A conjugate of pd-1 antibody and iRGD, a preparation method and application thereof in preparing immunotherapy drugs

Abstract

The application provides a conjugate of a PD-1 antibody and iRGD, a preparation method and application thereof in preparation of an immunotherapy drug. The conjugate of the PD-1 antibody and iRGD has the characteristics of site specificity and high uniformity, and compared with an unmodified PD-1 antibody, shows obviously enhanced penetration ability in a 3D cell ball model in vitro and a mouse tumor model, and effectively promotes the penetration of PD-1 positive T cells to a tumor part. The conjugate provided by the application enhances the connection between T cells and tumor cells, improves the targeting property, and further improves the killing ability of T cells to tumor cells. In different mouse tumor models, the conjugate of the PD-1 antibody and iRGD provided by the application can effectively inhibit tumor growth, and does not cause obvious adverse reactions such as inflammation damage of main organs and weight loss of mice. Compared with the combination of iRGD and the PD-1 antibody, the conjugate of the PD-1 antibody and iRGD provided by the application shows equivalent or better anti-tumor activity at a lower dose of iRGD.

Description

Technical Field

[0001] This invention relates to the field of antitumor immunotherapy drugs, specifically to a conjugate of PD-1 antibody and iRGD, its preparation method, and its application in the preparation of immunotherapy drugs. Background Technology

[0002] In recent years, with the increasing aging population, the burden of cancer in my country has significantly increased, seriously affecting the health and lives of the Chinese population. Immunotherapy is a new strategy for cancer treatment following surgery, chemotherapy, and radiotherapy. Immune checkpoint inhibitors are the most widely used immunotherapy methods in clinical practice, bringing significant survival benefits to cancer patients. However, the treatment response rate of immune checkpoint inhibitors is low, only about 20%, which greatly limits their clinical application. Currently, all clinically approved immune checkpoint inhibitors are monoclonal antibodies, which have a large molecular weight and are difficult to distribute rapidly and effectively in the tumor site. At the same time, abnormal angiogenesis, matrix proliferation, and suppressor cells in the tumor site together form a "barrier," further limiting the distribution of large molecule drugs. A small-sample clinical isotope tracing trial suggested that the uptake of PD-1 monoclonal antibodies in the tumor site is positively correlated with patient survival and immunotherapy response. Therefore, enhancing the penetration of immune checkpoint inhibitors is a potential strategy to improve the clinical treatment response rate.

[0003] iRGD is a tumor-penetrating cyclic peptide composed of 9 amino acids, with the sequence c(CRGDKGPDC). iRGD accumulates locally in the tumor by binding to integrins, and after being cleaved by various proteases in the tumor microenvironment, it exposes its Neuropilin-1 (NRP1) binding site, mediating secondary penetration. Co-administration of iRGD with albumin-bound paclitaxel, doxorubicin, and trastuzumab significantly increases drug distribution locally in the tumor. However, co-administration has certain limitations. Under non-conjugated conditions, high doses of free iRGD are required to achieve similar penetration improvement effects, posing certain safety risks. Furthermore, the short half-life of free iRGD necessitates frequent dosing, which is detrimental to treatment adherence. Conjugating iRGD with PD-1 monoclonal antibodies not only avoids the potential side effects of high-dose free iRGD but also allows for longer dosing intervals, improving treatment adherence. However, preparing PD-1 antibody-iRGD conjugates remains very challenging, especially for conjugates that are homogeneous, stable, and maintain the structural integrity of iRGD. Fusion expression methods struggle to control the cyclization state of iRGD. Traditional conjugation techniques typically rely on the amino and thiol groups on antibody amino acid residues, achieving drug-antibody conjugation through ester activation or addition reactions with maleimide. This usually yields heterogeneous products. The complex structure of iRGDs further complicates the process. Currently, there is no effective method to construct homogeneous and stable iRGD conjugates on PD-1 antibodies. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a conjugate of PD-1 antibody and iRGD, its preparation method, and its application in the preparation of immunotherapeutic drugs.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] On one hand, the present invention provides a conjugate of PD-1 antibody and iRGD.

[0007] An antibody-drug conjugate comprising a PD-1 antibody and iRGD, wherein the PD-1 antibody specifically binds to one or more epitopes of a human or mouse PD-1 protein, and the iRGD is a cyclic polypeptide with the amino acid sequence c(CRGDKGPDC).

[0008] Furthermore, the structure of the antibody conjugate is as follows:

[0009] In the formula: It is a PD-1 antibody;

[0010] GlcNAc is directly or indirectly linked to the amino acids of the PD-1 antibody;

[0011] GalX is galactose or its derivatives, and GalX is linked to GlcNAc via a β-1,4 glycosidic bond.

[0012] Fuc is fucose, and Fuc is linked to GlcNAc through an α1,6 glycosidic bond, where b is 0 or 1.

[0013] Fuc* is a fucose derivative containing iRGD, and Fuc* is linked to GlcNAc via an α1,3 glycosidic bond;

[0014] z is any integer from 1 to 8.

[0015] Furthermore, the structure of the antibody conjugate is as follows:

[0016]

[0017] In the formula: It is a PD-1 antibody;

[0018] The GlcNAc directly linked to the PD-1 antibody is the core GlcNAc, and the core GlcNAc is directly or indirectly linked to the amino acids of the PD-1 antibody.

[0019] Fuc is fucose, and Fuc is linked to the core GlcNAc via an α1,6 glycosidic bond.

[0020] Man refers to mannose;

[0021] GalX is galactose or its derivatives, and GalX is linked to GlcNAc via a β-1,4 glycosidic bond.

[0022] Fuc* is a fucose derivative containing iRGD, and Fuc* is linked to GlcNAc via an α1,3 glycosidic bond.

[0023] Furthermore, the structure of the antibody conjugate is as follows:

[0024]

[0025] In the formula: It is a PD-1 antibody;

[0026] GlcNAc is directly or indirectly linked to the amino acids of the PD-1 antibody;

[0027] GalX is galactose or its derivatives, and GalX is linked to GlcNAc via a β-1,4 glycosidic bond.

[0028] Fuc is fucose, and Fuc is linked to GlcNAc through an α1,6 glycosidic bond, where b is 0 or 1.

[0029] Fuc* is a fucose derivative containing iRGD, and Fuc* is linked to GlcNAc via an α1,3 glycosidic bond.

[0030] Furthermore, the PD-1 antibody comprises the following antibodies or their antigen-binding moieties: toripalimab, sintilimab, camrelizumab, tislelizumab, penaprilimab, cepalimumab, slulilimumab, pembrolizumab, and 2E5.

[0031] Further, the PD-1 antibody comprises HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1; HCDR3 comprises the amino acid sequence shown in SEQ ID NO 1; HCDR2 comprises the amino acid sequence shown in SEQ ID NO 2; HCDR1 comprises the amino acid sequence shown in SEQ ID NO 3; LCDR3 comprises the amino acid sequence shown in SEQ ID NO 4; LCDR2 comprises the amino acid sequence shown in SEQ ID NO 5; and LCDR1 comprises the amino acid sequence shown in SEQ ID NO 6.

[0032] Furthermore, the PD-1 antibody comprises VL and VH; the VL comprises the amino acid sequence shown in SEQ ID NO 7; and the VH comprises the amino acid sequence shown in SEQ ID NO 8.

[0033] Furthermore, the heavy chain of the PD-1 antibody contains the amino acid sequence shown in SEQ ID NO 9, and the light chain contains the amino acid sequence shown in SEQ ID NO 10.

[0034] Furthermore, the PD-1 antibody contains an Fc fragment, and the GlcNAc is directly or indirectly linked to the Asn in the Fc fragment.

[0035] Furthermore, the GlcNAc is directly or indirectly connected to Asn297 in the Fc segment, and the Fc segment is numbered according to the Kabat numbering system.

[0036] Furthermore, the Fuc* has an iRGD-L-Fuc' structure, where L is a connector.

[0037] Furthermore, in the iRGD-L-Fuc' structure, the iRGD has the following structure:

[0038]

[0039] The spiral on the right side of the formula indicates a connection with L.

[0040] Furthermore, in the iRGD-L-Fuc' structure, the structure of L is as follows:

[0041]

[0042] In the formula, FL is a spacer, and FL is selected from at least one of the following substances: polypeptides and their derivatives, polyethylene glycol and its derivatives, alkyl groups and their derivatives; s is 0 or 1.

[0043] Furthermore, in the iRGD-L-Fuc' structure, the structure of Fuc' is as follows:

[0044]

[0045] In the formula, the spiral on the right end indicates that Fuc' is connected to GlcNAc, and the spiral on the left end indicates that Fuc' is connected to L.

[0046] Furthermore, the structure of Fuc* is as follows:

[0047]

[0048] The spiral on the right side of the formula indicates that Fuc* is connected to GlcNAc.

[0049] Furthermore, the GalX is selected from at least one of the following structures:

[0050]

[0051] In the above structure, the spiral indicates a connection with GlcNAc.

[0052] A pharmaceutical composition comprising an antibody conjugate as described above, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0053] On the other hand, the present invention provides a method for preparing a conjugate of PD-1 antibody and iRGD.

[0054] A method for preparing antibody conjugates includes the following steps:

[0055] Step 1: Incubate 3-15 mg / mL of PD-1 antibody, 0.01-0.1 mg / mL of EndoS and 0.5-2 mg / mL of Alfc in 50 mM Tris-HCl buffer at pH 5.0-8.0 at 30-37°C.

[0056] Step 2: After 24 hours, add any one of UDP-Gal, UDP-GalNAz or UDP-GalNAc, bovine β1,4-GalT1 and MnCl2 to the reaction mixture from Step 1, and incubate at 30-37°C for 2-48 hours to obtain the antibody.

[0057] Step 3: Purify and modify the antibody obtained in Step 2 with protein A resin to obtain αPD-1-(GalXβ1,4)GlcNAc. Replace 3-15 mg / mL of αPD-1-(GalXβ1,4)GlcNAc with 50 mM Tris-HCl buffer (pH 5.0-8.0) by ultrafiltration. Incubate with 0.1-5 mM GDP-FAm-iRGD, 0.1-1 mg / mL Hp1,3-FucT, and 0-10 mM MgCl2 at 30-37°C for 2-48 hours to obtain the modified antibody.

[0058] Step 4: Purify the modified antibody obtained in Step 3 using protein A resin to obtain the PD-1 antibody iRGD conjugate.

[0059] A method for preparing antibody conjugates includes the following steps:

[0060] Step 1: Incubate 3-15 mg / mL of PD-1 antibody and 0.01-0.1 mg / mL of EndoS in 50 mM Tris-HCl buffer (pH 5.0-8.0) at 30-37°C for 0.1-1 hours.

[0061] Step 2: Add either UDP-Gal or UDP-GalNAz to the reaction mixture from Step 1 at a final concentration of 1-10 mM, bovine β1,4-GalT1 at a final concentration of 0.1-0.5 mg / mL, and MnCl2 at a final concentration of 0.1-5 mM. Incubate at 30-37°C for 2-48 hours to obtain the modified antibody.

[0062] Step 3: Purify the modified antibody with protein A resin to obtain αPD-1-(Fucα1,6)(GalNAzβ1,4)GlcNAc. Replace 3-15 mg / mL of αPD-1-(Fucα1,6)(GalNAzβ1,4)GlcNAc with 50 mM Tris-HCl buffer (pH 5.0-8.0) via ultrafiltration. Incubate with 0.1-5 mM GDP-FAm-iRGD, 0.1-1 mg / mL Hp1,3-FucT, and 0-10 mM MgCl2 at 30-37°C for 2-48 hours to obtain the PD-1 antibody iRGD conjugate: αPD-1-(Fucα1,6)(GalNAzβ1,4)GlcNAc-FAm-iRGD.

[0063] A method for preparing antibody conjugates includes the following steps:

[0064] Step 1: Incubate 3-15 mg / mL of PD-1 antibody, 1-10 mM of UDP-Gal, 0.1-1.0 mg / mL of bovine β1,4-GalT1, 0.1-5 mM of GDP-FAm-iRGD, 0.1-1 mg / mL of Hp1,3-FucT, 0-10 mM of MgCl2, and 50 mM of Tris-HCl buffer at pH 5.0-8.0 at 30-37°C for 24-72 hours to obtain the modified antibody.

[0065] Step 2: Purify the modified antibody obtained in Step 1 with protein A resin to obtain the PD-1 antibody iRGD conjugate: αPD-1-G2F-FAm-iRGD.

[0066] In another aspect, the present invention provides the application of a conjugate of PD-1 antibody and iRGD in the preparation of immunotherapeutic drugs, wherein the immunotherapies include diseases such as cancers, gastric cancer, and melanoma that have PD-1 / PD-L1 immune checkpoints.

[0067] The beneficial effects of this invention are:

[0068] 1) Traditional conjugation techniques are generally based on the amino and thiol groups on the amino acid residues of antibodies. Drug conjugation to antibodies is achieved by activating esters or by adding maleimide. Usually, heterogeneous products are obtained. This invention provides an effective method to construct a homogeneous and stable iRGD conjugate on PD-1 antibodies.

[0069] 2) The PD-1 antibody and iRGD conjugate provided by this invention have the characteristics of site specificity and high homogeneity;

[0070] 3) Compared with the unmodified PD-1 antibody, the PD-1 antibody and iRGD conjugate provided by the present invention showed significantly enhanced penetration ability in both the in vitro 3D cell spheroid model and the mouse tumor model, and effectively promoted the penetration of PD-1 positive T cells into the tumor site.

[0071] 4) The PD-1 antibody and iRGD conjugate provided by this invention enhances the binding of T cells to tumor cells, improves targeting, and further improves the killing ability of T cells against tumor cells;

[0072] 5) In different mouse tumor models, the conjugate of PD-1 antibody and iRGD provided by this invention can effectively inhibit tumor growth without causing significant inflammatory damage to major organs or weight loss in mice.

[0073] 6) Compared with the combination of iRGD and PD-1 antibody, the conjugate of PD-1 antibody and iRGD provided by the present invention shows comparable or superior antitumor activity at a lower dose of iRGD. Attached Figure Description

[0074] Figure 1 Schematic diagram and characterization of PD-1 antibody iRGD conjugation: (A) Schematic diagram of PD-1 antibody iRGD conjugation; (B) High-resolution mass spectrum of αPD-1-(iRGD)2; (C) ELISA detection of the binding affinity of αPD-1-(iRGD)2 and unmodified PD-1 antibody to human PD-1 protein; (D) ELISA detection of the binding affinity of αPD-1-(iRGD)2 and unmodified PD-1 antibody to mouse PD-1 protein; (E) Synthetic route of GDP-FAm-iRGD.

[0075] Figure 2To demonstrate how PD-1 antibody iRGD conjugates promote T cell-tumor cell binding and enhance cytotoxicity: (A) Illustration of PD-1 antibody iRGD conjugates promoting T cell-tumor cell binding; (B) Flow cytometry analysis of the changes in relative mean fluorescence intensity in GES-1, HGC27, NCIN87, and MFC cell lines co-cultured with αPD-1-(iRGD)2-Cy5 or αPD-1-Cy5; (C) Flow cytometry plots reflecting the expression of iRGD's main receptors, integrin αvβ5 and Nrp-1, in HGC27, N87, and GES-1 cell lines; (D) PD-1 antibody iRGD conjugates promoting T cell-tumor cell binding and enhancing cytotoxicity: (A) Illustration of PD-1 antibody iRGD conjugates promoting T cell-tumor cell binding; (B) Flow cytometry analysis of the changes in relative mean fluorescence intensity in GES-1, HGC27, NCIN87, and MFC cell lines co-cultured with αPD-1-(iRGD)2-Cy5; (C) Flow cytometry plots reflecting the expression of iRGD's main receptors, integrin αvβ5 and Nrp-1, in HGC27, N87, and GES-1 cell lines; (D) PD-1 antibody iRGD conjugates promoting T cell-tumor cell binding and enhancing cytotoxicity. (E) Changes in MFI folds of Jurkat cells after 1 h of incubation with αPD-1-(iRGD)2-Cy5 or αPD-1-Cy5; (E) Monolayers of HGC27 cells co-cultured with healthy donor PBMCs, incubated with αPD-1-(iRGD)2 (concentrations of 0.1, 0.5, 1.0, 2.5, 5.0 μg / 100 μl) or αPD-1 (concentrations of 0.1, 0.5, 1.0, 2.5, 5.0 μg / 100 μl) combined with 0.1 μg of free iRGD at an effector-target ratio of 10:1. (F) The lysis rate of tumor cells after 16 hours of incubation; (G) A simplified diagram of the experimental method for promoting T cell-tumor cell binding with αPD-1-(iRGD)2; (H) Co-incubation of Dye670-labeled HGC27 cells and CFSE-labeled OT-I cells at an effector-target ratio of 1:10, with the addition of αPD-1-(iRGD)2 (concentration of 1 μg / 100 μl) or αPD-1 (concentration of 1 μg / 100 μl) or αPD-1. -1 (concentration of 1 μg / 100 μl) combined with free iRGD (0.1 μg) for 1 hour, percentage of CFSE and Dye670 double-positive cells; (I) Representative flow cytometry results; (J) Schematic diagram of the experimental method for αPD-1-(iRGD)2 to promote the binding of Jurkat cells to tumor cells; (K) Percentage of CFSE and Dye670 double-positive cells after co-incubating Dye670-labeled HGC27 cells and CFSE-labeled Jurkat cells at an effector-target ratio of 1:10 for 1 hour; (L) Representative flow cytometry results, data are shown as mean ± sem; n = 3.

[0076] Figure 3 Permeability analysis of the PD-1 antibody iRGD conjugate: (A) Schematic diagram of the permeability assay of αPD-1-(iRGD)2-Cy5, in which the cell spheres are composed of HGC27 cells, each sphere containing approximately 5 × 10⁻⁶ cells. 4(a) Schematic diagram of αPD-1-Cy5 (1 μg), or αPD-1-(iRGD)2-Cy5 (1 μg), or αPD-1-Cy5 (1 μg) and low-dose free iRGD (L-iRGD, 0.01 μg), or αPD-1-Cy5 (1 μg) and high-dose free iRGD (H-iRGD, 0.1 μg) per well; (B) Schematic diagram of αPD-1-(iRGD)2 promoting the infiltration of CFSE-labeled Jurkat-PD-1 cells into HGC27 MCS, wherein the cell spheres are composed of CFSE fluorescently labeled HGC27 cells, each sphere containing approximately 5 × 10⁻⁶ cells. 4 (c) Cells, each well containing αPD-1 (1 μg), or αPD-1-(iRGD)2 (1 μg), or αPD-1 (1 μg) and a high dose of free iRGD (0.1 μg); (c) Confocal microscopy image showing the penetration of αPD-1-(iRGD)2-Cy5 in MCS; (d) Confocal microscopy image showing the penetration of CFSE-labeled Jurkat cells in MCS, magnification ×50; scale bar 200 μm; (e) Mean fluorescence intensity of MCS incubated with αPD-1-(iRGD)2-Cy5 or control reagent at a specified time; (f) Mean fluorescence intensity of MCS co-cultured with CFSE-labeled Jurkat cells and αPD-1-(iRGD)2 or control reagent, data shown as mean ± sem; n = 3.

[0077] Figure 4 Targeting analysis of PD-1 antibody-iRGD conjugate: (A) Mice subcutaneously inoculated with MFC cells were intraperitoneally injected with αPD-1-(iRGD)2-Cy5 (50 μg), αPD-1-Cy5 (50 μg), or αPD-1-Cy5 (50 μg) + free iRGD (1.25 μg), and Cy5 bioluminescence images were taken at regular intervals; (B) In vitro fluorescence images of tumors, liver, heart, kidneys, lungs, and spleen 24 hours after intraperitoneal injection; (C) Line graph of mean fluorescence intensity of tumor sites in each MFC tumor-bearing mouse after drug injection; (D) Mean fluorescence intensity of tumor volume excised 48 h after drug injection, data are shown as mean ± sem; n = 3.

[0078] Figure 5 Analysis of the antitumor effect of PD-1 antibody iRGD conjugate: (A) Schematic diagram of the treatment regimen in the MFC mouse gastric tumor model, 615 mice were subcutaneously injected with 1×10 6Starting on day 3 after MFC cell administration, mice were intraperitoneally injected every three days with PBS (100 μL control), αPD-1 (5 mg / kg), αPD-1 (5 mg / kg), free iRGD (1.25 μg), and αPD-1-(iRGD)2 (5 mg / kg); (B) Tumor volume graph and tumor weight collected at the treatment endpoint in the MFC animal model; (C) Schematic diagram of the treatment regimen in the B16F10 mouse melanoma model; mice were subcutaneously inoculated with 1×10 5 B16F10 cells were intraperitoneally injected every three days with PBS (100 μL control), αPD-1 (5 mg / kg), αPD-1 (5 mg / kg), free iRGD (1.25 μg), and αPD-1-(iRGD)2 (5 mg / kg); (D) Tumor volume graph and tumor weight collected at the treatment endpoint in the B16F10 animal model; (E) CD8+ in tumor tissue in the MFC mouse gastric tumor model. + T cell abundance; (F) endpoint of MFC mouse gastric tumor model, flow cytometry quantification of intratumoral CD8 + PD-1 expression on T cells; endpoint of (G)MFC mouse gastric tumor model; flow cytometry analysis of CD8+ in tumors. + Expression of GZMB and IFNγ on T cells; endpoint of (H)MFC mouse gastric tumor model, quantitative determination of CD4 in tumors by flow cytometry. + Expression of GZMB and IFNγ on T cells; (I) In the MFC mouse gastric tumor model, CD8+ from the tumor + Representative flow cytometry scatter plots of GZMB and IFNγ expression on T cells; (J) In the MFC mouse gastric tumor model, CD4+ expression from the tumor... + Representative flow cytometry results of GZMB and IFNγ expression on T cells.

[0079] Figure 6 Equivalent free iRGD dose determination for PD-1 antibody-iRGD conjugate: (A) shows a schematic diagram of the treatment regimen in an MFC allogeneic model, briefly described as using 1×10 6 MFC cells were used to treat tumor-bearing mice, which were injected intraperitoneally every three days with PBS (100 μl control), αPD-1 (5 mg / kg), free iRGD (iRGD-Hi) (50 μg) and αPD-1-(iRGD)2 (5 mg / kg); (B) Tumor volume line graph; (C) Tumor weight bar graph at the endpoint; (D) In ​​vitro imaging of mouse tumor tissue at the endpoint.

[0080] Figure 7Modulation of the tumor microenvironment by PD-1 antibody iRGD conjugate: (A) Single-cell analysis, schematic diagram of treatment regimen in MFC mouse gastric tumor model, subcutaneous injection of 1×10⁻⁶ cells into 615 mice. 6 Starting on day 3 after MFC cell administration, patients were intraperitoneally injected every three days with PBS (100 μl control), αPD-1 (5 mg / kg), and free iRGD (1.25 μg) and αPD-1-(iRGD)2 (5 mg / kg); (B) CD45 + Two-dimensional UMAP visualization of tumor-infiltrating immune cells; (C) CD45 as described in single-cell analysis + The ratio of different subsets within tumor-infiltrating immune cells; (D)CD45 + (E) Heatmap of mean relative expression of selected genes in NK cells of each treatment group within tumor-infiltrating immune cells; CD45 as described in single-cell analysis. + CD8 in the αPD-1-(iRGD)2 treatment group of tumor-infiltrating immune cells + (F) Average relative expression heatmap of selected genes in T cells; CD45 as described in single-cell analysis + CD4 in the αPD-1-(iRGD)2 treatment group of tumor-infiltrating immune cells + Mean relative expression heatmap of selected genes in T cells; KEGG analysis of differentially expressed genes (G)αPD-1 and αPD-1-(iRGD)2 in tumor dendritic cells (DCs); KEGG analysis of differentially expressed genes (H)αPD-1 and αPD-1-(iRGD)2 in macrophages.

[0081] Figure 8 The PD-1 antibody iRGD conjugate amplified stem cell-like effector CD8 cells in the tumor microenvironment. + T cell subsets: (A)CD3 + Two-dimensional UMAP visualization of T cells; (B) shows CD3 + The ratio of different subsets within tumor-infiltrating T cells; (C) six CD3 + Normalized expression heatmap of representative genes within T cell clusters; (D)CD3 + Expression levels of stem cells (Tcf7, Bach2), effector cells (Gzmb, Ifng), co-stimulatory molecules (CD7, CD27, CD28), exhaustion cells (PD-1, Tim-3, Lag3, Entpd1), and transcription factors (Icos, T-bet, Tgfbi) in the six clusters of T cells.

[0082] Figure 9Safety analysis of PD-1 antibody-iRGD conjugate: (A) Body weight of MFC tumor-bearing mice after αPD-1-(iRGD)2 treatment, 615 mice subcutaneously injected with 1×10 6 Starting from the third day after MFC cell treatment, PBS (100 μl control), αPD-1 (5 mg / kg), αPD-1 (5 mg / kg), free iRGD (1.25 μg), and αPD-1-(iRGD)2 (5 mg / kg) were injected intraperitoneally every three days; (B) H&E staining of major organs (heart, liver, spleen, lung, and kidney) in the MFC subcutaneous mouse model after αPD-1-(iRGD)2 treatment. Detailed Implementation

[0083] The present invention will now be described in detail with reference to the accompanying drawings.

[0084] I. Protein Expression and Preparation Experiments

[0085] 1.1 PD-1 antibody expression and purification

[0086] The heavy and light chain sequences of the PD-1 antibody are SEQ ID NOs 9 and 10. Gene sequences encoding the light and heavy chains of the PD-1 antibody were constructed and cloned into the PPT5 vector (Nanjing Genscript). 293F cells were cultured to approximately 2.5 × 10⁻⁶ cells / year. 6 Cells were introduced at a density of [number] cells / ml and transfected by directly adding 0.37 μg / ml and 0.66 μg / ml light and heavy chain expression plasmid DNA, respectively, and 2.2 μg / ml polyethyleneimine (linear 25 kDa PEI, Polysciences, Inc., Warrington, PA) to the suspension culture. Twenty-four hours after transfection, the culture was diluted 1:1 with 293F expression medium containing 4.4 mM valproic acid (final concentration 2.2 mM) and protein production was continued at 37°C for 4–5 days. The antibody was finally purified by protein A agarose gel.

[0087] 1.2 Expression and purification of bovine β1,4-GalT1(Y289L), EndoS, Alfc and Hp1,3-FucT

[0088] The cloning, expression, and purification of bovine β-1,4-GalT1 (Y289L) (Bovine β-1,4-galactosyltransferase I, bovine β-1,4-galactosyltransferase with the Y289L mutation, SEQ ID NO 15), EndoS (Streptococcus pyogenesendoglycosidase S, SEQ ID NO 17), and Alfc (Lactobacilluscasei α-1,6-fucosidase Alfc, Lactobacillus casei α-1,6-fucosidase, SEQ ID NO 19) were performed according to the methods reported by Qasba PK et al. (J. Biol. Chem. 2002, 277, 20833; Prot. Expr. Fur. 2003, 30, 219) and Collin, M. et al. (EMBO) respectively. J. 2001, 20, 3046; Infect. Immun. 2001, 69, 7187) and the method reported by Wang L. et al. (Methods Mol. Biol. 2018, 19, 367).

[0089] The expression and purification of Hp1,3FucT involved synthesizing the nucleic acid sequence encoding Hp1,3FucT (SEQ ID NO 14) and inserting it into the pET24b vector (Nanjing GenScript) via NdeI and BamHI restriction sites. The constructed recombinant plasmid was then transformed into E. coli BL21(DE3). The transformed recombinant bacteria were cultured in LB medium containing 50 μg / mL kanamycin at 37°C until the OD600 reached 0.6-0.8. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.2 mM, and the culture was continued at 25°C and 200 rpm for 16 hours to induce protein expression. After centrifugation, the induced bacterial cells were resuspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 500 mM sodium chloride, 20 mM imidazole, 1 mM PMSF (phenylmethylsulfonyl fluoride)). The suspended cells were lysed by sonication and then purified using Ni-NTA packing material (GE Health). The main fraction with a purity greater than 90% was collected and then dialyzed into storage buffer (25 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 5% glycerol).

[0090] II. GDP-FAm-iRGD Synthesis Experiment

[0091] Synthesis of GDP-FAmP4Pro: 1500 μL ddH2O, 500 μL NaHCO3 (200 mM), 1750 μL THF, and 750 μL NHS-PEG4-Prop (50 mM in tetrahydrofuran (THF)) were added to 500 μL GDP-FAm (100 mM) solution. The reaction was stirred at room temperature (rt) for 4 h, and the reaction was monitored by thin-layer chromatography (TLC). The solvent was removed under reduced pressure. The crude product was further purified by Prep-HPLC to obtain the desired product (27.1 mg, 64%) as a white solid. The calculated relative molecular mass was 845.2013. High-resolution mass spectrometry analysis of the product showed a molecular weight of approximately 845.1989, consistent with expectations.

[0092] Synthesis of GDP-FAm-iRGD: 64 μL CuSO4 (50 mM / ddH2O), 128 μL BTTP (50 mM-ddH2O), 1280 μL N3-iRGD (50 mM, DMSO, Nanjing Yuantai Biotechnology Co., Ltd., China), and 320 μL ascorbic acid (50 mM / ddH2O) were added to 640 μL of GDP-FAmP4Prop (50 mM, DMSO, Nanjing Yuantai Biotechnology Co., Ltd., China). The reaction was stirred at room temperature for 12 hours, and the reaction process was monitored by TLC. The crude product was further purified by Prep HPLC to obtain a white solid product (GDP-FAm-iRGD, 38.4 mg, 64%) with a calculated relative molecular mass of 936.7932. High-resolution mass spectrometry analysis showed that the molecular weight of the product was 936.7961, consistent with expectations.

[0093] III. Synthesis Experiment of αPD-1-RGD Couplings

[0094] 3.1 Synthesis of αPD-1-(GalXβ1,4)GlcNAc-FAm-iRGD conjugate

[0095] The synthesis process of αPD-1-(GalXβ1,4)GlcNAc-FAm-iRGD is as follows: Figure 1(A) PD-1 antibody (3-15 mg / mL) was incubated with EndoS (0.01-0.1 mg / mL) (SEQ.ID.NO: 17) and Alfc (SEQ.ID.NO: 19) (0.5-2 mg / mL) in 50 mM Tris-HCl buffer (pH 5.0-8.0) at 30-37°C. After 24 hours, UDP-Gal or UDP-GalNAz or UDP-GalNAc (final concentration 1-10 mM, Shanghai Baosen Biotechnology Co., Ltd., China), bovine β1,4-GalT1 (Y289L) (SEQ.ID.NO: 15) (final concentration 0.1-0.5 mg / mL) and MnCl2 (final concentration 0.1-5 mM) were added to the reaction mixture and incubated at 30-37°C for 2-48 hours. The modified antibody was purified using protein A resin to obtain αPD-1-(GalXβ1,4)GlcNAc, with the following structures: αPD-1-(Galβ1,4)GlcNAc (molecular weight 145964.00 Da by mass spectrometry), αPD-1-(GalNAzβ1,4)GlcNAc (molecular weight 146128.00 Da by mass spectrometry), and αPD-1-(GalNAcβ1,4)GlcNAc (molecular weight 146043.00 Da by mass spectrometry).

[0096] αPD-1-(GalXβ1,4)GlcNAc (3-15 mg / mL) was replaced with 50 mM Tris-HCl buffer (pH 5.0-8.0) by ultrafiltration and incubated with GDP-FAm-iRGD (final concentration 0.1-5 mM), Hp1,3-FucT (final concentration 0.1-1 mg / mL), and MgCl2 (final concentration 0-10 mM) at 30-37°C for 2-48 hours. The modified antibodies were purified using protein A resin to obtain PD-1 antibody iRGD conjugates, with the formulas αPD-1-(Galβ1,4)GlcNAc-FAm-iRGD (mass spectrometry-characterized molecular weight 148831.00 Da, drug-to-antibody ratio between 1.8 and 2.0), αPD-1-(GalNAcβ1,4)GlcNAc-FAm-iRGD (mass spectrometry-characterized molecular weight 148992.00 Da, drug-to-antibody ratio between 1.8 and 2.0), and αPD-1-(GalNAcβ1,4)GlcNAc-FAm-iRGD (mass spectrometry-characterized molecular weight 148910.00 Da, drug-to-antibody ratio between 1.8 and 2.0). In the figure and caption, αPD-1-(Galβ1,4)GlcNAc-FAm-iRGD is abbreviated as αPD-1-(iRGD)2.

[0097] 3.2 Synthesis of αPD-1-(Fucα1,6)(GalNAzβ1,4)GlcNAc-FAm-iRGD conjugate

[0098] The synthesis process of αPD-1-(Fucα1,6)(GalNAzβ1,4)GlcNAc-FAm-iRGD is as follows: PD-1 antibody (3-15 mg / mL) and EndoS (0.01-0.1 mg / mL) are incubated in 50 mM Tris-HCl buffer (pH 5.0-8.0) at 30-37℃ for 0.1-1 hours. Then, UDP-Gal or UDP-GalNAz (final concentration 1-10 mM, Shanghai Baosen Biotechnology Co., Ltd., China), bovine β1,4-GalT1 (Y289L) (final concentration 0.1-0.5 mg / mL) and MnCl2 (final concentration 0.1-5 mM) are added to the reaction mixture and incubated at 30-37℃ for 2-48 hours. The modified antibody was purified with protein A resin to obtain αPD-1-(Fucα1,6)(GalNAzβ1,4)GlcNAc (mass spectrometry characterization showed a molecular weight of 146417.00 Da).

[0099] αPD-1-(Fucα1,6)(GalNAzβ1,4)GlcNAc (3-15 mg / mL) was replaced with 50 mM Tris-HCl buffer (pH 5.0-8.0) by ultrafiltration and incubated with GDP-FAm-iRGD (final concentration 0.1-5 mM), Hp1,3-FucT (final concentration 0.1-1 mg / mL), and MgCl2 (final concentration 0-10 mM) at 30-37 °C for 2-48 hours. The product αPD-1-(Fucα1,6)(GalNAzβ1,4)GlcNAc-FAm-iRGD was obtained (mass spectrometry characterization showed a molecular weight of 149285.00 Da, and the drug-to-antibody ratio was between 1.8 and 2.0).

[0100] 3.3 Synthesis of αPD-1-G2F-FAm-iRGD conjugate

[0101] In the protein expression and preparation experiments, the synthesized human and mouse universal PD-1 antibody (3-15 mg / mL) was incubated with UDP-Gal (final concentration 1-10 mM, Shanghai Baosen Biotechnology Co., Ltd., China), bovine β1,4-GalT1 (Y289L) (final concentration 0.1-1.0 mg / mL), GDP-FAm-iRGD (final concentration 0.1-5 mM), Hp1,3-FucT (final concentration 0.1-1 mg / mL), MgCl2 (final concentration 0-10 mM), MnCl2 (final concentration 0.1-5 mM), and 50 mM Tris-HCl buffer (pH 5.0-8.0) at 30-37℃ for 24-72 hours. The modified antibody was purified using protein A resin to obtain the PD-1 antibody iRGD conjugate, with the formula αPD-1-G2F-FAm-iRGD (mass spectrometry characterization showed a molecular weight of 154540 Da, and a drug-to-antibody ratio between 3.5 and 4.0). Figure 1 As shown in (C).

[0102] One-pot synthesis of 3.4αPD-1-(GalXβ1,4)GlcNAc-FAm-iRGD conjugate

[0103] The synthesis process of αPD-1-(GalXβ1,4)GlcNAc-FAm-iRGD is as follows: Figure 1 (A): PD-1 antibody (3-15 mg / mL) was incubated with EndoS (0.01-0.1 mg / mL) (SEQ.ID.NO: 17) and Alfc (SEQ.ID.NO: 19) (0.5-2 mg / mL) in 50 mM Tris-HCl buffer (pH 5.0-8.0) at 30-37 °C. After 24 hours, UDP-GalNH2 (final concentration 1-10 mM, Shanghai Baosen Biotechnology Co., Ltd., China), bovine β1,4-GalT1 (Y289L) (SEQ.ID.NO: 15) (final concentration 0.1-0.5 mg / mL), GDP-FAm-iRGD (final concentration 0.1-5 mM), Hp1,3-FucT (SEQ.ID.NO: 14) (final concentration 0.1-1 mg / mL), MgCl2 (final concentration 0-10 mM), and MnCl2 (final concentration 0.1-5 mM) were added to the reaction mixture and incubated at 30-37°C for 2-48 hours. The modified antibody was purified using protein A resin to obtain the PD-1 antibody iRGD conjugate, with the formula αPD-1-(GalNH2β1,4)GlcNAc-FAm-iRGD (mass spectrometry characterization showed a molecular weight of 148831.00 Da and a drug-to-antibody ratio between 1.8 and 2.0).

[0104] IV. Characterization of the binding ability of PD-1 antibody iRGD conjugate

[0105] 4.1 Construction of Cy5 fluorescently labeled PD-1 antibody and PD-1 antibody-iRGD conjugate

[0106] The synthesis methods for αPD-1-(Fucα1,6)(GalNAzβ1,4BCN-Cy5)GlcNAc and αPD-1-(Fucα1,6)(GalNAzβ1,4BCN-Cy5)GlcNAc-FAm-iRGD are as follows: αPD-1-(Fucα1,6)(GalNAzβ1,4)GlcNAc (1-10 mg / mL), αPD-1-(Fucα1,6)(GalNAzβ1,4)GlcNAc-FAm-iRGD (1-10 mg / mL), and BCN-Cy5 (3-10 antibody equivalents) (Xi'an Ruixi Biotechnology Co., Ltd., China) were incubated in PBS (pH 7.0-7.5) buffer containing 10% DMF for 2-10 hours. The products were purified by desalting column chromatography. We obtain αPD-1-(Fucα1,6)(GalNAzβ1,4BCN-Cy5)GlcNAc (abbreviated as αPD-1-Cy5) and αPD-1-(Fucα1,6)(GalNAzβ1,4BCN-Cy5)GlcNAc-FAm-iRGD (abbreviated as αPD-1-(iRGD)2-Cy5).

[0107] 4.2 Characterization of the binding ability of the PD-1 antibody iRGD conjugate to the PD-1 protein

[0108] Recombinant human or mouse PD-1 extracellular domain (PD-1, nascent protein) was diluted to a final concentration of 250 ng / mL, coated with buffer, and plated in 96-well plates (100 μL / well) overnight at 4°C. After removing excess antigen solution, the plates were blocked with 3% (v / v) bovine serum albumin in PBS at 37°C for 2 hours. After washing three times with PBST (PBS containing 0.03% Tween-20), αPD-1 and αPD-1-(iRGD)2 were added to PBST (PBS containing 1% (v / v) bovine serum albumin) to achieve a series of final concentrations (3000 ng / mL, 1000 ng / mL, 333.33 ng / mL, 111.11 ng / mL, 37.04 ng / mL, 12.35 ng / mL, 4.12 ng / mL, 1.37 ng / mL, 0.46 ng / mL, 0.15 ng / mL, 0.05 ng / mL, 0 ng / mL), and then added to plates. After incubation for 1.5 hours, the plates were washed three times with PBST, and then horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody was added to each well, and the plates were incubated at 37°C for 1 hour. Finally, each well was washed three times with PBST and then co-treated with tetramethylbenzidine substrate to produce color for visualization. After incubation for 15 minutes, the reaction in each well was stopped by adding 100 μL of 3M HCl.

[0109] The absorbance was read at 450 nm on the Synergy™ LX flat panel reader, and nonlinear fitting of the absorbance was performed using Graphpad 7.0, such as... Figure 1 (C) and Figure 1 (D) shows the calculated KiRGD conjugate of PD-1 antibody with human PD-1. D It was 2.855 ng / ml, compared to the K of mouse PD-1. D The concentration was 3.944 ng / ml; the unmodified PD-1 antibody and human PD-1 K... D It was 2.599 ng / ml, compared to the K of mouse PD-1. D The concentration was 2.766 ng / ml, indicating that the PD-1 antibody before and after modification has considerable affinity for human and mouse PD-1.

[0110] 4.3 Characterization of the binding ability of PD-1 antibody iRGD conjugate to tumor cells

[0111] First, 2 μg of flow cytometry antibody against iRGD receptor NRP-1 and integrin avβ5 was used with 10 6GES-1, HGC27, and N87 human cells were co-incubated at 4°C for 30 minutes, washed twice with PBS, and then analyzed by flow cytometry using a BD Accuri C6 PLUS flow cytometer (BD Biosciences). Data processing was performed using FlowJo software (10.4, Tree Star). The results are as follows: Figure 2 As shown in (C), all the human cell lines tested expressed the iRGD receptor. 2 μg of fluorescently labeled PD-1 antibody-iRGD conjugate (αPD-1-(iRGD)2-Cy5) or fluorescently labeled PD-1 antibody (αPD-1-Cy5) was mixed with 10... 6 GES-1, HGC27, N87, and MFC cells were incubated at 4°C for 30 minutes, followed by two washes with PBS. Flow cytometry analysis was performed using a BD Accuri C6 PLUS flow cytometer (BD Biosciences), and data processing was performed using FlowJo software (10.4, Tree Star). Figure 2 As shown in (B), the affinity of the PD-1 antibody-iRGD conjugate to the corresponding cell lines was significantly higher than that of the unmodified PD-1 antibody.

[0112] 4.4 Characterization of the binding ability of PD-1 antibody iRGD conjugate to T cells

[0113] αPD-1-(iRGD)2-Cy5 (2 μg) or αPD-1-Cy5 (2 μg) with 10 6 Jurkat cells were co-incubated at 4°C for 30 minutes, washed twice with PBS, and then analyzed by flow cytometry using a BD Accuri C6 PLUS flow cytometer (BD Biosciences). Data processing was performed using FlowJo software (10.4, Tree Star). Figure 2 As shown in (D), the PD-1 antibody iRGD conjugate has a similar binding ability to the PD-1 antibody and the PD-1 protein on the cell surface.

[0114] V. Experiment on the promotion of T cell-tumor cell binding by PD-1 antibody iRGD conjugate

[0115] To verify that the PD-1 antibody iRGD conjugate promotes T cell-tumor cell binding, Jurkat cells were first transduced with lentivirus generated from PLV-EF1a-PD-1 to construct Jurkat cells expressing human PD-1 (hPD-1). + Jurkat cells). Then, tumor cells and T cells were labeled with fluorescent cell dyes, respectively. Figure 2As shown in (G) and (J), Jurkat cells or OT-I cells expressing human PD-1 protein were labeled with 2 μM CFSE. The dye Dye eFluor was used. TM HGC27 cells were labeled with 670 (Thermo Fisher Scientific). After washing three times with FACS buffer (DPBS containing 2% FBS), hPD-1+ Jurkat cells or OT-I cells were co-cultured with HGC27 cells at an E:T ratio of 10:1. αPD-1-(iRGD)2 (1 μg) or αPD-1 (1 μg) was added to the culture medium along with 0.1 μg of free iRGD (total volume 100 μL). After incubation at 37°C for 1 hour, flow cytometry analysis was performed directly using a BD DAC C6 PLUS flow cytometer (BD Biosciences). Data analysis was performed using FlowJo software (10.4, Tree Star). Figure 2 As shown in (H)-(L), αPD-1-(iRGD)2 significantly improved the binding ratio between T cells and tumor cells.

[0116] VI. Experiment on the effect of PD-1 antibody iRGD conjugate on promoting T cell killing of tumor cells

[0117] 6.1 Acquisition and Processing of PBMC

[0118] Peripheral blood mononuclear cells (PBMCs) from healthy donors were collected by Ficoll density centrifugation and added to AIM-V medium (Gibco, USA) containing 10% FBS. After culturing for 2 hours in a cell incubator at 37°C and 5% CO2, suspension T cells that were still not attached to the bottom of the culture dish were collected and cultured in AIM-V medium (Gibco, USA) containing 10% FBS, 100 IU interleukin-2 (IL2) (Peprotech, USA), 10 ng / ml IL7 (Peprotech, USA), and 10 ng / ml IL15 (PeproTech, USA).

[0119] 6.2 Construction of multicellular spheroids (MCS) of HGC27 cells

[0120] After trypsin digestion of HGC27 cells, they were resuspended in 1640 complete medium containing 10% FBS and 1% penicillin-dextrose antibody to a concentration of 1500 cells / ml. The medium was then added at 100 μl / well to each of 96-well ultra-low adsorption plates, and the medium was changed every other day. When the cell spheroids reached a diameter of approximately 500 μm, the MCS (metabolites and sclerosing cells) were collected for later use.

[0121] 6.3 Analysis of the killing effect of T cells on tumor cells

[0122] 1×105 T cells were co-cultured with HGC27 cells labeled with αPD-1-(iRGD)2 (1 μg), αPD-1 (1 μg), or αPD-1 (1 μg) + free iRGD (0.01 μg) at a 20:1 E:T ratio for 24 hours. After incubation, 100 ng / ml propidium iodide (PI, MedChemExpress) was added to the culture medium. Tumor cytotoxicity was measured by flow cytometry. CFSE and PI double-positive cells were considered lysed tumor cells. One-way ANOVA was used for statistical testing, and the results are as follows: Figure 2 As shown in (E) and (F), the antibody conjugate significantly demonstrated the killing ability of T cells against tumor cells under both planar culture and MCS conditions.

[0123] VII. Experiment on the enhanced penetration ability of PD-1 antibody iRGD conjugate

[0124] 7.1 Analysis of the direct penetration ability of PD-1 antibody iRGD conjugate to MCS

[0125] like Figure 3 As shown in (A), MCS was co-cultured with αPD-1-(iRGD)2-Cy5 (1 μg), αPD-1-Cy5 (1 μg), αPD-1-Cy5 (1 μg) + low-dose free iRGD (0.01 μg), or αPD-1 (1 μg) + high-dose free iRGD (0.1 μg) at 37°C for 24 hours at an effector-to-target ratio of 5:1. Then, the MCS was washed with PBS and fixed with 4% paraformaldehyde. Imaging was then performed using a ZEN710 confocal microscope (Zeiss, Jena, Germany) to obtain fluorescence images near the center height of the sphere. Fluorescence intensity was calculated using Leica Application Suite X (LAS X). The results are shown below. Figure 3 As shown in (C) and (E), the PD-1 antibody iRGD conjugate can penetrate the core of the MCS more effectively than the unmodified PD-1 antibody.

[0126] 7.2 PD-1 antibody iRGD conjugate enhances T cell penetration into MCS.

[0127] like Figure 3 As shown in (B), hPD-1 was labeled with carboxyfluorescein succinimide ester (CFSE, MedChemExpress). +Jurkat cells (Abcam, Cambridge, UK). After labeling, hPD-1+ Jurkat cells, MCS, and αPD-1-(iRGD)2 (1 μg), αPD-1 (1 μg), or αPD-1 (1 μg) + free iRGD (0.1 μg) were co-cultured at 37°C at a 5:1 effector-to-target ratio for 24 hours. Then, the MCS were washed with PBS and fixed with 4% paraformaldehyde, followed by imaging using a ZEN710 confocal microscope (Zeiss, Jena, Germany) to obtain fluorescence images near the center height of the spheres. Fluorescence intensity was calculated using Leica Application Suite X (LAS X). Results are shown below. Figure 3 As shown in (D) and (F), the PD-1 antibody iRGD conjugate is more effective than the unmodified PD-1 antibody in promoting T cell penetration of the MCS.

[0128] 7.3 In vivo penetration analysis of PD-1 antibody iRGD conjugate

[0129] Near-infrared in vivo imaging was used to track the distribution of αPD-1-(iRGD)2-Cy5 or αPD-1-Cy5. αPD-1-(iRGD)2-Cy5 (50 μg), αPD-1-Cy5 (50 μg), or αPD-1-Cy5 (50 μg) + free iRGD (1 μg) were injected intraperitoneally into tumors with an average volume of 200 mm². 3 In MFC tumor-bearing mice, after sevoflurane gas anesthesia, the mice were scanned using a CRi Maestro in vivo imaging system (Research Instruments, Cambridge, Massachusetts, USA) at 30 min, 1 h, 3 h, 6 h, 24 h, and 48 h post-injection. The results are as follows. Figure 4 As shown in (A) and (C), the PD-1 antibody iRGD conjugate can be distributed more rapidly and concentratedly in the tumor site. Forty-eight hours after administration, mice were euthanized humanely, and tumor tissue and major organs were removed. The in vitro mean radiation efficiency of the tumor and major organ tissues was analyzed using the CRi Maestro in vivo imaging system (Research and Instruments, Cambridge, Massachusetts, USA). Figure 4 As shown in (B) and (D), the PD-1 antibody iRGD conjugate is mainly concentrated in tumor tissue, with a lower distribution in organs.

[0130] VIII. Experiments on the inhibition of tumor growth in mouse models of gastric cancer and melanoma by PD-1 antibody iRGD conjugate

[0131] 8.1 Construction of a mouse gastric cancer tumor model

[0132] 615 mice weighing approximately 20g and aged 6-8 weeks were injected subcutaneously into the left groin with 1×10 6MFC cells were collected, and the major diameter of the subcutaneous tumor was measured with calipers the following day, denoted as a mm and the minor diameter as b mm. The tumor volume was calculated as a × b × b / 2 mm. 3 .

[0133] 8.2 Establishment of a mouse malignant melanoma model

[0134] C57BL / 6 mice, weighing approximately 20g and aged 6-8 weeks, were injected subcutaneously with 1×10⁻⁶ mol / L in the left groin. 5 B16F10 cells were used. The following day, the major diameter of the subcutaneous tumor was measured with calipers and recorded as a mm, and the minor diameter as b mm. The tumor volume was calculated as a × b × b / 2 mm. 3 .

[0135] 8.3 Antitumor drug delivery mode of PD-1 antibody iRGD conjugate

[0136] Treatment process such as Figure 5 As shown in (A) and (C), on the third day after the establishment of the mouse tumor model, mice were randomly divided into 5 groups of 6 mice each. On alternate days, each group received an intraperitoneal injection of 100 μL of physiological saline; 100 μg of PD-1 monoclonal antibody; 1.25 μg of free iRGD; 100 μg of PD-1 monoclonal antibody + 1.25 μg of iRGD; or 100 μg of αPD-1-(iRGD)2. After four administrations, the mice were euthanized humanitarianally, and tumor tissue and major organs (heart, liver, spleen, lung, and kidney) were dissected for relevant analysis.

[0137] 8.4 Analysis of the antitumor efficacy of PD-1 antibody iRGD conjugate

[0138] like Figure 5 As shown in (B) and (D), in the MFC and B16F10 subcutaneous tumor models, αPD-1-(iRGD)2 significantly delayed tumor growth and significantly reduced tumor weight. Unmodified PD-1 antibody also inhibited tumor growth to some extent, while the antitumor effect of PD-1 antibody combined with free iRGD was not significantly different from that of the PD-1 group. αPD-1-(iRGD)2 was more effective at inhibiting tumor growth than unmodified PD-1 monoclonal antibody.

[0139] 8.5 PD-1 antibody iRGD conjugate equivalent free iRGD dose analysis

[0140] like Figure 6As shown in (A), on the third day after the establishment of the mouse tumor model, mice were randomly divided into 5 groups of 6 mice each. Each group received an intraperitoneal injection every other day of the following: 100 μl of saline; 100 μg of PD-1 monoclonal antibody; 50 μg of free iRGD; 100 μg of PD-1 monoclonal antibody + 50 μg of iRGD; or 100 μg of αPD-1-(iRGD)2. After four administrations, the mice were euthanized, and tumor tissue was dissected and weighed for analysis. Figure 6 As shown in (BD), the equivalent free iRGD dose of the PD-1 antibody iRGD conjugate is approximately 50 μg, which is 40 times the actual amount of iRGD conjugated.

[0141] IX. Experiment on the enhancement of anti-tumor immunity by PD-1 antibody iRGD conjugate in a mouse gastric cancer model

[0142] 9.1 Preparation of single-cell suspension from tumor tissue

[0143] In experiments investigating the inhibition of tumor growth in mouse models of gastric cancer and melanoma using PD-1 antibody-iRGD conjugates, single-cell suspensions of tumor tissue from each group of mice were prepared as follows: Tumor tissue was minced and digested with 1 mg / ml collagenase IV (Sigma-Aldrich) in serum-free RMPI-1640 medium at 37°C for 2 h. Cells were filtered through a 40 μm nylon cell filter (Biosharp), washed twice with PBS, and then red blood cells were removed using erythrocyte lysis buffer (Biosharp).

[0144] 9.2 Flow cytometry staining and analysis

[0145] Extracellular proteins were detected by staining single-cell suspensions with specific antibodies against markers such as CD3, CD4, CD8, PD-1, CD51, and NRP-1 at 4°C for 30 minutes. For intracellular proteins, single-cell suspensions were treated with a fixation / permeabilization kit (BD Biosciences) and / or BD GolgiPlug's leukocyte activation combination solution (per 10 cells). 6 Each cell was injected at a volume of 2 μL (BD Biosciences), and then incubated with a flow cytometry antibody targeting IFN-γ and GZMB. Detection was performed using a BD Accuri C6 PLUS flow cytometer (BD Biosciences), and data analysis was conducted using FlowJo software (10.4, Tree Star). Results are as follows: Figure 5 As shown in (FJ), compared with other groups, αPD-1-(iRGD)2 recruited more CD8+ cells into the tumor microenvironment. + T cells, and significantly increased CD8 + and CD4 +The expression of interferon-γ (IFNγ) and granzyme B (GZMB) in T cells was affected, while PD-1 antibody alone or together with free iRGD had only a slight effect on the expression of IFNγ and GZMB.

[0146] 10. Safety analysis of PD-1 antibody iRGD conjugate in mice

[0147] 10.1 Mouse body weight analysis during treatment

[0148] Analysis results as follows Figure 9 (A) shows that the mice did not experience a significant decrease in body weight during the treatment period.

[0149] 10.2 HE staining analysis of sections from major organs (heart, liver, spleen, lung, kidney)

[0150] Freshly removed organs are fixed in fixative for at least 24 hours. The tissue is removed from the fixative and, in a fume hood, the tissue at the target site is trimmed smooth with a scalpel. The trimmed tissue and corresponding labels are placed in a dehydration box. The dehydration box is placed in a basket and dehydrated sequentially with alcohol in a dehydrator: 75% alcohol 4h - 85% alcohol 2h - 90% alcohol 2h - 95% alcohol 1h - anhydrous ethanol I 30min - anhydrous ethanol II 30min - xylene I 5-10min - xylene II 5-10min - 65°C melted paraffin I 1h - 65°C melted paraffin II 1h - 65°C melted paraffin III 1h. The paraffin-soaked tissue is then embedded in an embedding machine. First, the melted paraffin is placed in the embedding frame. Before the paraffin solidifies, the tissue is removed from the dehydration box, placed in the embedding frame according to the embedding surface requirements, and labeled accordingly. Cool at -20°C on a freezing stage. After the wax solidifies, remove the wax block from the embedding frame and trim it. Place the trimmed wax block on a paraffin microtome and section it to a thickness of 4μm. Float the sections on a 40°C warm water spreader to flatten the tissue. Lift the tissue onto a glass slide and bake it in a 60°C oven. After the wax melts from the water, remove it and store it at room temperature for later use. Sequentially immerse each organ section in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then wash with tap water. Stain the sections with hematoxylin for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, re-blue with blue solution, and rinse with running water. Dehydrate the sections sequentially with 85% and 95% graded ethanol solutions for 5 min each, then stain with eosin solution for 5 min. The sections were sequentially immersed in anhydrous ethanol I for 5 min, then anhydrous ethanol II for 5 min, then anhydrous ethanol III for 5 min, then xylene I for 5 min, and finally xylene II for 5 min, and then mounted with clear neutral resin. The sections of each major organ were observed using an optical microscope, and the results are as follows: Figure 9 (B) No abnormal inflammatory damage was recorded in the major organs.

[0151] XI. CD45 in the tumor microenvironment of mice after treatment with PD-1 antibody iRGD conjugate + Single-cell analysis experiment

[0152] 11.1CD45 + Cell sorting

[0153] The construction of mouse models using single-cell analysis is as described above, and the treatment strategies are as follows: Figure 7 As shown in (A). Single-cell suspensions were obtained in a mouse gastric cancer tumor model as previously described, and lymphocytes were enriched using a 67% Percoll gradient (800g, 20°C for 20 minutes). The cell suspensions were then stained with the Zombie Aquu™ Fixable Viability Kit (#423101, Biolegend) and anti-CD45-FITC (#157607, Biolegends) at 4°C for 30 minutes, and CD45-positive cells were sorted using the FACS AriaII (BD Biosciences) system.

[0154] 11.2CD45 + Cell labeling, single-cell sequencing library construction and sequencing

[0155] Preparation of the ClickTag label: 0.5 μl of NHS-TCO (1 mM in DMSO) and 20 μl of 25 μM Tz oligomer were thoroughly mixed and immediately pipetted into the sample. After mixing for 15 minutes at room temperature in the dark, the mixture was incubated with 10 μl of quenching buffer (300 μM acetylated polyethylene glycol tetrazine in FBS) for 5 minutes. Cells were then washed three times with DPBS to assess cell number and viability. The labeled cells were then mixed and loaded into a Singleton Matrix 15000-cell microarray (GEXSCOPE Single cell RNA seq Kit, Singleton Biotechnologies, Nanjing, China). Single-cell sequencing libraries were prepared according to the manufacturer's instructions (Singleron Biotechnologies, Nanjing, China). After amplification, cDNA and ClickTags were separated using 0.6× and 1.4× SPRI, respectively. The ClickTag library was then quantified (Qubit, Invitrogen) and amplified using primers SGR-beads-1 / SGR-tag-1, followed by indexing using additional PCR with primers SGR-beads-2 / SGR-tag-2. The final ClickTag and transcriptome libraries were analyzed using a BioAnalyzer High Sensitivity DNA Kit (Agilent) and sequenced on an Illumina NovaSeq 6000.

[0156] 11.3 Analysis of Single-Cell Sequencing Results

[0157] like Figure 7 As shown in (BC), single-cell sequencing data were processed, analyzed, and visualized using the Seurat v.4 package in the R (R Core Team, 2013) language environment. The results are shown in the figure. UMAP displayed 27 clusters, including 5 cell types. They are T cells (clusters 4, 7, 10, 11, 12, 15, 18, 23), DCs (clusters 6, 9, 19, 22), macrophages (clusters 0, 6, 8, 14, 16, 21, 24), monocytes (clusters 3, 5, 20), and NK cells (cluster 12, 26). Consistent with previous findings, CD8+ in mice treated with αPD-1-(iRGD)2 showed... + In T cells, the expression of several effector genes (Gzma, Ifng, Ifng1, etc.), inflammatory cytokine receptors (Il2ra, Il7r, Il12rb1, etc.), and co-stimulatory factors (Cd28, Cd27, etc.) is upregulated. In CD4+... + Similar transcriptional profile changes have also been observed in TILs. Figure 7As shown in (E), CD8 after αPD-1-(iRGD)2 administration... + In T cells, stem cell and memory-related genes (Lef1, Tcf7, Bach2, Ikzf2) were significantly upregulated. RNA levels of genes involved in migration and adhesion (Ccr2, Cxcr3, S1pr1, Itgb1, and Ly6c2) were also increased, indicating that αPD-1-(iRGD)2 recruits CD8+. + T cells infiltrate the tumor microenvironment (TME). Besides T cells, NK cells, dendritic cells (DCs), and macrophages also directly or indirectly participate in the tumor-suppressive effect of PD-1 antibodies. For example... Figure 7 As shown in (D) and (F), in mice treated with αPD-1-(iRGD)2, NK cells in the TME expressed higher levels of effector genes (Nkg7, Gzma, Prf1, Klrg1), co-stimulatory factors (Slamf7, Cd244a, Klrk1), migration genes (Itgb2, Itga2, Icam1), immunostimulatory cytokines, and receptors (Ifng, Ifng1, Il2ra, Il2rb). Differentially expressed genes in DCs and macrophages were analyzed using the Kyoto Encyclopedia of Genomes and Genomes (KEGG). The results showed... Figure 7 (GH) Mice treated with αPD-1-(iRGD)2 showed increased IFNγ response, antigen presentation, and DC presentation. Genes in macrophage cellular response pathways to lipopolysaccharide and biostimuli were significantly activated. These observations indicate that αPD-1-(iRGD)2 simultaneously improves the function of NK cells, DCs, and macrophages. In conclusion, αPD-1-(iRGD)2 improves immunosuppressive TME, thereby promoting CD8+. + T-cell cytotoxicity.

[0158] 12. Experiment on the promotion of expansion of specific T cell subsets with stem cell characteristics by PD-1 antibody iRGD conjugate

[0159] like Figure 8 As shown in (A), UMAP analysis of T cells was performed again. UMAP revealed 10 clusters, which were annotated into 6 subtypes based on cellular markers, including the better effector CD8. + T cells (cluster 4), effector CD8 + T cells (cluster 3), intermediate depleted CD8 + T cells (clusters 0, 2, 5), terminally exhausted CD8 + T cells (cluster 1, 9), memory CD8 + T cells (cluster 6), CD4 + T cells (clusters 7 and 8). For example... Figure 8 As shown in (B), CD8 + A unique subset of T cells (better CD8)+ T effector cells were significantly increased in αPD-1-(iRGD)2-treated mice. This subset of T cells highly expressed stem cell and memory-related genes (Tcf7, Il7r, Lef1, Bach2) and effector genes (Gzma, Gzmb, Ifg). Furthermore, as... Figure 8 (D) shows the CD8 + The T cell population also expressed low levels of exhaustion markers such as PD-1, Lag3, Havcr2 (TIM-3), and EntPD-1. αPD-1-(iRGD)2 enhanced the effector function of T cells within the tumor while preventing exhaustion and maintaining CD8 levels. + T cell subsets and their stem cell and memory properties.

[0160] In some embodiments of this application, the fucosyltransferase is α1,3-fucosyltransferase or a functional variant or fragment thereof.

[0161] In some embodiments of this application, the fucosyltransferase is derived from bacteria.

[0162] In some embodiments of this application, the fucosyltransferase is derived from Helicobacter pylori.

[0163] In some embodiments of this application, the fucosyltransferase is derived from Helicobacter pylori 26695.

[0164] In some embodiments of this application, the fucosyltransferase is Helicobacter pylori α1,3-fucosyltransferase with GenBank accession number AAD07710.1.

[0165] In some embodiments of this application, the fucosylation enzyme comprises a catalytically active region and at least one heptapeptide repeat fragment, the catalytically active region comprising the amino acid sequence shown in SEQ ID NO 11, and the heptapeptide repeat fragment comprising the amino acid sequence shown in SEQ ID NO 12.

[0166] In some embodiments of this application, the fucosyltransferase is α1,3-fucosyltransferase or a functional variant or fragment thereof, and it contains the amino acid sequence shown in SEQ ID NO 13.

[0167] In some embodiments of this application, the fucosyltransferase comprises the fucosyltransferase described in this application and a tag sequence.

[0168] In some embodiments of this application, the fucosyltransferase comprises the amino acid sequence shown in any one of SEQ ID NO 13 and 14.

[0169] In some embodiments of this application, β-1,4-galactosyltransferase and its functional variants comprise bovine β-1,4-galactosyltransferase, human β-1,4-galactosyltransferase or its functional variants or fragments.

[0170] In some embodiments of this application, β-1,4-galactosyltransferase comprises human β(1,4)-GalT1 with the Y285L mutation or bovine β(1,4)-GalT1 with the Y289L mutation.

[0171] In some embodiments of this application, β-1,4-galactosyltransferase comprises the amino acid sequence shown in any one of SEQ ID NO 15 and SEQ ID NO 16.

[0172] In some embodiments of this application, the glycoside endonuclease comprises Endo S, Endo S2, Endo A, Endo F, Endo M, Endo D, Endo H and / or their functional variants.

[0173] In some embodiments of this application, the glycoside endonuclease comprises Endo S.

[0174] In some embodiments of this application, the glycoside endonuclease comprises the amino acid sequence shown in any one of SEQ ID NO 17 and SEQ ID NO 18.

[0175] In some embodiments of this application, α1,6-fucosidase may be BfFucH, Alfc, BKF and / or their functional variants.

[0176] In some embodiments of this application, α1,6-fucosidase comprises Alfc.

[0177] In some embodiments of this application, α1,6-fucosidase comprises the amino acid sequence shown in any one of SEQ ID NO 19 and SEQ ID NO 20.

[0178] The method of this application can be carried out in a suitable buffer solution. Suitable buffer solutions are known in the art. For example, the buffer solution may be Tris-HCl buffer, citrate buffer, HEPES buffer, glycine buffer, phosphate buffer, or acetate buffer, etc. The method of this application can be carried out at a suitable pH. Suitable pH is known in the art. For example, the suitable pH range is 4-10. In some embodiments, the suitable pH range is 5-9. In some embodiments, the suitable pH range is 6-8. In some embodiments, the suitable pH range is 7-8. The method of this application can be carried out at a suitable temperature. Suitable temperatures are known in the art. For example, a suitable temperature is 0-50°C. In some embodiments, a suitable temperature is 10-45°C. In some embodiments, a suitable temperature is 20-40°C. In some embodiments, a suitable temperature is 25-37°C. For example, the method can be carried out at a temperature of about 30°C. For example, the method can be carried out at a temperature of about 37°C. The method of this application can be carried out in the presence of a suitable metal ion. Suitable metal ions are known in the art. For example, a suitable metal ion may be Mn. 2+ Mg 2+ wait.

[0179] In this application, the term "antibody" generally refers to a protein or antigen-binding fragment thereof produced by the immune system that recognizes and binds to a specific antigen. The term "antibody" is used in its broadest sense in this application and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., multispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. Antibodies also include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. In this application, the term "antibody" generally includes complete antibodies, but also includes antibody fragments, such as antibody Fab fragments, (Fab')2, Fv fragments, or Fc fragments from cleaved antibodies, scFv-Fc fragments, minibody, single-domain antibody (also known as nanobody), multispecific antibody (diabody), affibody, or scFv. Furthermore, the term "antibody" also includes engineered or genetically modified antibodies and / or antibody derivatives. In some embodiments, antibodies are referred to as immunoglobulins and include various classes and isotypes, such as IgA (IgA1 and IgA2), IgD, IgE, IgM, and IgG (IgG1, IgG3, and IgG4), etc. In this application, the term "antibody" can include polyclonal antibodies and monoclonal antibodies and their functional fragments. Antibodies include modified or derived antibody variants that retain the ability to specifically bind epitopes. Antibodies are capable of selectively binding target antigens or epitopes. In this application, antibodies can be derived from any source, such as mice or humans, including chimeric antibodies thereof; for example, antibodies can be humanized.

[0180] In this application, the term "Fc region" generally refers to the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally contains two constant domains, namely a CH2 domain and a CH3 domain, and optionally includes a CH4 domain and / or a hinge region. In this application, the term "Fc region" includes any polypeptide (or nucleic acid encoding such a polypeptide), regardless of its production method. As used herein, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous antibody population, meaning that the individual antibodies comprising the population are identical except for possible minor natural mutations and / or post-translational modifications (e.g., isomerization, amidation).

[0181] In this application, the term "IgG" generally refers to various classes of polypeptides or proteins that can be distinguished by biochemical methods. Those skilled in the art will understand that immunoglobulin heavy chains are classified as γ, μ, α, δ, or ε (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4 or α1-α2). It is the nature of this chain that determines the "isotype" of an antibody as IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (subtypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc., have been well characterized and are known to confer functional specialization. A typical characteristic of human IgG is the glycosylation at position Asn297 (according to Kabat numbering) in the CH2 region of the heavy chain in the Fc region.

[0182] In this application, the term "HCDR" refers to the antibody heavy chain complementarity-determining region, and "LCDR" refers to the antibody heavy chain complementarity-determining region. The complementarity-determining region can form a precise complementarity with the antigenic determinant in terms of spatial structure.

[0183] In this application, the term "VH" refers to the variable region of the antibody heavy chain, and "VL" refers to the variable region of the antibody light chain. The regions of the antibody light and heavy chains near the N-terminus where the amino acid sequence changes significantly are called variable regions.

[0184] In this application, the term "HC" refers to the antibody heavy chain and "LC" refers to the antibody light chain.

[0185] In this application, the term "sugar" generally refers to a monosaccharide, such as glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc). The term "sugar derivative" generally refers to a derivative of a monosaccharide, i.e., a monosaccharide containing substituents and / or functional groups. Examples of sugar derivatives include amino sugars and sugar acids, such as glucosamine (GlcNH2), galactosamine (GalNH2), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) (also known as N-acetylneuraminic acid (NeuNAc)), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). Examples of sugar derivatives also include compounds represented as GalX in this application, which can be galactose or galactose derivatives. Examples of sugar derivatives also include compounds represented as Fuc* in this application, which can be fucose derivatives.

[0186] In this application, the term "direct link" generally refers to a linking site that does not contain any other compound (e.g., other glycosyl groups) or linker. For example, a direct link between one molecule or entity and another can mean that there are no other molecules or entities between them. For example, a direct link can mean that one part is linked to another without any intermediate part or linker. For example, a direct link between GlcNAc and an amino acid residue of a protein (such as an antibody) generally means that GlcNAc is linked to the amino acid residue of the protein via a covalent bond, for example, via an N-glycosidic bond to an amide nitrogen bond connecting an atom on the side chain of the protein's amino acid (such as an asparagine amino acid). In this application, when GlcNAc is "indirectly linked" to an amino acid of a protein, there is generally at least one monosaccharide moiety between GlcNAc and the amino acid of the protein.

[0187] In this application, the term "nucleotide" generally refers to a molecule consisting of a nucleobase, a pentose sugar (ribose or 2-deoxyribose), and one, two, or three phosphate groups. Without the phosphate group, the nucleobase and sugar constitute a nucleoside. Therefore, nucleotides are also referred to as nucleoside monophosphates, nucleoside diphosphates, or nucleoside triphosphates. The nucleobase can be adenine, guanine, cytosine, uracil, or thymine. Examples of nucleotides include diphosphate ribonucleotides, such as uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP), and cytidine monophosphate (CMP).

[0188] In this application, "Asn297" or "N297" may be used interchangeably, referring to asparagine at site 297 (numbered according to the Kabat numbering system) of the antibody Fc fragment. Asn297 may be attached with one or more oligosaccharides.

[0189] In this application, the terms “GlcNAc” or “N-acetylglucosamine” are used interchangeably and generally refer to an amide derivative of the monosaccharide glucose.

[0190] In this application, the term "humanized antibody" generally refers to an antibody containing some or all of the CDRs derived from a non-human animal, and the frame and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

[0191] In this application, the terms "α1,6 glycosidic bond," "α1,3 glycosidic bond," and "β1,4 glycosidic bond" refer to the linkage between sugars. For example, Fuc can be linked at its C1 position to the C6 position of GlcNAc via an α1,6 glycosidic bond. Similarly, GalX can be linked at its C1 position to the C6 position of GlcNAc via a β1,4 glycosidic bond. Furthermore, Fuc* can be linked at its C1 position to the C3 position of GlcNAc via an α1,3 glycosidic bond.

[0192] In this application, the term "treatment" generally refers to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing the disease and / or the side effects caused by it. As used in this application, "treatment" encompasses any treatment of a disease in mammals (particularly humans) and includes preventing the occurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with it; inhibiting the disease, i.e., preventing its development; and alleviating the disease, i.e., causing its remission.

[0193] In this application, the term "PD-1" generally refers to programmed death receptor 1, an important immunosuppressive molecule belonging to the immunoglobulin superfamily. The PD-1 in this application can be human PD-1, mouse PD-1, or PD-1 from other species. In some embodiments, the PD-1 is human PD-1.

[0194] In this application, the term "iRGD" generally refers to a cyclic polypeptide with the sequence H2N-Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys-COOH from N-terminus to C-terminus, where the first and last Cys (cysteine ​​residues) form a disulfide bond.

[0195] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. An antibody conjugate, characterized in that, The antibody conjugate comprises a PD-1 antibody and an iRGD, wherein the PD-1 antibody specifically binds to one or more epitopes of the human or mouse PD-1 protein, and the iRGD is a cyclic polypeptide with the amino acid sequence c(CRGDKGPDC). The structure of the antibody conjugate is as follows: In the formula: It is a PD-1 antibody; The GlcNAc directly linked to the PD-1 antibody is the core GlcNAc, and the core GlcNAc is directly or indirectly linked to the amino acids of the PD-1 antibody. Fuc is fucose, and Fuc is linked to the core GlcNAc via an α1,6 glycosidic bond. Man refers to mannose; GalX is galactose or its derivatives, and GalX is linked to GlcNAc via a β-1,4 glycosidic bond. Fuc Fucoidan is a fucose derivative containing iRGD. Linked to GlcNAc via α1,3-glycosidic bonds. The Fuc The structure is as follows: 。 2. The antibody conjugate according to claim 1, characterized in that, The PD-1 antibody comprises the following antibodies or their antigen-binding moieties: toripalimab, sintilimab, camrelizumab, tislelizumab, penapralimab, cepalimumab, slulizumab, pembrolizumab, or 2E5.

3. The antibody conjugate according to claim 1, characterized in that, The PD-1 antibody comprises HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1; The HCDR3 has the amino acid sequence shown in SEQ ID NO 1; The HCDR2 has the amino acid sequence shown in SEQ ID NO 2; The HCDR1 has the amino acid sequence shown in SEQ ID NO 3; The LCDR3 has the amino acid sequence shown in SEQ ID NO 4; The LCDR2 has the amino acid sequence shown in SEQ ID NO 5; The LCDR1 is the amino acid sequence shown in SEQ ID NO 6.

4. The antibody conjugate according to claim 1, characterized in that, The PD-1 antibody comprises VL and VH; The VL contains the amino acid sequence shown in SEQ ID NO 7; The VH contains the amino acid sequence shown in SEQ ID NO 8.

5. The antibody conjugate according to claim 1, characterized in that, The heavy chain of the PD-1 antibody contains the amino acid sequence shown in SEQ ID NO 9, and the light chain contains the amino acid sequence shown in SEQ ID NO 10.

6. The antibody conjugate according to claim 1, characterized in that, The PD-1 antibody contains an Fc fragment, and the GlcNAc is directly or indirectly linked to the Asn in the Fc fragment.

7. The antibody conjugate according to claim 1, characterized in that, The GlcNAc is directly or indirectly connected to Asn297 in the Fc segment, and the Fc segment is numbered according to the Kabat numbering system.

8. The antibody conjugate according to claim 1, characterized in that, The GalX is selected from at least one of the following structures: 、 、 、 ； In the above structure, the spiral indicates a connection with GlcNAc.

9. A pharmaceutical composition, characterized in that, It comprises the antibody conjugate as described in any one of claims 1-3, and at least one pharmaceutically acceptable excipient or carrier.

10. A method for preparing the antibody conjugate as described in claim 1, characterized in that, Includes the following steps: Step 1: Incubate 3-15 mg / mL of PD-1 antibody, 0.01-0.1 mg / mL of EndoS and 0.5-2 mg / mL of Alfc in 50 mM Tris-HCl buffer at pH 5.0-8.0 at 30-37°C. Step 2: After 24 hours, add any one of UDP-Gal, UDP-GalNAz or UDP-GalNAc, bovine β1,4-GalT1 and MnCl2 to the reaction mixture from Step 1, and incubate at 30-37°C for 2-48 hours to obtain the antibody. Step 3: Purify the antibody obtained in Step 2 using protein A resin to obtain αPD-1-(GalXβ1,4)GlcNAc. Replace 3-15 mg / mL of αPD-1-(GalXβ1,4)GlcNAc with 50 mM Tris-HCl buffer (pH 5.0-8.0) via ultrafiltration. Incubate with 0.1-5 mM GDP-FAm-iRGD, 0.1-1 mg / mL Hp1,3-FucT, and 0-10 mM MgCl2 at 30-37°C for 2-48 hours to obtain the modified antibody. Step 4: Purify the modified antibody obtained in Step 3 using protein A resin to obtain the antibody conjugate; Among them, EndoS is Streptococcus pyogenes endosidase S; Alfc is Lactobacillus casei α-1,6-fucosidase; bovine β1,4-GalT1 is bovine β-1,4-galactosyltransferase 1; UDP-Gal is uridine diphosphate-galactose; UDP-GalNAz is uridine diphosphate-N-azidoacetylgalactosamine; UDP-GalNAc is uridine diphosphate-N-acetylgalactosamine; Hp1,3-FucT is Helicobacter pylori α1,3-fucosidase; The structural formula for GDP-FAm-iRGD is: 。 11. The use of the antibody-drug conjugate according to any one of claims 1-8 in the preparation of antitumor drugs.

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