed a consistent effect, and the glycoengineered ADCs generated by co-overexpression of B4GALT1 and ST6GAL1 exhibited stronger condensate rejection and tumor cell killing activity compared to the other schemes. This change was also accompanied by enhanced LysoLight reporter signaling, supporting improved lysosomal processing and linker cleavage.

[0019] Compared with the corresponding unmodified control ADC, the glycoengineered ADC obtained in this invention exhibits an increased level of sialylation in the N-glycan of the antibody Fc segment, reduced partitioning in glycogen condensates, and enhanced lysosomal processing, payload release, and antitumor activity. (Note: The corresponding unmodified control ADC is identical to the glycoengineered ADC in terms of antibody amino acid sequence, linker, payload, and other preparation and detection conditions, except for the absence of glycoengineering modification. The increased sialylation level refers to the increased sialylation level compared with the control antibody. The control antibody is identical in terms of antibody amino acid sequence and other preparation conditions, except for the absence of glycoengineering modification.)

[0020] Compared with the prior art, the present invention has at least the following beneficial effects: (1) It is proposed to regulate the distribution behavior of ADC in glycogen condensates by increasing the sialylation level of N-glycan chain of antibody Fc fragment, so as to provide a new glycoengineering strategy for improving the lysosomal processing efficiency of cleavable peptide linker ADC.

[0021] (2) The glycoengineered ADC prepared by the present invention can reduce the capture of glycogen condensates, improve the LysoLight reporter signal and payload release, and enhance the killing activity of tumor cells.

[0022] (3) This invention does not rely on changing the ADC target antigen, linker or payload itself, but improves the processing efficiency of ADC in tumor cells through antibody glycan engineering, and has the potential for platform application.

[0023] (4) The technical effects of the present invention have been verified in HER2-targeted ADC and TROP2-targeted ADC models, indicating that the strategy can be applied to cleavable peptide linker ADCs with different target antigens.

[0024] (5) The screening method provided by the present invention uses the corresponding unmodified ADC as a direct control and combines glycogen aggregate distribution, lysosome processing, payload release and cytotoxicity for stratified verification. It can be used to screen glycoengineered ADC candidates with improved lysosome processing efficiency. Attached Figure Description

[0025] Figure 1 This diagram illustrates the ADC glycosylation editing strategy and shows the aggregate distribution and cytotoxicity analysis results of deglycosylated ADCs. Figure 1 The 'a' in the diagram represents an ADC glycosylation editing strategy (including enzymatic deglycosylation and genetic glycoengineering). Figure 1 In this context, b represents the in vitro phase separation analysis of T-DXd partitioning in glycogen aggregates after PNGase F or EndoS treatment; Figure 1 In this context, ch represents the effect of deglycosylation of Dato-DXd or T-DXd on the survival and cell death of MDA-MB-231 and BT474 cells.

[0026] Figure 2 Results of in vitro glycogen aggregate partitioning, LysoLight fluorescence, and cytotoxicity analysis of the glycoengineered ADC. Figure 2 In this context, 'a' represents the in vitro phase separation analysis of glycoengineered T-DXd partitioning in glycogen aggregates; Figure 2 b in the figure represents the LysoLight fluorescence flow cytometry analysis of glycoengineered T-DXd-LysoLight in BT474 cells; Figure 2 In this context, ch represents the effect of glycoengineered Dato-DXd or T-DXd on the survival and cell death of MDA-MB-231 and BT474 cells.

[0027] Figure 3 This presents the characterization, in vitro phase separation analysis, and cytotoxicity evaluation results of highly sialylated ADCs. Figure 3 In the figure, 'a' represents the sialylation efficiency of different polysaccharide engineering strategies as determined by Sambucus nigra lectin (SNA) blot analysis. Figure 3 In the figure, b represents the in vitro phase separation analysis of the hypersialylated ADC partitioning in glycogen condensates; Figure 3 In the figure, ch represents the effect of hypersialylated Dato-DXd or T-DXd on the survival and cell death of MDA-MB-231 and BT474 cells.

[0028] Figure 4 The results show the antitumor activity of highly sialylated ADCs in the PDX tumor model. Among them, Figure 4 In the diagram, 'a' represents the experimental treatment scheme (divided into six groups: control IgG, unmodified T, sialylated T, control IgG-DXd, unmodified T-DXd, and sialylated T-DXd). Figure 4 In this context, 'b' represents the tumor growth curve. Figure 4 In this context, 'c' represents survival analysis. Figure 4In the figure, d represents weight change; the legends for control IgG, unmodified T, sialylated T, control IgG-DXd, unmodified T-DXd, and sialylated T-DXd are “IgG”, “T”, “Sia-T”, “IgG-DXd”, “T-DXd”, and “Sia-T-DXd”, respectively. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0030] Example 1: Preparation of Glycoengineered Antibody-Drug Conjugates This embodiment provides a method for preparing glycoengineered antibody-drug conjugates.

[0031] Step 1: Glycoengineering of antibody expression host cells. CHO cells (Chinese hamster ovary cells, commercially available) were used as the antibody expression host. The following four groups of single-gene glycoengineered cells were established through lentiviral transduction, electroporation transfection, and / or CRISPR / Cas9 gene editing: (1) Overexpression of ST6GAL1 (α2,6-sialyltransferase); (2) Overexpression of ST3GAL4 (α2,3-sialyltransferase); (3) Overexpression of B4GALT1 (β1,4-galactosyltransferase); (4) Knock out FUT8 (fucosyltransferase).

[0032] Meanwhile, three groups of multi-saccharide-engineered CHO cell lines with overexpression combinations (B4GALT1+ST6GAL1, B4GALT1+ST3GAL4, and FUT8 knockout+ST6GAL1 overexpression) were established, for a total of seven groups.

[0033] Step 2: Expression and purification of glycoengineered antibodies. Expression vectors encoding anti-HER2 antibody (trastuzumab) or anti-TROP2 antibody (datopotamab) were transfected into the aforementioned glycoengineered CHO cells. After culturing to an appropriate density, the cell culture supernatant was collected, and the antibody was purified by Protein A affinity chromatography.

[0034] Step 3: Preparation of antibody-drug conjugates. The purified glycoengineered antibody was conjugated to DXd via a cleavable peptide linker (such as the GGFG linker) to obtain a glycoengineered ADC. During conjugation, 6-8 molar equivalents of the linker-drug were added; after the reaction, unconjugated linker-drug and aggregates were removed. Drug loading distribution and weighted average drug-antibody ratio (DAR) were determined by HIC-HPLC to confirm the comparability of the DAR between the glycoengineered ADC and the corresponding unmodified control ADC.

[0035] Example 2: Effects of deglycosylation on ADC glycogen aggregate partitioning and cytotoxicity To detect whether antibody glycans affect the distribution of ADCs in glycogen aggregates, Dato-DXd and T-DXd were treated with PNGase F or EndoS, respectively. PNGase F was used to remove N-glycans, while EndoS was used to specifically hydrolyze the chitobiose core of the Fc segment N-glycans.

[0036] In vitro phase separation analysis showed that the degree to which T-DXd entered glycogen aggregates was significantly reduced after treatment with PNGase F or EndoS. Figure 1 (b) Subsequently, MDA-MB-231 cells were treated with deglycosylated Dato-DXd, and BT474 cells were treated with deglycosylated T-DXd. CCK-8 analysis showed that deglycosylated ADCs reduced the survival rate of the corresponding tumor cells (b). Figure 1 (c, d); PI flow cytometry analysis showed that deglycosylated ADCs induced increased cell death ( Figure 1 (eh in the middle).

[0037] The above results suggest that the Fc segment N-glycan structure can affect the interaction between ADCs and glycogen condensates. However, complete deglycosylation may simultaneously alter Fc receptor binding, complement-related functions, stability, or in vivo behavior. Therefore, this invention further employs a refined glycoengineering strategy that preserves the Fc segment N-glycan backbone.

[0038] Example 3: In vitro glycogen aggregate distribution and cytotoxicity of glycoengineered ADCs More refined glycosylation editing is performed in antibody-producing CHO cells. Antibodies generated by engineered cells are coupled to DXd via a cleavable linker to generate glycoengineered ADCs. Specific glycoengineering strategies include: (1) Knock out UGT (remove galactose); (2) Overexpression of B4GALT1 (increases galactosylation); (3) Knock out FUT8 (remove fucose); (4) Overexpression of GnTIII (increases bifurcation GlcNAc); And (5) overexpression of ST6GAL1 (increases α2,6-linked sialylation).

[0039] In vitro phase separation analysis showed that UGT knockout, B4GALT1 overexpression, FUT8 knockout, or GnTIII overexpression did not produce the same consistent effect as ST6GAL1 overexpression; ST6GAL1-mediated sialylation significantly reduced the degree of T-DXd entry into glycogen condensates. Figure 2 (a) in the middle.

[0040] LysoLight-labeled ADCs further revealed that ST6GAL1-engineered T-DXd produced a stronger LysoLight reporter signal in BT474 cells, suggesting enhanced lysosomal ADC degradation and peptide linker cleavage. Figure 2 (b) In the tested glycoengineered ADCs, ST6GAL1-mediated sialylation produced a relatively consistent decrease in tumor cell viability and an increase in cell death. Figure 2 (ch in the middle).

[0041] These results support the conclusion that increasing ADC sialylation levels can reduce its entry into glycogen aggregates and enhance lysosomal processing and tumor cell killing activity.

[0042] Example 4: Optimization and characterization of hypersialylated ADC Considering that enhanced sialylation modification can be detected by Sambucus nigra lectin (SNA, which recognizes α2,6-linked sialic acid) and / or Maackia amurensis lectin II (MAL-II, which recognizes α2,3-linked sialic acid), this embodiment further optimizes ADC sialylation by performing multisaccharide engineering in CHO cells, including the following four groups: (1) ST6GAL1 overexpression alone; (2) Co-expression of B4GALT1 and ST3GAL4; (3) Co-expression of B4GALT1 and ST6GAL1; (4) FUT8 knockout combined with ST6GAL1 overexpression.

[0043] SNA lectin blot analysis confirmed glycosylation editing efficiency: ST6GAL1 overexpression increased SNA reactivity by approximately 2.5-fold, and co-expression of B4GALT1 and ST6GAL1 further increased it by approximately 4.2-fold. Figure 3 (a) Compared to other multi-strategy approaches, the B4GALT1 / ST6GAL1 engineered ADC exhibited the strongest glycogen condensate removal in vitro ( Figure 3 (b) and the most potent tumor cell killing activity ( Figure 3 (ch in the middle).

[0044] Therefore, the co-expression of B4GALT1 and ST6GAL1 was chosen as the preferred glycoengineering strategy for subsequent in vivo evaluation.

[0045] Example 5: Antitumor activity of hypersialylated ADCs in a PDX tumor model A HER2-positive breast cancer PDX tumor model was established in immunized humanized NCG mice. When the tumor reached 80-100 mm³, the mice were randomly divided into six groups and administered the following intravenous treatments: (1) Control IgG; (2) Unmodified trastuzumab (T); (3) sialylated trastuzumab (Sia-T); (4) Isotype control IgG-DXd; (5) Unmodified T-DXd; (6) Sialylated T-DXd (Sia-T-DXd).

[0046] All antibodies or ADCs were administered at a dose of 50 nmol / kg every 4 days for a total of 5 cycles. Figure 4 (a) in the middle.

[0047] Tumors in both the IgG and IgG-DXd treatment groups showed exponential growth, with no significant difference between the two groups, indicating that non-targeted payload delivery did not contribute to antitumor activity. Figure 4 (b) Uncoupled T and Sia-T only produced mild tumor suppression, with no significant difference in tumor volume and survival, indicating that sialylation did not significantly enhance the activity of uncoupled antibodies. Figure 4 (b, c in the text).

[0048] In contrast, T-DXd inhibited tumor growth, while Sia-T-DXd produced the strongest anti-tumor effect, with significantly smaller tumor volume compared to the T-DXd group, and the difference increased over time. Figure 4 (b) Sia-T-DXd also significantly prolonged median survival, while the unconjugated antibody and control group showed no survival benefit. Figure 4 (c) The changes in body weight were similar across groups, indicating that no significant systemic toxicity was observed. Figure 4 (d in the text)

[0049] These results support the conclusion that B4GALT1 and ST6GAL1-mediated antibody sialylation enhances the antitumor activity of T-DXd with cleavable peptide linkers in this HER2-positive breast cancer PDX model, without additional weight loss observed during the observed cycles.

[0050] In the above embodiments, this invention sequentially compared enzymatic deglycosylation, single-gene glycan engineering, and multiple-glycan engineering, and validated these methods using complementary indicators such as in vitro glycogen condensate partitioning, LysoLight reporter assay, cell viability, and cell death. Ultimately, increasing sialylation levels was identified as an effective approach. This approach preserves the Fc segment N-glycan and its fundamental role in antibody structure and effector function, while also identifying specific terminal glycan characteristics that can reduce the interaction between the ADC and glycogen condensate.

[0051] Example 6: Screening for candidate antibody-drug conjugates with improved lysosomal processing efficiency This embodiment provides a screening method using glycoengineered antibodies and their corresponding unmodified antibodies as a paired candidate set; the lysosomal processing of glycoengineered ADCs is evaluated by LysoLight reporter signal, and the payload release is quantitatively evaluated by free DXd.

[0052] Step 1: Construction of the candidate antibody set. Candidate antibodies can be selected from antibodies obtained by different single-gene or multiple sugar engineering strategies; each candidate antibody is accompanied by an unmodified control antibody with the same amino acid sequence and other preparation conditions, except that the corresponding sugar engineering modification was not performed.

[0053] Step 2: In vitro glycogen aggregate partition coefficient detection. Candidate antibodies and corresponding unmodified control antibodies were labeled with the same fluorescent dye (e.g., Cy3, Cy5, or FITC) under identical conditions, and normalized according to antibody concentration and fluorescence labeling intensity to ensure identical fluorescent labeling levels for both antibodies. Equal amounts of the fluorescently labeled candidate antibody and unmodified control antibody were then mixed with an equal amount of glycogen (oyster glycogen in this example) in independent but identical reaction systems. The phase separation buffer contained 20 mM sodium acetate and 150 mM NaCl, pH 5.0, simulating a lysosomal environment. After incubating each reaction mixture at room temperature for 30 minutes, droplet formation and molecular partitioning behavior were observed using a confocal microscope. The fluorescence intensity of each antibody in the dense phase and dilute phase was quantified, and the partition coefficient was calculated using the following formula: Partition coefficient = Dense phase fluorescence intensity / Dilute phase fluorescence intensity.

[0054] Candidate antibodies with a lower allocation coefficient than the corresponding unmodified control were selected as pre-selected antibodies and proceeded to the subsequent ADC conjugation and cell validation steps.

[0055] Step 3: Intracellular LysoLight reporter assay. Candidate antibodies and corresponding unmodified control antibodies were conjugated to DXd using the same cleavable peptide linker to prepare paired ADCs. The ADCs were labeled with LysoLight and incubated with target antigen-positive tumor cells. LysoLight fluorescence intensity was detected by flow cytometry. Increased signal intensity suggests enhanced ADC degradation and peptide linker cleavage in lysosomes, but this does not equate to direct quantification of free DXd.

[0056] Step 4: Detection of free DXd release. After ADC treatment of cells, cell culture supernatant was collected, filtered by 10 kDa centrifugation, and the level of free DXd was detected using a DXd ELISA kit and compared with the corresponding unmodified control ADC.

[0057] Step 5: Cytotoxicity validation. The effects of the candidate ADC and the corresponding unmodified control ADC on cell viability and cell death of tumor cells were detected by CCK-8 assay and PI flow cytometry.

[0058] This screening method can be used to screen ADC candidates with improved lysosomal processing efficiency from a collection of glycoengineered antibodies.

[0059] Example 7: Method for detecting sialic acidification levels This embodiment provides a method for detecting the sialylation level of glycoengineered antibodies.

[0060] Method 1: Lectin Blot Analysis. The purified glycoengineered antibody was separated by SDS-PAGE and transferred to a PVDF membrane, which was then blocked with 3 wt% BSA solution. α2,6-linked sialic acid was detected using biotin-labeled Sambucus nigra lectin (SNA, 1:1,000), and α2,3-linked sialic acid was detected using biotin-labeled Maackia amurensis lectin II (MAL-II, 1:1,000). Subsequent color development was performed using streptavidin-HRP and a chemiluminescent substrate.

[0061] Method 2: N-glycan chromatography. Purified glycan-engineered antibodies were treated with PNGase F to release N-glycans; they were then reductively labeled with 2-aminobenzamide (2-AB) and detected by HILIC-UPLC fluorescence. Based on the standard retention times of the 2-AB-labeled dextran ladder and combined with exonuclease sequencing, the glycan structures were identified, and the relative proportions of sialylated glycans were compared.

[0062] Method 3: Characterization of key quality attributes of ADCs. Surface plasmon resonance was used to detect the binding kinetics of T-DXd-related samples with HER2 and Dato-DXd-related samples with TROP2; HIC-HPLC was used to detect drug loading distribution and calculate the weighted average DAR; SEC-HPLC was used to evaluate monomer and aggregate levels. Glycoengineered ADCs should be comparable to corresponding unmodified control ADCs in terms of target antigen binding, drug loading, and aggregate levels.

[0063] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements 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 a glycoengineered antibody-drug conjugate, characterized in that, Includes the following steps: (1) Prepare antibodies; (2) The Fc segment N-glycan chain of the antibody is engineered to give the antibody enhanced sialylation modification, thereby obtaining a glycoengineered antibody; (3) The glycoengineered antibody obtained in step (2) is coupled with a linker and a cytotoxic payload to obtain a glycoengineered antibody-conjugated drug.

2. The preparation method according to claim 1, characterized in that, In step (2), the Fc segment N-glycan of the antibody is engineered, specifically including: (i) Overexpression of sialyltransferase; And optionally (ii) further overexpression of galactosyltransferase and / or knockout of fucosyltransferase; This increases the sialylation level of the Fc segment N-glycan chain of the antibody; The enhanced sialylation modification includes increased levels of α2,6-linked sialic acid and / or α2,3-linked sialic acid. The enhancement refers to an increase in the corresponding sialylation level compared to the control antibody, which has the same amino acid sequence and other preparation conditions as the control antibody except for the one that did not undergo the corresponding glycoengineering modification in step (2).

3. The preparation method according to claim 2, characterized in that, The host cell for antibody expression is Chinese hamster ovary (CHO) cells; The sialyl transferase includes ST6GAL1 and / or ST3GAL4; The galactosyltransferase includes B4GALT1; The fucotransferase includes FUT8.

4. The preparation method according to claim 1, characterized in that, In step (1), the antibody is capable of targeting HER2 or TROP2; preferably, the antibody is trastuzumab or datopotamab. In step (3), the linker is a cleavable peptide linker; preferably, the linker is a GGFG linker. The cytotoxic payload is deruxtecan (DXd) or a derivative thereof.

5. A glycoengineered antibody-drug conjugate prepared by the preparation method according to any one of claims 1-4.

6. Use of the glycoengineered antibody-drug conjugate as described in claim 5 in the preparation of a medicament for treating tumors.

7. The use as described in claim 6, characterized in that, The tumor is either HER2-positive or TROP2-positive breast cancer.

8. A method for screening glycoengineered antibody-drug conjugate candidates with improved lysosomal processing efficiency, characterized in that, Includes the following steps: S1: Provides candidate glycoengineered antibodies and their corresponding unmodified control antibodies; S2: Under the same conditions, detect the partition coefficients of the candidate glycogen engineered antibody and the unmodified control antibody in glycogen aggregates; S3: Select candidate glycoengineered antibodies with a partition coefficient lower than that of the unmodified control antibody as pre-selected antibodies; S4: The preselected antibody and the unmodified control antibody are conjugated with the same cleavable peptide linker and cytotoxic payload, respectively, to obtain the candidate antibody-drug conjugate and the corresponding unmodified control antibody-drug conjugate.

9. The method as described in claim 8, characterized in that, In step S2, equal amounts of fluorescently labeled candidate glycogen engineered antibody and unmodified control antibody are mixed with glycogen under the same in vitro phase separation conditions. The fluorescence intensity of each antibody in the aggregate phase and dilution phase is quantified, and the partition coefficient of each antibody is calculated using the following formula. Distribution coefficient = fluorescence intensity of aggregate phase / fluorescence intensity of dilute phase.

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