CD54-targeted antibody coupling medicine and application thereof in preparation of medicine for treating nasopharynx cancer

Abstract

The invention relates to a CD54-targeted antibody coupling medicine and application of the CD54-targeted antibody coupling medicine in preparation of a medicine for treating nasopharynx cancer. The CD54 is identified as a nasopharynx cancer ADC target with clinical transformation potential. On the basis, the ADC drug targeting CD54 is designed, prepared and represented, the in-vivo and in-vitro anti-tumor activity and biological safety of the drug are measured, and a potential targeted therapy candidate drug is provided for targeted therapy of nasopharynx cancer. The research explores the possibility of developing a novel ADC (Analog to Digital Converter) in nasopharyngeal carcinoma by using a new CD54 target spot, meanwhile, the CD54-MMAE is proved to have a remarkable killing effect on nasopharyngeal carcinoma in both in-vivo and in-vitro levels, and a promising targeted therapy candidate drug is provided for targeted therapy of nasopharyngeal carcinoma.

Description

Technical Field

[0001] The present disclosure relates to the field of bioengineering technology, and in particular to an antibody-drug conjugate targeting CD54 and its preparation and use in a drug for treating nasopharyngeal carcinoma. Background Art

[0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor that originates in the nasopharyngeal mucosal epithelium and is a common malignancy in my country and Southeast Asia. In 2022, the global incidence of NPC was 120,416, accounting for 0.6% of all malignant tumor diagnoses, and the number of deaths was 73,476. Asia accounted for 83.3% (100,298 cases) of the global NPC incidence and 83.6% (61,442 deaths). The development and progression of NPC is significantly associated with infection with the Epstein-Barr virus (EBV). EBV DNA can integrate into the host cell genome, initiating carcinogenesis. The mechanisms by which EBV induces NPC include genetic integration, expression of oncogenic proteins, modulation of the immune response, epigenetic influences, and regulation of host cell signaling pathways through viral miRNAs. More than 90% of NPC patients have positive plasma EBV-DNA, and the EBV-DNA load significantly negatively affects the immune function, radiotherapy efficacy and prognosis of NPC patients. Due to the hidden location of the disease, more than 70% of NPC patients are diagnosed in the locally advanced stage. Currently, the main treatment for NPC is a combination of radiotherapy and chemotherapy. Although certain progress has been made in the comprehensive treatment of NPC, 20-30% of NPC patients still experience recurrence and metastasis after radiotherapy and chemotherapy, resulting in treatment failure and incurable. Therefore, breaking through the bottleneck of NPC radiotherapy efficacy and improving the cure rate of NPC patients are still the current clinical bottlenecks.

[0003] Antibody-drug conjugates (ADCs) are novel drug delivery systems based on high-molecular-weight antibodies. They consist of a monoclonal antibody that binds to a highly expressed tumor-specific antigen, conjugated to a small molecule cytotoxin via a chemical linker. Once administered, the ADC specifically binds to the target antigen on tumor cells, mediating endocytosis and allowing the ADC drug to enter the target cell. The cytotoxic drug is then released and exerts its cytotoxic effect, killing the tumor cells. This provides targeted therapy and anti-tumor efficacy unattainable by traditional anti-tumor drugs. Previous studies have reported that trastuzumab deruxtecan (T-DXd, a HER2-targeting ADC) has an objective response rate of 60.9% and a disease control rate of 97.3% for the treatment of HER2-positive advanced breast cancer, demonstrating excellent and durable anti-tumor activity in patients with HER2-positive breast cancer. The unique drug structure and targeting specificity of ADCs offer a novel therapeutic option for the precision medicine treatment of advanced solid tumors. However, no ADCs are currently approved for the treatment of nasopharyngeal carcinoma. In previous research, the applicant conducted a systematic and unbiased screening of nasopharyngeal carcinoma membrane protein targets. The results showed that CD54 is significantly overexpressed in nasopharyngeal carcinoma tumor tissue, making it a novel drug target for nasopharyngeal carcinoma. Based on this, the applicant synthesized an antibody-drug conjugate targeting CD54. CD54-MMAE demonstrated excellent anti-tumor efficacy both in vitro and in vivo in nasopharyngeal carcinoma cells. Summary of the Invention

[0004] In one aspect, the present application provides an antibody-drug conjugate targeting CD54, the structure of which is shown below: Ab-(LD)n, in: Ab is anti-CD54 antibody, L is a linker, D is a cytotoxic warhead, n is the number of warheads coupled to the antibody (range 1-40), The anti-CD54 antibody is coupled to the cytotoxic warhead via the linker.

[0005] As an optional embodiment of the present application, optionally, the anti-CD54 antibody is a monoclonal antibody, including a full-length antibody having an immunoglobulin Fc region.

[0006] As an optional embodiment of the present application, optionally, the heavy chain and light chain amino acid sequences of the anti-CD54 antibody are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0007] As an optional embodiment of the present application, optionally, the linker is valine-citrulline.

[0008] As an optional embodiment of the present application, optionally, the cytotoxic warhead is a tubulin inhibitor (monomethyl auristatin peptide E: MMAE).

[0009] On the other hand, the present application provides a method for preparing an antibody-drug conjugate targeting CD54, comprising the following steps: (1) Mixing a disulfide bond reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] with the anti-CD54 antibody to reduce all or part of the disulfide bonds in the cysteine ​​of the anti-CD54 antibody to sulfhydryl groups; (2) The linker-cytotoxic warhead is mixed with the product of step (1), and the anti-CD54 antibody is coupled to the linker to obtain the antibody-drug conjugate: CD54-MMAE.

[0010] As an optional embodiment of the present application, the reaction process of steps (1) and (2) is optionally carried out in a solvent. The solvent can be selected from any one or more of borate buffer and phosphate buffer.

[0011] As an optional embodiment of the present application, optionally, the reaction temperature of steps (1) and (2) is 10-37°C, preferably the reaction temperature is 15-30°C.

[0012] As an optional embodiment of the present application, optionally, in step (1), the amount of the disulfide bond reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] is greater than or equal to the amount of the anti-CD54 antibody calculated on a molar basis.

[0013] As an optional embodiment of the present application, optionally, the molar ratio of the disulfide bond reducing agent to the anti-CD54 antibody is 10-50:1.

[0014] As an optional embodiment of the present application, optionally, the molar ratio of the disulfide bond reducing agent to the anti-CD66c antibody is 15-20:1.

[0015] As an optional embodiment of the present application, optionally, the amount of the linker-cytotoxic warhead is greater than the amount of the product of step (1) calculated on a molar basis.

[0016] As an optional embodiment of the present application, optionally, the molar ratio of the linker-cytotoxic warhead to the product of step (1) is 10-50:1.

[0017] As an optional embodiment of the present application, optionally, the molar ratio of the linker-cytotoxic warhead to the product of step (1) is 15-25:1.

[0018] As an optional embodiment of the present application, optionally, the DAR value of the antibody-drug conjugate: CD54-MMAE is 4.

[0019] On the other hand, the present application also proposes the use of the above-mentioned CD54-targeting antibody-drug conjugate, or the CD54-targeting antibody-drug conjugate prepared by the above-mentioned preparation method, in the treatment of nasopharyngeal carcinoma.

[0020] Technical effects of the present invention: In this study, we identified CD54 as a potential ADC target for nasopharyngeal carcinoma (NPC). Based on this, we designed, prepared, and characterized an ADC targeting CD54, and determined its in vitro and in vivo antitumor activity and biosafety, providing a promising candidate for the targeted treatment of NPC.

[0021] Experimental results revealed that multi-omics data from a large sample of NPC tumors indicated that CD54 is significantly overexpressed in NPC tumor tissue, making it a potential ADC target. Clinical specimen immunohistochemistry (IHC) confirmed that the novel target CD54 is significantly overexpressed in NPC tissue. Among eight NPC cell lines, six showed significant CD54 overexpression, one showed moderate CD54 expression, and one showed low CD54 expression. Normal epithelial cells did not express CD54. Flow cytometry and confocal fluorescence imaging revealed that NPC cells with high CD54 expression significantly internalized the CD54 antibody. The half-maximal inhibitory concentration (IC50) of CD54-MMAE on cells was determined using a CCK-8 assay. The IC50 values ​​for CD54-MMAE were 9.2 nM, 52.6 nM, 62.4 nM, and 7.2 nM in C666, HK1, HNE1, and SUNE1 cells, respectively, demonstrating promising antitumor activity. Furthermore, CD54-MMAE showed effective and durable tumor regression in a subcutaneous nasopharyngeal carcinoma xenograft model without significant toxicity.

[0022] Further features and aspects of the present disclosure will become apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments, features, and aspects of the disclosure and, together with the description, serve to explain the principles of the disclosure.

[0024] Figure 1 CD54 is overexpressed in the membrane of nasopharyngeal carcinoma cells. (A) Venn diagram of differentially expressed genes in five datasets; (B) Venn diagram of differentially expressed genes and membrane protein datasets; (C) Heat map of the expression of selected genes in different datasets; Figure 2Immunohistochemical analysis shows that CD54 is overexpressed in nasopharyngeal carcinoma tissues and is significantly positively correlated with EBV. (A) Immunohistochemical analysis shows that CD54 is highly expressed in nasopharyngeal carcinoma specimens and is significantly correlated with EBV. (B) CD54 expression levels are significantly correlated with TNM stage. (C) CD54 expression levels are significantly correlated with EBV DNA copy number. (D) High CD54 expression is a significant adverse prognostic factor for nasopharyngeal carcinoma. Figure 3 Figure 2 shows the expression of CD54 in multiple NPC cell lines. (A) Flow cytometric analysis of CD54 expression in eight NPC cell lines and 293T normal cells; (B) Representative flow cytometric analysis of NPC cell lines with high CD54 expression. Figure 4 Shown are fluorescence confocal staining images of multiple nasopharyngeal carcinoma cell lines, indicating that CD54 is highly overexpressed on the membrane of nasopharyngeal carcinoma cells but not on normal 293T cells; Figure 5 This shows that CD54 can be significantly internalized by NPC cells. (A) CD54 internalization of different NPC cell lines at 0, 30, 60, 120, and 240 minutes; (B) Flow cytometry histogram of CD54 internalization of each NPC cell line at different time points; (C) CD54 internalization efficiency curve of each NPC cell line; Figure 6 Shown is a diagram of the drug structure of CD54-MMAE; Figure 7 Shown are IC50 curves of CD54-MMAE and the chemotherapy drug gemcitabine in treating different nasopharyngeal carcinoma cell lines; Figure 8 Figure 3 shows the in vivo efficacy of CD54-MMAE in a NPC subcutaneous tumor model. (A) Construction of the SUNE1 subcutaneous tumor model and administration dose and interval; (B) Growth curves of NPC subcutaneous tumors treated with different treatment groups (PBS, CD54 naked antibody, gemcitabine, and CD54-MMAE); (C) Tumor images from different groups; (D) Comparison of tumor tissue weights among different treatment groups; (E) Changes in mouse weights among different groups; (F) Comparison of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, creatinine, and urea nitrogen levels among mice in different groups; (G) HE staining of major normal organs from different treatment groups. DETAILED DESCRIPTION

[0025] Various exemplary embodiments, features, and aspects of the present disclosure will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise indicated.

[0026] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0027] In addition, numerous specific details are provided in the following detailed description to better illustrate the present disclosure. Those skilled in the art will appreciate that the present disclosure can be practiced without certain specific details. In some instances, methods, components, and circuits well known to those skilled in the art are not described in detail in order to highlight the main points of the present disclosure.

[0028] The reagents and experimental equipment of this embodiment can be purchased from the laboratory or on the market.

[0029] Design, preparation and characterization of CD54-MMAE The structural composition of ADC drugs determines their high targeting and tumor cell killing properties. Therefore, based on the identification of the CD54 target, this application selected the appropriate antibody, linker, and warhead, determined the drug / antibody ratio (DAR), designed CD54-MMAE, and prepared and characterized it.

[0030] In the present invention, the anti-CD54 antibody is selected from monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions). The anti-CD54 antibody can be selected from any antibody or antigen-binding fragment thereof that can bind to CD54 in the prior art, such as the CD54 antibody from MabPlex.

[0031] In one embodiment, the amino acid sequences of the heavy chain and light chain of the anti-CD54 antibody are shown as SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0032] The anti-CD54 antibody was coupled to a tubulin inhibitor (monomethyl auristatin E, MMAE) via a linker (valine-citrulline, vc) as a cytotoxic warhead.

[0033] In one embodiment, the antibody-drug conjugate is anti-CD54 Ab-vc-MMAE (abbreviated as CD54-MMAE).

[0034] In one embodiment, the structure of the antibody-drug conjugate is as follows: Ab-(LD)n, wherein Ab is an anti-CD54 antibody, L is a linker, D is a cytotoxic warhead, and n is the number of warheads conjugated to the antibody (ranging from 1 to 40).

[0035] Preparation process: (1) mixing a disulfide bond reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] with an anti-CD54 antibody to reduce all or part of the disulfide bonds in the cysteine ​​of the anti-CD54 antibody to sulfhydryl groups; (2) Mixing the linker-cytotoxic agent with the product of step 1) to couple the anti-CD54 antibody to the linker to obtain the antibody-drug conjugate.

[0036] The reaction processes of steps (1) and (2) are carried out in a solvent. The solvent can be selected from any one or more of borate buffer and phosphate buffer.

[0037] In certain embodiments of the present invention, the reaction temperature of steps (1) and (2) is 10-37° C. Preferably, the reaction temperature is 15-30° C.

[0038] In step (1), the amount of the disulfide bond reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] is generally equal to or in excess of the anti-CD54 antibody on a molar basis. In certain embodiments of the present invention, the molar ratio of the disulfide bond reducing agent to the anti-CD54 antibody is 10-50:1. In a preferred embodiment, the molar ratio of the disulfide bond reducing agent to the anti-CD66c antibody is 15-20:1. The linker and the cytotoxic agent can be a commercially available reagent that has been successfully linked, i.e., linker-cytotoxic agent (mc-vc-PAB-MMAE); the linker and the cytotoxic agent can also be homemade.

[0039] In certain embodiments of the present invention, the amount of linker-cytotoxic agent (toxic warhead) is generally in excess relative to the product of step (1) on a molar basis. In certain embodiments of the present invention, the molar ratio of linker-cytotoxic agent (mc-vc-PAB-MMAE) to the product of step (1) is 10-50:1. In a more preferred embodiment, the molar ratio of linker-cytotoxic agent (mc-vc-PAB-MMAE) to the product of step (1) is 15-25:1. In certain embodiments of the present invention, step (2) further comprises removing unreacted linker and cytotoxic agent.

[0040] In one embodiment, the DAR value of the CD54-MMAE is 4.

[0041] The above specific ratios or condition values ​​can be adjusted by the test personnel according to their needs and are not limited here.

[0042] In order to verify the therapeutic effect of this drug on nasopharyngeal carcinoma, the following experimental studies were conducted: 1 Materials and Methods 1.1 Clinical NPC samples, NPC cell lines, and main reagents Human nasopharyngeal carcinoma cell lines (C666; HK1; HNE1; CNE1; CNE2; 5-8F; 6-10B; and SUNE1) and normal epithelial cells (293T) were obtained from our laboratory cell bank. RPMI-1640 culture medium and fetal bovine serum were purchased from Gibco, USA.

[0043] The main reagents are as follows:

[0044]

[0045]

[0046]

[0047] 1.2 Experimental Methods 1.2.1 Immunohistochemistry (IHC) Immunohistochemistry was used to examine CD54 expression in 76 human nasopharyngeal carcinoma tissues. Histochemistry scores were expressed using the H-Score (H-score = 0 × (percentage of negative cells) + 1 × (percentage of weakly positive cells) + 2 × (percentage of moderately positive cells) + 3 × (percentage of strongly positive cells), ranging from 0 to 300.

[0048] 1.2.2 Cell culture Nasopharyngeal carcinoma cells and normal human epithelial cells were routinely cultured in RMPI-1640 medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin in a 37°C, 5% CO2 incubator. The medium was changed every 2-3 days. Cells reaching a density of approximately 90% and in the logarithmic growth phase were trypsinized for passage and subsequent experiments.

[0049] 1.2.3 Flow cytometry Flow cytometry was used to examine the expression of CD54 on the cell membrane of eight human nasopharyngeal carcinoma cell lines and one normal epithelial cell line, 293T. The procedure was as follows: 1×106 cells were collected and washed twice with PBS. The cells were then blocked with 1% bovine serum albumin (BSA) for 30 minutes. After blocking, the cells were incubated with PE-CD54 antibody for 1 hour at room temperature, with PE-IgG serving as a control. Following incubation, the cells were washed three times with PBS containing 1% BSA and resuspended in PBS. The intensity of CD54 expression in each cell line was assessed by flow cytometry.

[0050] Flow cytometry was used to examine the internalization efficiency of CD54 in different cell lines. The following procedures were performed: 1 × 106 cells were collected, washed twice with PBS, and incubated with mouse anti-human CD54 antibody in an ice bath for 30 minutes. The incubated cells were then rinsed with PBS buffer containing 1% BSA and resuspended in PBS. The primary anti-CD54 antibody was incubated with the cells for different time periods (0 min, 30 min, 60 min, 120 min, and 240 min). The cells were then rinsed three times with PBS. The secondary antibody PE-IgG was added to the endowed cells for 30 minutes, followed by PBS washing and fixation with paraformaldehyde (4%) for flow cytometry. The internalization efficiency was calculated using the established formula (1 - mean cell fluorescence intensity (t = incubation time) / mean cell fluorescence intensity (t = 0 min)) × 100%. An internalization curve was generated based on the internalization efficiency at different time points. 1.2.4 Immunofluorescence confocal microscopy (IF) The subcellular localization of CD54 in four nasopharyngeal carcinoma cell lines and normal epithelial 293T cells was observed under single-photon confocal microscopy. The procedure was as follows: 1×106 cells were seeded into three confocal dishes, each containing 1 mL of culture medium, and incubated overnight at 37°C. The culture medium was removed, and the cells were washed once with PBS. On ice, the cells were blocked with 1 mL of PBS containing 1% BSA for 15 minutes. After blocking, the liquid was aspirated, and the cells were incubated with antibodies at 37°C in three groups: Group 1: Incubate cells with 1 mL of PBS for 1 hour; Group 2: Incubate cells with 2 μL of PE-IgG in 1 mL of PBS for 1 hour; Group 3: Incubate cells with 2 μL of PE-CD54 in 1 mL of PBS for 1 hour. Wash cells once with 1 mL of PBS, then add 1 mL of Hoechst nuclear stain and stain at 37°C for 20–30 minutes. Wash cells one to two times with 1 mL of PBS, and observe under single-photon confocal microscopy.

[0051] Confocal immunofluorescence was used to examine whether the CD54 antibody selectively enters NPC cells and is rapidly transported to lysosomes. The procedure was as follows: 1×106 cells were seeded onto five confocal microplates, each containing 1 ml of culture medium, and incubated at 37°C overnight. The culture medium was removed, and 2 μL of PE-CD54 in 1 ml of PBS was added on ice. After staining on ice for 30 minutes, the cells were washed once with cold PBS. The cells were then added with 1 ml of PBS and allowed to internalize at 37°C for 0, 30, 60, 120, and 240 minutes. At the end of each time period, the cells were washed once or twice with PBS, fixed with 4% paraformaldehyde for 10 minutes, and washed once or twice with PBS. The PBS was aspirated, and 1 ml of Hoechst staining solution was added. The cells were stained at 37°C for 20-30 minutes, the stain was aspirated, and the cells were washed once or twice with PBS. Finally, images were taken, analyzed, and stored using a confocal microscope.

[0052] 1.2.5 In vitro cytotoxicity test (CCK-8 test) The IC50 values ​​of four human nasopharyngeal carcinoma (NPC) cell lines and normal epithelial 293T cells were determined using a CCK-8 assay. The assay was performed as follows: cells were seeded at a density of 3,000–5,000 cells per well in 96-well culture dishes and cultured overnight. After adherence, the ADC and chemotherapeutic agents were added. The cell culture medium was replaced with medium containing either the chemotherapeutic agent (maximum concentration: 117.11 μmol / L) or serially diluted concentrations of CD54-MMAE (maximum concentration: 0.67 μmol / L). 10-fold serial dilutions were performed, totaling eight concentrations, in triplicate wells. After 72 hours of incubation with the cells and drugs, the culture medium was discarded, and the CCK-8 assay reagent was diluted 10-fold in fresh cell culture medium at 100 μL / well. The plates were incubated at 37°C, and absorbance (OD) was read at 450 nm using a microplate reader. Cell viability was determined by comparing the absorbance of drug-treated cells with that of untreated control cells. The assay was repeated three times.

[0053] 1.2.6 Statistical analysis Experimental data were analyzed using GraphPad Prism 9.0 and FlowJo V10. Data are expressed as mean ± SD. Differences between groups were analyzed using the t-test, with P values ​​< 0.05 considered statistically significant. * p < 0.05, ** p < 0.01, and *** p < 0.001. GraphPad Prism 9.0 was used to plot the data.

[0054] 2 Results 2.1 CD54 is significantly overexpressed in nasopharyngeal carcinoma tumor tissues.

[0055] The applicant first screened the nasopharyngeal carcinoma gene expression dataset in the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ) with the following inclusion criteria: (1) the dataset is the nasopharyngeal carcinoma mRNA expression profile; (2) it includes nasopharyngeal carcinoma tissue and normal nasopharyngeal tissue datasets; (3) both nasopharyngeal carcinoma tissue and normal nasopharyngeal tissue are confirmed by histopathology and the number of nasopharyngeal carcinoma tissues is greater than 10. A total of 5 datasets that meet the above conditions were included, including 128 nasopharyngeal carcinoma tissues and 37 normal nasopharyngeal tissues (GSE12452; GSE13597; GSE53819; GSE64634; GSE68799). Based on the above 5 datasets, differentially expressed genes were screened according to p < 0.05 and |logFC| > 1. A total of 38 differentially expressed genes were found in the 5 datasets ( Figure 1 A), then the applicant compared the 38 differentially expressed genes with the cell membrane protein database and found that 14 differentially expressed genes were located in the cell membrane ( Figure 1 B). Among these 14 differentially expressed genes, 4 genes were significantly upregulated in nasopharyngeal tumors (FPR3; CNTNAP2; GJA1; CD54), and 10 genes were downregulated in nasopharyngeal tumors. Subsequently, the applicant searched the human protein atlas database and found that the GJA1 gene was highly expressed in 13 normal organs, with high off-target toxicity, making it unsuitable as an ADC target. Among the remaining three highly expressed genes, CD54 was the most highly expressed in all four data sets, and its expression level was significantly higher than that of CNTNAP2 and FPR3 ( Figure 1 C). Immunohistochemistry results further verified the high expression of CD54 in nasopharyngeal carcinoma. Immunohistochemistry results showed that among 76 nasopharyngeal carcinoma samples, 48 ​​patients had high expression of CD54, 20 patients had moderate expression, and the high expression site was located at the cell membrane ( Figure 2 A). In addition, CD54 expression was significantly positively correlated with PD-L1 and Epstein-Barr virus-encoded RNA (EBER; EBV immunohistochemical marker). CD54 expression increased significantly with the increase of TNM stage ( Figure 2 B); CD54 expression in patients with EBV DNA copy number ≥ 500 was significantly higher than that in patients with EBV DNA copy number < 500 ( Figure 2 C). In addition, patients with high CD54 expression had a significantly poorer prognosis ( Figure 2 D). It can be seen from this that the CD54 target protein is specifically expressed in nasopharyngeal carcinoma tissue and is significantly associated with Epstein-Barr virus, and can be used as a potential target for ADC drugs to treat nasopharyngeal carcinoma.

[0056] 2.2 Flow cytometry detection of CD54 expression in eight nasopharyngeal carcinoma cell lines and normal epithelial cells like Figure 2 As shown in A, 6 out of 8 nasopharyngeal carcinoma cell lines significantly overexpressed CD54 protein, 1 nasopharyngeal carcinoma cell line moderately expressed CD54 protein, and 1 nasopharyngeal carcinoma cell line lowly expressed CD54 protein ( Figure 3 A). Streaming results ( Figure 3 B) shows that CD54 peak (red) and nonspecific IgG peak (blue) are significantly separated in nasopharyngeal carcinoma cell lines, while CD54 is lowly expressed in normal 293T cells. In addition, the applicant visually demonstrated through immunofluorescence imaging that CD54 is overexpressed in multiple nasopharyngeal carcinoma cell lines, and the overexpression location is at the cell membrane, while it is not expressed in 293T normal epithelial cells ( Figure 4This validates the high specificity of the CD54 target and provides feasibility for the successful development of an antibody-drug conjugate targeting CD54.

[0057] 2.3 Quantification of CD54 antibody cellular internalization efficiency by imaging flow cytometry Considering that the internalization of target antibodies by tumor cells is one of the important considerations for the efficacy of ADC drugs, the applicant visually demonstrated through immunofluorescence imaging results at different time points that PE-CD54 antibodies can be effectively internalized by nasopharyngeal cancer cells over time ( Figure 5 A). At the same time, the applicant quantified the internalization efficiency of CD54 antibodies in different nasopharyngeal carcinoma cell lines at different time points by flow cytometry. The results showed that after 4 hours, all nasopharyngeal carcinoma cell lines could significantly internalize CD54 antibodies, with the internalization efficiency of HK1 cells: 38.2%; C666 cells: 59.1%; HNE1 cells: 41.8%; SUNE1 cells: 68.0%. Among them, SUNE1 cells had the highest internalization efficiency of CD54 antibodies ( Figure 5 BC).

[0058] 2.4 Design, Preparation, and Characterization of CD54-Targeted Antibody-Drug Conjugates The applicant synthesized an ADC drug targeting CD54: CD54-MC-Vc-Pab-MMAE (CD54-MMAE). Figure 6 The molecular structures of the drug linker and warhead are shown. The drug-to-antibody ratio (DAR) of CD54-MMAE, as determined by hydrophobic interaction chromatography (HIC), is 4.

[0059] 2.5 CCK-8 assay to evaluate the in vitro inhibitory activity IC50 of CD54-MMAE against different nasopharyngeal carcinoma cell lines The applicant evaluated the killing effect of CD54-MMAE and nasopharyngeal carcinoma guideline-recommended chemotherapy drugs (gemcitabine) on nasopharyngeal carcinoma cell lines through CCK-8 cytotoxicity experiments ( Figure 6 The results showed that CD54-MMAE had a significant killing effect on nasopharyngeal carcinoma tumor cells, which was significantly better than gemcitabine. In addition, CD54-MMAE had no killing effect on normal 293T cells that did not express CD54 ( Figure 6 The IC50 values ​​of CD54-MMAE in C666, HK1, HNE1 and SUNE1 were 9.2nM, 52.6nM, 62.4nM and 7.2nM, respectively.

[0060] 2.6 In vivo studies to evaluate the inhibitory activity of ADC drugs against NPC In the in vivo experiment, the applicant used the highly metastatic nasopharyngeal carcinoma cell line SUNE1 to construct a subcutaneous tumor model ( Figure 7 A). The applicant compared the therapeutic efficacy of CD54-MMAE with gemcitabine and other drugs. The results showed that CD54-MMAE significantly killed subcutaneous tumors of nasopharyngeal carcinoma, and the killing effect was significantly better than gemcitabine (the first-line chemotherapy drug for nasopharyngeal carcinoma recommended by the guidelines) ( Figure 7 BD). There was no significant difference in the body weight change curves of mice among the treatment groups ( Figure 7 E). There was no statistical difference in serum liver and kidney function indicators (ALT, AST, ALKP, TB, BUN, Cre) among the treatment groups ( Figure 7 F). The applicant also performed HE staining on the important normal organs (heart, lung, liver, spleen, and kidney) of each drug-treated group. The HE staining results showed that there was no morphological manifestation of drug toxicity such as apoptosis in each organ ( Figure 7 G). The above results indicate that CD54-MMAE exhibits excellent anti-tumor efficacy in an in vivo NPC tumor model with good tolerability and no significant off-target toxicity.

[0061] 3 Conclusion Antibody-drug conjugates (ADCs) are novel drug delivery systems based on high-molecular-weight antibodies. They consist of monoclonal antibodies that bind to highly expressed tumor-specific antigens, conjugated to small molecule cytotoxins via chemical linkers. As of 2024, no ADCs have been clinically approved for the treatment of nasopharyngeal carcinoma.

[0062] In this study, the applicant identified CD54 as a new ADC target for the treatment of NPC through a large-scale, unbiased screening of potential cell membrane targets in NPC. An ADC drug candidate targeting CD54 was designed, prepared, and characterized. The results of this application demonstrate that CD54-MMAE exhibits significant antitumor efficacy in a NPC model with limited toxicity, confirming its excellent in vitro antitumor activity and biosafety, providing a new direction for the development of a new class of ADC drugs for NPC.

[0063] CD54 is a transmembrane glycoprotein that binds to integrins CD11a / CD18 or CD11b / CD18 and participates in immune and inflammatory responses. In addition, CD54 is also a receptor for rhinovirus, and its soluble form can promote angiogenesis and serve as a biomarker for activation or damage of vascular endothelial cells. CD54 is upregulated in a variety of cancers, such as multiple myeloma and triple-negative breast cancer, and is associated with tumor progression and poor prognosis. This study explored the expression level of CD54 in nasopharyngeal carcinoma tumor tissue for the first time and demonstrated that CD54 is a promising ADC target for the treatment of nasopharyngeal carcinoma. In addition, this application also introduces a comprehensive strategy for identifying ADC targets and efficiently selecting payloads using large-scale data.

[0064] While various embodiments of the present disclosure have been described above, the foregoing description is intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. An antibody-drug conjugate targeting CD54, characterized in that: The structure of the antibody-drug conjugate is shown below: Ab-(LD)n, in: Ab is anti-CD54 antibody, L is a linker, D is a cytotoxic warhead, n is the number of warheads coupled to the antibody (range 1-40), The anti-CD54 antibody is coupled to the cytotoxic warhead via the linker.

2. The CD54-targeting antibody-drug conjugate according to claim 1, characterized in that: The anti-CD54 antibody is a monoclonal antibody, including a full-length antibody having an immunoglobulin Fc region.

3. The CD54-targeting antibody-drug conjugate according to claim 1, characterized in that: The amino acid sequences of the heavy chain and light chain of the anti-CD54 antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

4. The CD54-targeting antibody-drug conjugate according to claim 1, characterized in that: The linker is valine-citrulline.

5. The CD54-targeting antibody-drug conjugate according to claim 1, characterized in that: The cytotoxic warhead is selected from the microtubule protein inhibitor (monomethyl auristatin peptide E: MMAE).

6. The method for preparing an antibody-drug conjugate targeting CD54 according to any one of claims 1 to 5, characterized in that: The steps include: (1) Mixing a disulfide bond reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] with the anti-CD54 antibody to reduce all or part of the disulfide bonds in the cysteine ​​of the anti-CD54 antibody to sulfhydryl groups; (2) The linker-cytotoxic warhead is mixed with the product of step (1), and the anti-CD54 antibody is coupled to the linker to obtain the antibody-drug conjugate: CD54-MMAE.

7. The preparation method according to claim 6, characterized in that The reaction process of steps (1) and (2) is carried out in a solvent. The solvent can be selected from any one or more of borate buffer and phosphate buffer.

8. The preparation method according to claim 6, characterized in that The reaction temperature of steps (1) and (2) is 10-37°C, preferably 15-30°C.

9. The preparation method according to claim 6, characterized in that In step (1), the amount of the disulfide bond reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] is greater than or equal to the amount of the anti-CD54 antibody calculated on a molar basis.

10. The preparation method according to claim 9, characterized in that The molar ratio of the disulfide bond reducing agent to the anti-CD54 antibody is 10-50:

1.

11. The preparation method according to claim 9, characterized in that The molar ratio of the disulfide bond reducing agent to the anti-CD66c antibody is 15-20:

1.

12. The preparation method according to claim 6, characterized in that The amount of linker-cytotoxic warhead is greater than the amount of the product of step (1) calculated on a molar basis.

13. The preparation method according to claim 12, characterized in that The molar ratio of the linker-cytotoxic warhead to the product of step (1) is 10-50:

1.

14. The preparation method according to claim 12, characterized in that The molar ratio of the linker-cytotoxic warhead to the product of step (1) is 15-25:

1.

15. The preparation method according to claim 6, characterized in that The DAR value of the antibody-drug conjugate: CD54-MMAE is 4.

16. Use of the CD54-targeting antibody-drug conjugate according to any one of claims 1 to 5, or the CD54-targeting antibody-drug conjugate prepared by the method according to any one of claims 6 to 15, in the treatment of nasopharyngeal carcinoma.

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