5t4 antibody-drug conjugates and uses thereof

Abstract

The present application relates to the technical field of biomedical or biopharmaceutical technology, and more particularly to a 5T4 antibody-drug conjugate and application thereof. The present application provides a novel humanized nanobody sequence and site-specific conjugation of drugs, which breaks through the inherent shortcomings of traditional full-length 5T4 antibody, such as weak tumor penetration, poor stability of conventional random conjugation ADC, low uniformity, high off-target toxicity, and simultaneously realizes the multi-dimensional synchronous improvement of affinity, tumor penetration, in-vivo tumor inhibition and safety. The constructed ADC molecule has a significant tumor inhibition effect in various mouse tumor models.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine or biopharmaceutical technology, and more specifically to a 5T4 antibody-drug conjugate and its applications. Background Technology

[0002] 5T4 (trophoblast glycoprotein, TPBG) is highly specifically overexpressed in various solid tumors, making it an ideal target for ADCs (anti-cancer drug delivery systems). ADCs significantly improve efficacy and reduce systemic toxicity by targeting highly active cytotoxic drugs into tumor cells. However, most current 5T4 ADCs in development use traditional full-length monoclonal antibodies as targeting vectors. Traditional antibodies have large molecular weights and limited penetration into solid tumor tissues, making it difficult to effectively deliver the payload deep into the tumor and limiting the overall efficacy of ADCs.

[0003] Using nanobodies as targeting carriers for ADCs holds promise for overcoming this inherent limitation. Nanobodies (Nb) have a molecular weight of only about 12-15 kDa. Their size advantage allows for faster and deeper penetration into tumor tissue and more uniform distribution compared to traditional antibodies, enabling efficient delivery of conjugated cytotoxic drugs into the tumor cavity within a short time. Furthermore, nanobodies can be easily genetically engineered to create multivalent or multispecific forms and can be site-specifically conjugated with optimized linker-loador systems to generate highly homogeneous ADC molecules. Therefore, constructing next-generation 5T4 ADCs based on nanobodies is an important direction for improving the therapeutic efficacy of solid tumors.

[0004] In antibody-drug conjugate (ADC) technology, currently available 5T4 ADCs generally use cysteine ​​residues as the conjugation site. This disrupts the disulfide bonds that maintain antibody stability, often resulting in high immunogenicity, poor plasma stability, and off-target toxicity, thus affecting the therapeutic window. Therefore, developing novel ADCs with higher homogeneity, better plasma stability, and a wider therapeutic window is an inevitable requirement for technological iteration.

[0005] Therefore, there is an urgent need in this field to develop a novel 5T4 ADC based on nanobodies that can efficiently penetrate tumors, and to combine it with optimized conjugation techniques and appropriate cytotoxic payloads to achieve higher efficacy, lower off-target toxicity, and a better therapeutic window. Simultaneously, developing ADCs carrying payloads with different mechanisms of action on the same target vector can provide a wider range of clinical treatment options to overcome tumor heterogeneity and drug resistance. Summary of the Invention

[0006] Antibody:

[0007] This invention provides an anti-5T4 single-domain antibody, which comprises CDR1, CDR2 and CDR3 as shown below: (1) CDR1 as shown in SEQ ID NO: 4, CDR2 as shown in SEQ ID NO: 5, and CDR3 as shown in SEQ ID NO: 6; or (2) CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9; or (3) CDR1 as shown in SEQ ID NO:10, CDR2 as shown in SEQ ID NO:11 and CDR3 as shown in SEQ ID NO:12.

[0008] In some embodiments, the anti-5T4 single-domain antibody is an alpaca antibody or a humanized antibody.

[0009] In some embodiments, the anti-5T4 single-domain antibody comprises a VHH sequence as shown in any one of SEQ ID NO: 1-3 and SEQ ID NO: 13-15.

[0010] In some embodiments, the anti-5T4 single-domain antibody further includes an Fc fragment.

[0011] In some specific embodiments, the anti-5T4 single-domain antibody sequence is shown in SEQ ID NO: 17~19.

[0012] The present invention also provides a monoclonal antibody comprising the aforementioned anti-5T4 single-domain antibody.

[0013] The present invention also provides a bispecific antibody, characterized in that the bispecific antibody comprises the aforementioned anti-5T4 single-domain antibody.

[0014] The present invention also provides a nucleic acid molecule that encodes the aforementioned anti-5T4 single-domain antibody.

[0015] The present invention also provides a host cell that is transformed with the aforementioned nucleic acid molecules, wherein the host cell is selected from prokaryotic cells and eukaryotic cells.

[0016] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0017] The present invention also provides a kit comprising the aforementioned anti-5T4 single-domain antibody.

[0018] The present invention also provides a pharmaceutical composition comprising the aforementioned anti-5T4 single-domain antibody.

[0019] In some embodiments, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0020] The present invention also provides the use of the aforementioned anti-5T4 single-domain antibody, the aforementioned monoclonal antibody, the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned host cell, and the aforementioned pharmaceutical composition in the preparation of medicaments for the prevention, relief, or treatment of cancer.

[0021] In some implementations, the cancer includes non-small cell lung cancer, breast cancer, stomach cancer, colorectal cancer, pancreatic cancer, or ovarian cancer.

[0022] Antibody-drug conjugates: This invention provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, the antibody-drug conjugate comprising an antibody, a linker fragment, and a loaded drug; The antibody is an anti-5T4 single-domain antibody, which contains CDR1, CDR2, and CDR3 as shown below: (1) CDR1 as shown in SEQ ID NO: 4, CDR2 as shown in SEQ ID NO: 5, and CDR3 as shown in SEQ ID NO: 6; or (2) CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9; or (3) CDR1 as shown in SEQ ID NO:10, CDR2 as shown in SEQ ID NO:11 and CDR3 as shown in SEQ ID NO:12; The loaded drug is coupled to the N198 site of the antibody Fc region via the linker fragment, which includes VC-PAB.

[0023] In some embodiments, the anti-5T4 single-domain antibody is an alpaca antibody or a humanized antibody.

[0024] In some embodiments, the anti-5T4 single-domain antibody comprises a VHH sequence as shown in any one of SEQ ID NO: 1-3 and SEQ ID NO: 13-15.

[0025] In some embodiments, the anti-5T4 single-domain antibody further includes an Fc fragment.

[0026] In some specific embodiments, the anti-5T4 single-domain antibody sequence is shown in SEQ ID NO: 17~19.

[0027] In some embodiments, the linker fragment includes a linker and a sugar structure, the sugar structure being linked to a conserved glycosylation site in the antibody Fc region, and the linker being linked to the drug payload.

[0028] In some embodiments, the connector-drug-load structure after the connector is connected to the drug-loaded substance is as follows: .

[0029] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof has the following structural formula: ; The MMAE structure is as follows: .

[0030] The present invention also provides a pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

[0031] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0032] The present invention also provides the use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof in the preparation of drugs, diagnostic and therapeutic reagents, and kits, characterized in that the drugs, diagnostic and therapeutic reagents, and kits are used to treat tumors, diseases caused by inflammation, diseases caused by viral infections, or immune diseases.

[0033] In some embodiments, the tumor is selected from any one of ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, bile duct cancer, osteosarcoma, cervical cancer, head and neck tumors, germ cell and embryonic cancer, esophageal cancer, malignant glioma, Ewing sarcoma, melanoma, bile duct cancer, prostate cancer, small cell lung cancer, liver cancer, non-small cell lung cancer, lymphoma, and hematologic malignancies.

[0034] The beneficial effects of this invention are: This invention provides a novel humanized nanobody sequence for site-specific drug conjugation, overcoming the inherent shortcomings of traditional full-length 5T4 antibodies, such as weak tumor penetration, poor stability, low uniformity, and high off-target toxicity of conventional random-conjugated ADCs. Simultaneously, it achieves multi-dimensional improvements in affinity, tumor penetration, in vivo tumor suppression, and safety. The constructed ADC molecule exhibits significant tumor-suppressive effects in various mouse tumor models. Attached Figure Description

[0035] Figure 1 This is a flowchart of the experimental process for the discovery of 5T4 single-domain antibody molecules.

[0036] Figure 2 The graph shows the binding curve of the 5T4 single-domain antibody in A431 cells.

[0037] Figure 3 The graph shows the binding curve of the 5T4 fusion antibody in A431 cells.

[0038] Figure 4 The graph shows the binding curve of the 5T4 fusion antibody in BxPC3 cells.

[0039] Figure 5 This image shows the detection of endocytosis of the 5T4 fusion antibody in A431 cells.

[0040] Figure 6 This is a schematic diagram of the preparation reaction of the MMAE-PEG0-DAR2 ADC molecule.

[0041] Figure 7 The figure shows the in vivo tumor suppression results of the MMAE-PEG0-DAR2 ADC molecule in the pancreatic cancer SU.86.86 CDX model.

[0042] Figure 8 The figure shows the in vivo tumor suppression results of the MMAE-PEG0-DAR2 ADC molecule in the SW756 CDX model of cervical cancer.

[0043] Figure 9 The figure shows the in vivo tumor suppression results of the MMAE-PEG0-DAR2 ADC molecule in the PC-3 CDX model of prostate cancer.

[0044] Figure 10 The figure shows the in vivo tumor suppression results of the MMAE-PEG0-DAR2 ADC molecule in the PDXM-223Pa PDX model of pancreatic cancer.

[0045] Figure 11 The figure shows the in vivo tumor suppression results of the MMAE-PEG0-DAR2 ADC molecule in the PDXM-214Li PDX model of liver cancer.

[0046] Figure 12 The figure shows the in vivo tumor suppression results of the MMAE-PEG0-DAR2 ADC molecule in the non-small cell lung cancer PDXM-563LuPDX model. Detailed Implementation

[0047] definition: To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0049] "VHH" is also known as heavy chain single-domain antibody, VHH, V H The H domain, VHH antibody fragment, VHH antibody, and nanobody are variable domains of antigen-binding immunoglobulins called "heavy chain antibodies" (i.e., "antibodies lacking light chains"). The term "VHH" is used to distinguish these variable domains from the heavy chain variable domains ("VH domain" or VH) and light chain variable domains ("VL domain" or VL) present in conventional tetrapeptide chain antibody structures. The VHH domain specifically binds to epitopes without the need for other antigen-binding domains; this binding behavior differs from that of the VH or VL domains in conventional tetrapeptide chain antibody structures, where the VL domain recognizes epitopes together with the VH domain.

[0050] The terms “complementarity-determining region” and “CDR” refer to the hypervariable region within the variable domain of an antibody that primarily facilitates antigen binding. The amino acid sequence boundaries of the CDR can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc.

[0051] The following specific embodiments further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0052] The initial gene for the anti-5T4 single-domain antibody of this invention was derived from Bactrian camels in Xinjiang and obtained using a well-established camel-derived single-domain antibody technology platform. The entire experimental procedure is as follows: Figure 1 As shown.

[0053] Example 1: Camel Immunization and Library Screening High-purity human 5T4 extracellular protein was used as an immunogen to immunize Bactrian camels in Xinjiang, with a total of 7 immunizations. After immunization, peripheral blood was collected from the camels, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted from these cells and cDNA was prepared by reverse transcription. Using this cDNA as a template, the VHH gene fragment was amplified by nested PCR. The amplified product was cloned into a phage display vector to construct a nanobody phage display library. Using human 5T4 extracellular protein as a solid-phase antigen, the library was subjected to multiple rounds of affinity panning using phage display technology, achieving an antigen-specific phage enrichment of more than 30-fold. Single colonies were randomly selected, and antigen-binding activity was identified using PE-ELISA. Positive clones were sequenced, and single-domain antibodies from different families were expressed using E. coli. Antibodies Nb7-3, Nb7-14, and Nb10-65 were obtained through screening, with sequences shown below: Nb7-3: QVQLQESGGGSVQPGGSLRLSCVVS GYTSCRTEWHL MYWYRQPPGKEREFVSF IDFNRRT SYADAVKGRFTVSQDNAKNTLYLQMNSLKPEDTAIYYC KIEGSSRCTT WGQGTQVTVSS SEQ ID NO: 1 Nb7-14: QVQLQESGGGSVQPGGSLRLSCTVS GYTYCSFFV MNWYRQAPGKEREFVSF IDNNGRT SYADSVKGRFTISQDNAKNTMYLQMNSLKPEDTALYYC KREDCSGRSWGQGTQVTVSS SEQ ID NO: 2 Nb10-65: QVQLQESGGGSVQSGGSLRLSCVVS RYTSCGSV TYWYRQAPGLEREFVSF IDSNGRT SYADSVKGRFTISRDNAKNTVYLQMNRLNPEDTAMYYC KRDQWGYSNCRGSY WGQGTQVTVSS SEQ ID NO: 3 The CDR sequences of the above antibodies are shown in Table 1: Table 1. Antibody CDR Sequences (IMGT Numbering Rules)

[0054] Example 2: FACS detection of binding activity of 5T4 single-domain antibody A431 cells were digested with trypsin, counted, and their density was adjusted to 2E6 / mL with PBS buffer containing 2% FBS. 100 μL of cells were then added to each well of a 96-well plate. Antibodies were serially diluted, with 100 μL added to each well and mixed thoroughly. The plates were incubated at 4°C for 1 hour. After washing with PBS, the prepared anti-human Fc-FITC secondary antibody dilution buffer was added, and the plates were incubated at 4°C for 0.5 hours. After washing with PBS, the cells were resuspended in PBS, and fluorescence values ​​were read using flow cytometry. Results are as follows: Figure 2 As shown, antibodies Nb7-3, Nb7-14, and Nb10-65 all exhibited good A431 cell binding activity.

[0055] Example 3: Humanization of Single-Domain Antibodies Single-domain antibodies Nb7-3, Nb7-14, and Nb10-65 were humanized while maintaining their CDR region sequence. The FR region was replaced with a human template, followed by a small number of site-specific reversion mutations. The final humanized sequences are shown below. The CDR sequence of the humanized sequence is identical to that of the camel-derived antibody.

[0056] HuNb7-3: EVQLLESGGGLVQPGGSLRLSCVVS GYTSCRTEWHL MYWYRQPPGKGLEFVSF IDFNRRT SYADAVKGRFTVSQDNAKNTLYLQMNSLKPEDTAIYYC KIEGSSRCTT WGQGTLVTVSS SEQ ID NO:13 HuNb7-14: EVQLLESGGGLVQPGGSLRLSCTVS GYTYCSFFV MNWYRQAPGKGLEFVSF IDNNGRT SYADSVKGRFTISQDNAKNTMYLQMNSLKPEDTALYYC KREDCSGRS WGQGTLVTVSS SEQ ID NO:14 HuNb10-65: EVQLLESGGGLVQPGGSLRLSCVVS RYTSCGSV TYWYRQAPGLGLEFVSF IDSNGRT SYADSVKGRFTISRDNAKNTVYLQMNRLNPEDTAMYYC KRDQWGYSNCRGSY WGQGTLVTVSS SEQ ID NO:15 Example 4: Binding activity of humanized antibodies Three humanized antibodies, VHH sequences HuNb7-3, HuNb7-14, and HuNb10-65, were fused with human Fc protein (Fc sequence shown in SEQ ID NO: 16) for expression. The resulting antibodies were numbered MY6796 (HuNb10-65-Fc, sequence shown in SEQ ID NO: 17), MY6798 (HuNb7-3-Fc, sequence shown in SEQ ID NO: 18), and MY6800 (HuNb7-14-Fc, sequence shown in SEQ ID NO: 19), respectively. Plasmids carrying the fusion antibodies were mixed with PEI and transfected into HEK293 cells. After 5 days of culture, the supernatant was collected and purified using a protein A affinity column to obtain high-purity fusion antibodies. Flow cytometry was used to detect their binding activity with A431 and BxPC3 cells. After trypsin digestion and cell counting, the cell density was adjusted to 2 × 10⁻⁶ cells using PBS containing 2% FBS. 6 Cells were seeded at 100 μL per well in a 96-well plate. The fusion antibody was serially diluted, and 100 μL was added to each well of the cell suspension and mixed well. The cells were incubated at 4°C for 1 hour. After washing with PBS, diluted anti-human Fc-FITC secondary antibody was added, and the cells were incubated at 4°C in the dark for 0.5 hours. Cells were washed with PBS and resuspended, and fluorescence signals were detected by flow cytometry. Results are shown below. Figure 3 and Figure 4 As shown, the three fusion antibodies maintained good cell-binding activity in both A431 and BxPC3x cells.

[0057] Fc sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 16 MY6796(HuNb10-65-Fc) EVQLLESGGGLVQPGGSLRLSCVVSRYTSCGSVTYWYRQAPGLGLEFVSFIDSNGRTSYADSVKGRFTISRDNAKNTVYLQMNRLNPEDTAMYYCKRDQWGYSNCRGSYWGQGTLVTVSSRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17 MY6798(HuNb7-3-Fc) EVQLLESGGGLVQPGGSLRLSCVVSGYTSCRTEWHLMYWYRQPPGKGLEFVSFIDFNRRTSYADAVKGRFTVSQDNAKNTLYLQMNSLKPEDTAIYYCKIEGSSRCTTWGQGTLVTVSSRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 18 MY6800 (HuNb7-14-Fc) EVQLLESGGGLVQPGGSLRLSCTVSGYTYCSFFVMNWYRQAPGKGLEFVSFIDNNGRTSYADSVKGRFTISQDNAKNTMYLQMNSLKPEDTALYYCKREDCSGRSWGQGTLVTVSSRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 19 Example 5: Endocytosis activity of humanized single-domain antibodies A431 cells were prepared by trypsin digestion, centrifugation at 4°C, and resuspending in PBS buffer containing 2% FBS. Cell counts were performed, and the cell density was adjusted to 3E6 / mL. 100 μL of cells were then transferred to each well of a 96-well plate. Fusion antibodies (MY6796, MY6798, and MY6800) were adjusted to a concentration of 40 μg / mL and added to each well of the plate. The plates were mixed and incubated on ice for 0.5 hours. The plates were then washed with pre-chilled PBS, and the prepared anti-huFc FITC secondary antibody dilution buffer was added. The plates were incubated on ice for 0.5 hours, washed again with pre-chilled PBS, and resuspended in pre-warmed DMEM medium containing 2% FBS at 37°C. The plates were then placed in a cell culture incubator, and cells were harvested at 0, 0.25, 0.5, and 1 hour. Cells were then treated with elution buffer to remove antibodies bound to the cell surface. After resuspending the cells in PBS, fluorescence signals were detected by flow cytometry. Results are as follows: Figure 5 As shown, the endocytosis rates of the three fusion antibodies in A431 cells after 1 hour were 54%, 58%, and 67%, respectively, all of which showed good endocytosis activity.

[0058] Example 6: Preparation of MMAE-PEG0-DAR2 ADC molecule The nanobody fusion protein MY6798 was used as the antibody moiety of the MMAE-PEG0-DAR2 ADC molecule, with the variable region being VHH HuNb7-3 and the remaining sequence being the human IgG1 Fc sequence. The conjugation site was N198 (N297 according to Kabat's EU number). The preparation reaction is described below. Figure 6 .

[0059] The specific preparation process of ADC molecules is as follows: In a 15 mL centrifuge tube, add 25 mM His-HCl, pH 5.0 buffer, fusion antibody MY6798 (Final Con. 3 mg / mL), and 10 mM MMAE-LacNAc-ox (prepared according to the method of compound 11 in the article "One-step synthesis of site-specific antibody–drug conjugates by reprogramming IgG glycoengineering with LacNAc-based substrates" by Wei Shi et al., batch number HY-X1719, 30 eq, Final Con. 1.9 mM), mix well, adjust the pH to 6.5 with H3PO4, then add TL-E1 glycoside endonuclease (prepared according to the sequence SEQ ID NO:3 in patent WO2025067404, batch number 20231009, Final Con. 0.24 mg / mL), mix well, and react in a 30°C metal bath for 2 seconds. h, sampling was performed and monitored by LC-MS until the reaction endpoint was reached, yielding MMAE-PEG0-DAR2 ADC.

[0060] MMAE-PEG0-DAR2-ADC was purified using Protein A resin. AOGMA Protein A resin was loaded into an empty affinity chromatography column, and flow-through was performed using preservation buffer (preserved with 20% ethanol according to the instructions). Residual preservation buffer was removed using 1×PBS, pH 7.4 buffer (2 column volumes * 3 times). The pH of the flow-through was checked with pH paper and found to be weakly alkaline. The loading was repeated 12 times. The final flow-through was measured using a Nanodrop instrument to confirm complete antibody adsorption onto the Protein A resin. The column was then washed with 20 mM Citric-Buffer, pH 6.2 (2 column volumes * 15 times). The first 5 washes were collected separately (to prevent premature elution of the ADC due to weak binding to the resin). Elution was performed using 0.1N Glycine, pH 3.0 (1 column volume * 16 times). The antibody-containing eluent was confirmed using Nanodrop or SDS-PAGE. After incubation on ice for 45 min, the column was neutralized to pH 5.0 using 0.2N Tris-HCl, pH 8.5. Add the eluent to the inner tube of an Amicon-4 / 30k ultrafiltration tube. Using a low-temperature centrifuge, concentrate the eluent to 0.5 mL at 4°C, 4500 rpm, and 6.5 min. Gently agitate the liquid in the inner tube with a pipette to prevent protein sedimentation. Repeat this process until all target eluent is added to the inner tube. Add 1.5 mL of 20 mM Citric-Buffer, pH 6.2 solution to the inner tube and ultrafilter to concentrate to 0.1 mL at 4°C, 4500 rpm, and 6.5 min. Repeat this process three times to ensure complete buffer replacement.

[0061] Purification with Protein A yielded 9 mg of MMAE-PEG0-DAR2-ADC (75%). The molecular weight of MMAE-PEG0-DAR2-ADC was determined by LC-MS, confirming its accuracy. The DAR value of the ADCs was determined by HPLC-RP, showing a DAR value of 1.73. The purity of the ADCs was determined by HPLC-SEC, indicating a purity >90%. Free drug residues in the ADCs were determined by HPLC-RP, showing a drug residue rate <5%.

[0062] Example 7: Antitumor effect of ADC molecules in the SU.86.86 pancreatic cancer CDX model SU.86.86 cells were cultured in RPMI 1640 medium containing 10% FBS and maintained in a 37°C, humidified cell culture incubator with 5% CO2. SU.86.86 cells in the logarithmic growth phase were collected and resuspended in RPMI 1640 basal medium containing 50% Matrigel, adjusting the cell concentration to 4... 10 7 / mL. Under aseptic conditions, 0.1 mL of cell suspension was injected subcutaneously into the right side of mice, at an inoculation concentration of 4. 10 6 / 0.1 mL / mouse.

[0063] When the average tumor volume reaches 150 mm 3 At approximately 10:00 AM, animals were randomly divided into groups based on tumor volume, ensuring that the difference in tumor volume between groups was less than 10% of the mean. The grouping date was designated Day 0, and medication was initiated according to the animal's body weight. During the medication period, if any animal's body weight decreased by more than 15% compared to Day 0 (BWL ≥ 15%), medication was discontinued until the animal's body weight recovered (BWL < 15%), at which point medication was resumed. The control group received either the solvent or gemcitabine.

[0064] Animal body weight and tumor volume were measured twice weekly during the experiment. Tumor length and width were measured using a digital caliper, and tumor volume was estimated using the length and width measurements. The calculation formula was: TV = 1 / 2 a b 2 Where a and b are the measured length and width of the tumor, respectively. The experimental results are as follows: Figure 7 As shown, the data indicate that MMAE-PEG0-DAR2-ADC has a significant tumor-suppressing effect in the SU.86.86 CDX model compared with the solvent control group, and is superior to the control molecule gemcitabine.

[0065] Example 8: Antitumor effect of ADC molecules in the SW756 cervical cancer CDX model SW756 cells were cultured in DMEM medium containing 10% FBS and maintained in a 37°C, humidified cell culture incubator with 5% CO2. SW756 cells in the logarithmic growth phase were collected and resuspended in DMEM basal medium containing 50% Matrigel, adjusting the cell concentration to 50%. 10 7 / mL. Under aseptic conditions, 0.1 mL of cell suspension was injected subcutaneously into the right side of mice at an inoculation concentration of 5. 10 6 / 0.1 mL / mouse.

[0066] When the average tumor volume reaches 200 mm 3At approximately 10:00 AM, animals were randomly divided into groups based on tumor volume, ensuring that the difference in tumor volume between groups was less than 10% of the mean. The grouping date was designated Day 0, and medication was initiated according to the animal's body weight. During the medication period, if any animal's body weight decreased by more than 15% compared to Day 0 (BWL ≥ 15%), medication was discontinued until the animal's body weight recovered (BWL < 15%), at which point medication was resumed. The control group received either the solvent or paclitaxel.

[0067] Animal body weight and tumor volume were measured twice weekly during the experiment. Tumor length and width were measured using a digital caliper, and tumor volume was estimated using the length and width measurements. The calculation formula was: TV = 1 / 2 a b 2 Where a and b are the measured length and width of the tumor, respectively. The experimental results are as follows: Figure 8 As shown, the data indicate that MMAE-PEG0-DAR2-ADC has a significant tumor-suppressing effect in the SW756 cervical cancer CDX model compared with the solvent control group, and is superior to the control molecule paclitaxel.

[0068] Example 9: Antitumor effect of ADC molecules in a PC-3 prostate cancer CDX model PC-3 cells were cultured in DMEM medium containing 10% FBS and maintained in a humidified cell culture incubator at 37°C with 5% CO2. Logarithmic growth phase PC-3 cells were collected and resuspended in DMEM basal medium containing 50% Matrigel, adjusting the cell concentration to 50%. 10 7 / mL. Under aseptic conditions, 0.1 mL of cell suspension was injected subcutaneously into the right side of mice at an inoculation concentration of 5. 10 6 / 0.1 mL / mouse.

[0069] With an average tumor volume of 180 mm 3 At approximately 10:00 AM, animals were randomly divided into groups based on tumor volume, ensuring that the difference in tumor volume between groups was less than 10% of the mean. The grouping date was designated Day 0, and medication was initiated according to the animal's body weight. During the medication period, if any animal's body weight decreased by more than 15% compared to Day 0 (BWL ≥ 15%), medication was discontinued until the animal's body weight recovered (BWL < 15%), at which point medication was resumed. The control group received either the solvent or paclitaxel.

[0070] Animal body weight and tumor volume were measured twice weekly during the experiment. Tumor length and width were measured using a digital caliper, and tumor volume was estimated using the length and width measurements. The calculation formula was: TV = 1 / 2 a b 2Where a and b are the measured length and width of the tumor, respectively. The experimental results are as follows: Figure 9 As shown, the data indicate that MMAE-PEG0-DAR2-ADC has a significant tumor-suppressing effect in the PC-3 prostate cancer CDX model compared to the solvent control group, but it is weaker than the paclitaxel control molecule.

[0071] Example 10: Antitumor effect of ADC molecules in PDXM-223Pa pancreatic cancer, PDXM-214Li liver cancer, and PDXM-563 Lu lung cancer PDX models. Select tumor tissue in good growth condition, remove the mouse fascia and internal necrotic tissue, and cut it into 2×2×3mm pieces. 3 Small tissue blocks were stored in RPMI 1640 basal medium for later use. After anesthesia with isoflurane, the tissue blocks were subcutaneously inoculated into the right side of mice under aseptic conditions using an 11G cannula. The inoculation was completed when the average tumor volume reached 150 mm². 3 When the tumor volume was around 10%, the animals were randomly grouped according to their tumor volume, so that the difference in tumor volume between the groups was less than 10% of the mean.

[0072] Day 0 was designated as the grouping day, and medication was initiated based on animal body weight. In the PDXM-223Pa pancreatic cancer, PDXM-214Li liver cancer, and PDXM-563Lu lung cancer models, the control molecules were paclitaxel, lenvatinib, and DS8201, respectively. During the administration period, if any animal's body weight decreased by more than 15% compared to Day 0 (BWL ≥ 15%), medication was discontinued until the animal's body weight recovered (BWL < 15%), at which point administration was resumed.

[0073] Animal body weight and tumor volume were measured twice weekly during the experiment. Tumor length and width were measured using a digital caliper, and tumor volume was estimated using the length and width measurements. The calculation formula was: TV = 1 / 2 a b 2 Where a and b are the measured length and width of the tumor, respectively. The experimental results are as follows: Figure 10-12 As shown, the data indicate that, compared with the solvent control group, MMAE-PEG0-DAR2-ADC has significant tumor-suppressing effects in the PDXM-223a pancreatic cancer model, the PDXM-214Li liver cancer model, and the PDXM-563Lu lung cancer model, and is superior to the control molecules paclitaxel and lenvatinib. However, its tumor-suppressing effect in the lung cancer model is comparable to that of DS8201.

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that, The antibody-drug conjugate comprises an antibody, a linker fragment, and a drug payload; The antibody is an anti-5T4 single-domain antibody, which contains CDR1, CDR2, and CDR3 as shown below: (1) CDR1 as shown in SEQ ID NO: 4, CDR2 as shown in SEQ ID NO: 5, and CDR3 as shown in SEQ ID NO: 6; or (2) CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9; or (3) CDR1 as shown in SEQ ID NO:10, CDR2 as shown in SEQ ID NO:11 and CDR3 as shown in SEQ ID NO:12; The loaded drug is coupled to the N198 site of the antibody Fc region via the linker fragment; The connection segment includes VC-PAB.

2. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The anti-5T4 single-domain antibody is an alpaca antibody or a humanized antibody.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The anti-5T4 single-domain antibody comprises a VHH sequence as shown in any one of SEQ ID NO: 1~3 and SEQ ID NO: 13~15; Preferably, the anti-5T4 single-domain antibody further includes an Fc fragment; More preferably, the anti-5T4 single-domain antibody sequence is shown in SEQ ID NO: 17~19.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The linker fragment includes a linker and a sugar structure, the sugar structure being linked to a conserved glycosylation site in the antibody Fc region, and the linker being linked to the drug payload.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that, The structure of the connector-drug-loaded component after the connector is connected to the drug-loaded component is shown below: 。 6. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that, It has the following structural formula: ； The MMAE structure is as follows: 。 7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody-drug conjugate of any one of claims 1-6 or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

9. The use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-6 in the preparation of pharmaceuticals, diagnostic and therapeutic reagents, and kits, characterized in that, The drugs, diagnostic and therapeutic reagents, and kits mentioned are used to treat tumors, diseases caused by inflammation, diseases caused by viral infections, or immune diseases.

10. The use according to claim 9, characterized in that, The tumor is selected from any one of the following: ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, bile duct cancer, osteosarcoma, cervical cancer, head and neck tumors, germ cell and embryonic cancer, esophageal cancer, malignant glioma, Ewing sarcoma, melanoma, bile duct cancer, prostate cancer, small cell lung cancer, liver cancer, non-small cell lung cancer, lymphoma, and hematologic malignancies.

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