A nanobody conjugated toxin reagent for detecting antibody endocytosis internalization efficiency, and a preparation method and application thereof

Abstract

This invention discloses a nanobody-conjugated toxin reagent for detecting antibody endocytosis and internalization efficiency, its preparation method, and its application, belonging to the field of biopharmaceutical technology. This invention provides an alpaca-derived nanobody, whose amino acid sequence is shown in SEQ ID NO:1, and its CDR region is shown in SEQ ID NO:2-4. This nanobody binds with high specificity to the Fc fragment of human IgG1 / IgG4, and after conjugation with a toxin drug, forms the nanobody-conjugated toxin reagent HK-D4-MMAE. This reagent has a small molecular weight, high affinity, stable complex, a DAR value up to 4, and is non-cytotoxic within the working concentration range. After binding to the antibody to be tested, it has virtually no impact on its internalization process. The method for detecting antibody endocytosis and internalization efficiency using this reagent is simple to operate, yields accurate results, and is comparable to the internalization effect of real ADC drugs. It can be used for screening and evaluating the internalization efficiency of large quantities of antibodies in the early stages of ADC drug development, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a nano-antibody-conjugated toxin reagent for detecting antibody endocytosis efficiency, its preparation method, and its application. Background Technology

[0002] Antibody-drug conjugates (ADCs), as a new generation of targeted anti-tumor drugs, use antibodies as carriers and small molecule cytotoxic drugs as payloads. They are formed through linker conjugation. With the targeted specificity of antibodies, they recognize tumor cell surface antigens, enter tumor cells through clathrin-mediated endocytosis, and release cytotoxic drugs to kill tumor cells. They have the characteristics of high targeting, high specificity, high activity and low systemic toxicity, and have become a research hotspot for targeted anti-tumor therapy.

[0003] The selection of monoclonal antibodies is a core factor determining the efficacy of ADCs. The antigen targets for ADC drugs also require rigorous screening. The match between the antibody and target must meet several requirements: the antigen must be specifically expressed on tumor cells and expressed at low levels or not at all on normal cells; the antibody must have high affinity for the antigen-binding epitope; and after binding to the antigen, the antibody must be effectively internalized / endocytosed by target cells, thereby releasing toxins intracellularly to exert its activity. Therefore, in the early stages of ADC drug development, large-scale, precise evaluation of the internalization efficiency of naked antibodies is crucial. The evaluation method must be simple and rapid to operate, suitable for batch testing, and provide accurate and reliable results, precisely quantifying internalization efficiency and cytotoxicity to improve the success rate of drug development.

[0004] Currently, antibody internalization efficiency is primarily assessed using cell proliferation activity assays, with commonly used tools including Mab-ZAP and DT-3C, which are internalization assays based on indirect conjugation with immunotoxins. These assays form mAb-immunotoxin complexes after incubation with the target antibody. The specific recognition of the target antibody induces internalization / endocytosis, releasing toxins (ZAP or DT) into the cell and causing cytotoxicity. However, Mab-ZAP contains a complete antibody macromolecule and the ribosome-inactivating protein saponin (ZAP), typically bound in a 1:2 ratio of target antibody to Mab-ZAP, forming a large molecule of approximately 360 kDa. This composition can negatively impact the binding and internalization of the target antibody to its receptor target. While the target antibody-DT-3C complex is slightly smaller, its total molecular weight still exceeds 210 kDa. Furthermore, the low affinity of the target antibody for DT-3C and insufficient complex stability can also affect the internalization efficiency of the target antibody. In addition, existing detection reagents usually require high concentrations to obtain detectable signals in practical applications, resulting in large reagent consumption. At the same time, some reagents have certain cytotoxicity or insufficient complex stability, requiring additional steps such as washing after dosing, which further increases the complexity and cost of the detection process. Summary of the Invention

[0005] This invention provides a nano-antibody-conjugated toxin reagent for detecting antibody endocytosis internalization efficiency, its preparation method, and its application. It aims to solve the problem of internalization detection deviation caused by the large molecular size and insufficient affinity of traditional detection reagents, while overcoming the technical defects of existing reagents such as large dosage and high detection cost.

[0006] In a first aspect, the present invention provides a nanobody-conjugated toxin reagent for detecting antibody endocytosis and internalization efficiency, wherein the reagent is formed by conjugating a nanobody and a toxin drug through a linker. The amino acid sequence of the nanobody is shown in SEQ ID NO:1, and the amino acid sequence of its complementarity-determining region (CDR) is shown below: CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4.

[0007] Furthermore, in the complementarity-determining region (CDR), at least one CDR has an amino acid sequence that is the sequence shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, or a sequence that has at least 80% sequence identity with the sequence shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; preferably 85%, 90%, 95%, 98% or 99%.

[0008] Furthermore, the nanobody is selected from at least one of monovalent nanobody, multivalent nanobody, multispecific antibody and fusion nanobody.

[0009] Furthermore, the toxin drug is selected from at least one of microtubule inhibitors, DNA synthesis inhibitors, and cell-killing toxins.

[0010] Furthermore, the tubulin inhibitor is selected from at least one of MMAE, MMAF, DM1, DM4, tubulosyntheticin, cryptomycin, and antimitotic EG5 inhibitor.

[0011] Furthermore, the DNA synthesis inhibitor is selected from at least one of Ducamycin, pyrrolobenzodiazepines, pyrrolobenzodiazepines (PBD), indolechlorobenzodiazepines, enediynes, and topoisomerase inhibitors.

[0012] Furthermore, the cytotoxic toxin is selected from at least one of apoptosis inducers, telostatin and its analogues, amatoxins, nicotinamide phosphoribosyltransferase inhibitors, diphtheria toxin and its derivatives, and carbamycin.

[0013] Furthermore, the molar ratio of the nanobody to the toxin drug is 1:(1-10), preferably 1:4.

[0014] Further, the linker is selected from cleavable linkers or non-cleavable linkers; the cleavable linker is selected from at least one of chemically cleavable linkers and enzyme-cleavable linkers; preferably VC (valine-citrulline (Val-Cit) dipeptide), the VC linker can be pre-assembled with a toxin drug to form a VC-toxin module, and then coupled with a nanobody to form the reagent; The incisional linker is selected from at least one of MCC, SMCC, MC, pyrophosphate diester group, PEG, or negatively charged sulfonate group.

[0015] Secondly, the present invention provides a method for preparing the above-mentioned nano-antibody-conjugated toxin reagent, comprising the following steps: S1. Synthesize the gene encoding the above-mentioned nanobody, construct a recombinant plasmid, transform it into an expression host cell for fermentation expression, and purify to obtain the nanobody; S2. Coupling nanobodies with toxic drugs via a chemical reaction; S3. Purify the conjugated product, identify it by HPLC and mass spectrometry, detect the drug-antibody ratio, and obtain the nanobody-conjugated toxin reagent.

[0016] Thirdly, the present invention provides the application of the above-mentioned nano-antibody-conjugated toxin reagent in the detection of antibody endocytosis and internalization efficiency.

[0017] Furthermore, the application is implemented through the detection method of the following steps: S1. Prepare a solution of nano-antibody-conjugated toxin reagent using cell culture medium as an antibody diluent; S2. The antibody to be tested is added to the antibody diluent for serial dilution and incubated at room temperature to form the antibody-nanobody conjugate toxin reagent complex. S3. Seed the target cells into cell culture plates and culture overnight. Add different dilutions of the complex to each well, set up a blank control group and a positive control group, and continue culturing. S4. Add cell proliferation and toxicity detection reagents, incubate, and measure the absorbance at OD450 to calculate cell viability. S5. Plot the proliferation inhibition curve based on cell viability, calculate the IC50 value, and quantify the endocytosis and internalization efficiency of the antibody to be tested.

[0018] Further, in step S1, the concentration of the solution is 0.1 to 1 μg / mL.

[0019] Further, in step S2, the gradient dilution is a 5-10 fold gradient dilution.

[0020] Furthermore, in step S3, the overnight culture time is 16-18 hours.

[0021] Further, in step S3, the blank control group consists of only cell culture medium and nanobody-conjugated toxin reagent, without the addition of target cells; the positive control group consists of target cells, cell culture medium and nanobody-conjugated toxin reagent, without the addition of the antibody to be tested.

[0022] Furthermore, in step S3, the continued cultivation time is 3 to 7 days.

[0023] The beneficial effects of this invention are: 1. High affinity and stable complex: The nanobody of this invention binds to the Fc segment of human IgG1 / IgG4 with high specificity and high affinity, and the resulting complex has good stability, avoiding the problem of complex dissociation caused by insufficient affinity.

[0024] 2. Small molecular weight and low steric hindrance: The nano-antibody conjugate toxin HK-D4-MMAE formed by this invention has a molecular weight of only 87kDa. The overall molecular weight of the complex is small compared to the molecular weight of the antibody to be tested, and it does not affect the internalization efficiency of the antibody (mAb) to be tested.

[0025] 3. High drug-antibody ratio and wide detection window: The reagent of this invention has a DAR value of up to 4, and the unit complex carries a large number of toxin molecules, resulting in high detection sensitivity; at the same time, the reagent is non-cytotoxic within the working concentration range, and there is no need to wash cells after adding the drug, which reduces operation steps and experimental errors, and lowers time and material costs.

[0026] 4. Accurate detection results and high predictive value: Using trastuzumab as a model for verification, the IC50 value measured by the reagent of this invention is comparable to the IC50 value measured by the real ADC drug, indicating that the reagent of this invention can accurately predict the internalization and target cell killing effect of the antibody after it is loaded as an ADC, and has good application prospects. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the working principle of the nano-antibody-conjugated toxin reagent HK-D4-MMAE of the present invention. Figure 2 This is an image showing the SDS-PAGE detection results of the nanobody VHH-Fc of this invention; Figure 3 This is the HIC-HPLC chromatogram of the nanobody-conjugated toxin reagent HK-D4-MMAE of this invention; Figure 4 The image shows the results of VHH-HRP's specific binding to the human IgG1 Fc and IgG4 Fc fragments. Figure 5 This is a schematic diagram of the 96-well plate cell deposition of the present invention; Figure 6 This is a diagram showing the cytotoxicity verification results of HK-D4-MMAE of the present invention; Figure 7 This is a schematic diagram of the drug dosing layout for the 96-well plate of the present invention; Figure 8 This is a graph showing the results of detecting the internalization efficiency of trastuzumab using HK-D4-MMAE according to the present invention. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0029] In a first aspect, the present invention provides a nanobody-conjugated toxin reagent for detecting antibody endocytosis and internalization efficiency, wherein the reagent is formed by conjugating a nanobody and a toxin drug through a linker. The amino acid sequence of the nanobody is shown in SEQ ID NO:1, and the amino acid sequences of its complementarity-determining region (CDR) are as follows: CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4. The nanobody-conjugated toxin reagent provided by this invention has a small molecular weight, high affinity, high DAR value, and no cytotoxicity within the working concentration range. After binding with the antibody to be tested, it essentially does not affect the internalization process, accurately quantifying the antibody endocytosis efficiency. The detection results are comparable to those of real ADC drugs, providing a reliable detection tool for efficient screening of large quantities of antibodies in the early stages of ADC drug development.

[0030] In some embodiments, in the complementarity-determining region (CDR), at least one CDR has an amino acid sequence that is the sequence shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or a sequence that has at least 80% sequence identity with the sequence shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; preferably 85%, 90%, 95%, 98%, or 99%. This limitation retains the core performance of the nanobody in binding with the human IgG1 / IgG4 Fc fragment with high specificity and high affinity, while also broadening the sequence protection range to cover homologous sequences that still possess equivalent binding activity after minor modifications.

[0031] In some embodiments, the nanobody is selected from at least one of monovalent nanobody, multivalent nanobody, multispecific antibody, and fusion nanobody. This configuration enriches the selection of nanobody types, allowing for flexible selection based on the actual needs of antibody endocytosis detection, adapting to different detection scenarios while maintaining high specificity and high affinity binding to the Fc fragment.

[0032] In some embodiments, the toxin is selected from at least one of microtubule inhibitors, DNA synthesis inhibitors, and cell-killing toxins. This multi-category selection of toxins allows for flexible adaptation to the sensitivity of antibody endocytosis detection and the requirements of the detection system, broadening the applicable scenarios and detection range of the reagents.

[0033] In some embodiments, the tubulin inhibitor is selected from at least one of MMAE, MMAF, DM1, DM4, tubulosyntheticin, cryptomycin, and antimitotic EG5 inhibitors. Clearly defining the specific types of tubulin inhibitors provides a clear range of preferred toxins for reagent preparation, and the selected toxins are all mature cytotoxic substances in the field of antitumor detection, ensuring the effectiveness and reliability of the detection.

[0034] In some embodiments, the DNA synthesis inhibitor is selected from at least one of ducamycin, pyrrolobenzodiazepines, pyrrolobenzodiazepines (PBD), indolechlorobenzodiazepines, enediyne derivatives, and topoisomerase inhibitors. Limiting the specific type of DNA synthesis inhibitor enriches the selection dimensions of toxin drugs, allowing for the selection of suitable toxins for different target cells and detection needs, thereby improving the detection adaptability of the reagent.

[0035] In some embodiments, the cytotoxic toxoids are selected from at least one of apoptosis inducers, telolanlustatin and its analogues, amatoxins, nicotinamide phosphoribosyltransferase inhibitors, diphtheria toxin and its derivatives, and carbamycin. Listing the specific types of cytotoxic toxoids further expands the selection space for toxin drugs. This class of toxoids covers different types, including natural toxins and synthetic inducers, with diverse killing mechanisms. Some toxoids possess high toxicity and high specificity, meeting the antibody endocytosis detection needs of different detection precisions and target types. For example, for test antibodies with low internalization efficiency, highly toxic diphtheria toxin derivatives can be used to amplify the detection signal; for detection systems requiring high sensitivity, specific apoptosis inducers can be used to reduce non-specific cell killing. Furthermore, the conjugation applications of this class of toxoids have mature technical references, ensuring stable biological activity after conjugation with nanobodies.

[0036] In some embodiments, the molar ratio of the nanobody to the toxin drug is 1:(1-10), preferably 1:4. Defining the range of the molar ratio of the nanobody to the toxin drug and specifying the preferred ratio is a key design consideration that balances detection sensitivity, reagent specificity, and stability: a range of 1:(1-10) adapts to the toxicity intensity of different toxins, allowing for flexible adjustment of the coupling ratio based on the activity of the toxin, avoiding insufficient coupling of low-activity toxins leading to weak detection signals, or excessive coupling of high-activity toxins increasing the difficulty of reagent preparation.

[0037] In some embodiments, the linker is selected from cleavable linkers or non-cleavable linkers; the cleavable linker is selected from at least one of chemically cleavable linkers and enzyme-cleavable linkers; preferably, it is VC (valine-citrulline (Val-Cit) dipeptide), wherein the VC linker can be pre-assembled with a toxin drug to form a VC-toxin module, and then coupled with a nanobody to form the reagent; the non-cleavable linker is selected from at least one of MCC, SMCC, MC, pyrophosphate diester group, PEG, or negatively charged sulfonate group. This invention uses a cleavable linker (preferably VC (valine-citrulline dipeptide)) to couple nanobodies with a toxin drug, wherein the VC linker can be pre-assembled with a toxin to form a VC-toxin module, which can be efficiently coupled with the reduced nanobody, achieving a high drug-to-antibody ratio in the reagent, and the coupling reaction conditions are mild, the product is easy to purify, and the uniformity and activity of the reagent are guaranteed.

[0038] Secondly, the present invention provides a method for preparing the above-mentioned nano-antibody-conjugated toxin reagent, comprising the following steps: S1. Synthesize the gene encoding the above-mentioned nanobody, construct a recombinant plasmid, transform it into an expression host cell for fermentation expression, and purify to obtain the nanobody; The above steps, through a standardized process from gene synthesis and plasmid construction to fermentation and purification, can efficiently prepare nanobodies with correct structure, high purity, and high specificity and affinity binding activity to the Fc segment, laying a high-quality raw material foundation for subsequent conjugation reactions.

[0039] S2. Coupling nanobodies with toxic drugs via a chemical reaction; The above steps, by selecting VC-MMAE as the load-linker and using a chemical method to achieve the coupling of nanobodies and toxin drugs, can ensure the high efficiency and stability of the coupling reaction, so that the two are firmly bound together and retain their respective biological activities.

[0040] S3. Purify the conjugated product, identify it by HPLC and mass spectrometry, detect the drug-antibody ratio, and obtain the nanobody-conjugated toxin reagent.

[0041] The above steps can purify the conjugated product by removing unconjugated free nanobodies, toxins, and reaction byproducts. Combined with HIC-HPLC identification and drug-antibody ratio detection, the purity, uniformity, and conjugation efficiency of the reagents can be precisely controlled, ultimately obtaining nanobodies-conjugated toxin reagents that meet the performance standards and can be directly used for detection.

[0042] Thirdly, the present invention provides the application of the above-mentioned nano-antibody-conjugated toxin reagent in the detection of antibody endocytosis and internalization efficiency.

[0043] In some embodiments, the application is implemented through a detection method comprising the following steps: S1. Prepare a solution of nano-antibody-conjugated toxin reagent using cell culture medium as an antibody diluent; Using cell culture medium to directly prepare reagents as antibody diluents is simple to operate, requires no additional washing, and can stably form the antibody complex to be tested, ensuring accurate and reliable test results.

[0044] S2. The antibody to be tested is added to the antibody diluent for serial dilution and incubated at room temperature to form the antibody-nanobody conjugate toxin reagent complex. Gradual dilution and room temperature incubation can rapidly form a stable complex, ensuring accurate detection concentration, sufficient binding, and improved internalization efficiency, thus enhancing the accuracy and repeatability of the detection.

[0045] S3. Seed the target cells into cell culture plates and culture overnight. Add different dilutions of the complex to each well, set up a blank control group and a positive control group, and continue culturing. Setting up a dual-control system can eliminate non-specific interference, ensure that the experimental results are true, reliable, and quantitatively accurate, and improve the credibility of the detection.

[0046] S4. Add cell proliferation and toxicity detection reagents, incubate, and measure the absorbance at OD450 to calculate cell viability. The above steps involve adding cell proliferation and toxicity detection reagents and measuring the OD450 absorbance value to calculate cell viability. By using a mature absorbance detection method, cytotoxicity is converted into a quantifiable numerical signal, enabling accurate and objective measurement of cell viability.

[0047] S5. Plot the proliferation inhibition curve based on cell viability, calculate the IC50 value, and quantify the endocytosis and internalization efficiency of the antibody to be tested.

[0048] The above steps involve plotting a proliferation inhibition curve based on cell viability and calculating the IC50 value, converting qualitative cell viability data into quantitative IC50 values. This allows for precise and intuitive quantification of the endocytosis and internalization efficiency of the antibody under test, providing a comparable evaluation index for the internalization capabilities of different antibodies.

[0049] In some embodiments, in step S1, the concentration of the solution is 0.1–1 μg / mL. This concentration range represents the experimentally validated optimal working concentration of the nanobody-conjugated toxin reagent. At this concentration, the reagent ensures sufficient and efficient binding with the antibody to be tested during incubation, forming a stable antibody-nanobody-conjugated toxin reagent complex to meet the requirements of subsequent gradient dilution detection. It also effectively avoids non-specific cytotoxicity caused by excessively high reagent concentrations, preventing unbound free reagent from directly killing target cells, thereby eliminating detection errors caused by the reagent's own concentration. Furthermore, this low concentration range allows for control over reagent usage, reducing the cost of the detection experiment and balancing the effectiveness, accuracy, and economy of the detection.

[0050] In some embodiments, in step S2, the gradient dilution is a 5-10 fold gradient dilution. This effectively covers a wide dynamic range of the antibody from high to low concentrations while ensuring detection accuracy and repeatability. It avoids sparse data points due to excessively large dilution factors or redundancy in concentration ranges due to excessively small dilution factors, thereby improving the accuracy of IC50 value fitting and detection efficiency.

[0051] In some embodiments, in step S3, the overnight culture time is 16-18 hours. This ensures that the target cells recover to a stable physiological state, while avoiding excessive cell proliferation or state changes due to excessive culture time, thereby providing a uniform and stable cellular basis for subsequent drug treatment and improving the reliability and repeatability of the detection results.

[0052] In some embodiments, in step S3, the blank control group consists of only cell culture medium and nanobody-conjugated toxin reagent, without the addition of target cells; the positive control group consists of target cells, cell culture medium, and nanobody-conjugated toxin reagent, without the addition of the antibody to be tested. Clearly defining the specific settings of the blank and positive control groups can precisely eliminate the influence of background factors such as the culture medium and reagents themselves, while providing an accurate benchmark for cell viability calculation, significantly improving the accuracy and reliability of the detection results.

[0053] In some embodiments, the culture time in step S3 is 3 to 7 days. This time range ensures that the antibody-reagent complex completes endocytosis and that the toxin exerts its full cytotoxic effect, ensuring that significant changes in cell activity can be detected and providing effective data support for subsequent quantitative analysis.

[0054] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0055] Example 1: Preparation of nanobody-conjugated toxin reagent HK-D4-MMAE 1. Construction of nanobody libraries (1) Immunization and cell collection: Take 500 μg of human IgG1 and IgG4 Fc segment antigen, emulsify with Freund's complete adjuvant, and inject alpaca subcutaneously at multiple points. Use the same antigen and Freund's incomplete adjuvant, and perform booster immunization every two weeks. After four immunizations, collect 80 mL of blood 7 days later to obtain immunized alpaca PBMC cells and prepare RNA. (2) VHH amplification: First-strand cDNA was synthesized by reverse transcription and the VHH fragment was amplified by nested PCR; (3) Vector construction: The VHH fragment and pNB101 vector were digested with restriction endonuclease Sfi I, and the ligation product was obtained by T4 ligase; (4) Library construction: The ligation product was electroporated into TG1 competent cells to construct a nanobody phage display library targeting human IgG1 and IgG4 Fc fragment.

[0056] 2. Selection of Nanobody Libraries (1) Selection: Selecting the titers with a titer of 1×10 12 The PFU phage library was added to the enzyme-labeled wells coated with the antigen and incubated at room temperature for 2 hours. The cells were washed 5 times with PBST (PBS + 0.05% Tween-20), and the phages were dissociated with glycine-HCl (pH=2.7) and neutralization solution was added. (2) Amplification: Infect TG1 cells in logarithmic growth phase, culture at 37°C, and collect the generated phages for the next round of screening. Repeat the same screening process 3 times. (3) Single clone screening: After three rounds of screening, the cells were spread on culture dishes, single clones were selected and placed into deep well plates, cultured at 37°C to the logarithmic phase, and 1 mM IPTG was added for induction for 5 h. (4) ELISA identification: Centrifuge to collect the supernatant, add it to the enzyme-labeled plate coated with antigen, and incubate at room temperature for 1 h. After washing 4 times with PBST, add mouse anti myc-HRP and incubate at room temperature for 1 h. Wash 3 times with PBST, add TMB colorimetric solution, and incubate at room temperature for 20 minutes. Add 2M sulfuric acid to stop the colorimetric process. Read the absorbance value at OD450nm using an enzyme-labeled plate. (5) Sequencing: Select positive monoclonal samples for sequencing to obtain nanobody gene sequences.

[0057] 3. Expression and purification of nanobodies The codon-optimized positive nanobody gene sequence was constructed into the pCDNA3.4-hFc vector, transfected into HEK293 cells using PEI, and cultured at 37℃ and 5% CO2 for 6 days. After affinity purification and buffer replacement, the target nanobody VHH-Fc was obtained. SDS-PAGE results are shown below. Figure 2 As shown, lane M is a molecular weight marker for the protein; lane 1 (non-reducing conditions) shows a clear main band at approximately 87 kDa, consistent with the molecular weight of the homodimer expected to be formed by disulfide bonds in VHH-Fc, indicating that the protein folded correctly and formed its native conformation; lane 2 (reducing conditions) shows the main band migrating to approximately 45 kDa, consistent with the molecular weight of the depolymerized monomeric VHH-Fc polypeptide chain after reduction, and the band is single with no obvious impurities, indicating good protein purity and no degradation. These results demonstrate that this invention successfully obtained high-purity VHH-Fc nanobody protein.

[0058] 4. Preparation and identification of nanobody-conjugated toxin reagent HK-D4-MMAE (1) Reduction: Dilute the anti-human IgG Fc fragment nanobody to 1 mg / mL with PBS, add TCEP (tris(2-carboxyethyl)phosphine) to a final concentration of 1 mM, and reduce at 37°C for 3 h; (2) Coupling: Dissolve VC-MMAE in DMSO to a final concentration of 10 mg / mL, and add it to the reduced nanobody at a molar ratio of vc-MMAE:VHH-Fc=3:1. React at room temperature for 2 h. (3) Purification: After the reaction, the solution was changed using a buffer solution (PBS containing 20 mM L-histidine, pH 5.5) to remove uncoupled small molecules; (4) Quality control: The final product is concentrated, sterilized, filtered, and packaged before being stored at -80℃. The coupling products are analyzed by hydrophobic interaction chromatography, and the results are as follows: Figure 3 As shown, Figure 3 The results showed that the HPLC purity of the conjugated product was >99%, with good homogeneity. Based on the chromatographic peak assignment and peak area weighting, the conjugated component with a DAR value of 4 corresponding to the main peak accounted for more than 95% of the peak area, and the average drug-to-antibody ratio (DAR) was 4, indicating that the nanobody-conjugated toxin reagent HK-D4-MMAE with good homogeneity was successfully prepared.

[0059] Example 2: Verification of the binding ability of nanobodies to the Fc fragment of human IgG Horseradish peroxidase (HRP) was used to label VHH, constructing VHH-HRP for later use. ELISA plates were coated with human IgG1 or IgG4 Fc fragment protein at a concentration of 2 μg / mL. After blocking with skim milk, serially diluted VHH-HRP was added to the wells of the plates, and the mixture was incubated at 37°C for 1 h. The binding affinity of VHH-HRP to IgG1 or IgG4 Fc fragments was then measured. Results are as follows: Figure 4 As shown, Figure 4 The binding signals of VHH-HRP to both the Fc and Fc fragments of human IgG1 and IgG4 increased significantly with increasing VHH-HRP concentration, exhibiting a typical S-shaped binding curve, indicating that the binding is specific and dose-dependent. VHH-HRP has high binding capacity to both human IgG1 and IgG4 Fc fragment proteins.

[0060] Example 3: Cytotoxicity validation of HK-D4-MMAE 1. Cell plating: SKBR3 cells were digested, collected, and counted, at a density of 4 × 10⁶ cells / cells. 3 Seeds were placed in columns 3-11 of a 96-well plate at 90 μL / well; 90 μL of blank cell culture medium was added to column 2 as a blank control; the wells were sealed with 200 μL of blank culture medium or PBS. A detailed plating diagram is shown below. Figure 5As shown, the plate was placed in a 37℃, 6% CO2 incubator and incubated overnight for 16 hours.

[0061] 2. HK-D4-MMAE sample preparation: The next day, HK-D4-MMAE was diluted to a stock solution concentration of 80 ug / mL with DMEM medium without serum and antibiotics, and then serially diluted 2 times.

[0062] 3. Drug Addition Treatment: Remove the cultured 96-well plate and add 10 μL of HK-D4-MMAE at different dilutions to the wells in columns 3-10, ensuring a working concentration of HK-D4-MMAE starting from 8 μg / mL and serially diluted 2-fold in each well. Add 10 μL of serum- and antibiotic-free DMEM medium to column 2 as a blank control (cell-free), and add 10 μL of serum- and antibiotic-free DMEM medium to column 11 as a positive control (containing cells, but without HK-D4-MMAE). 4. Culture and detection: The 96-well plates with added reagent were placed in an incubator at 37℃ and 6% CO2 for 4 days. 10 μL of CCK8 reagent was added to each well, and the plates were incubated at 37℃ for 1.5 h. The absorbance at OD450 was measured using a microplate reader.

[0063] The formula for calculating cell viability is: Cell viability (%) = (As - Ab) / (Ac - Ab) × 100% Where: As: absorbance of the experimental group (including cells, culture medium and different concentrations of HK-D4-MMAE). Ac: Absorbance of the positive control group (containing cells and culture medium, but excluding HK-D4-MMAE). Ab: Absorbance of blank control group (including culture medium, excluding cells and HK-D4-MMAE).

[0064] 5. Results: like Figure 6 As shown, when HK-D4-MMAE is used alone within the normal operating concentration range (0.1-1 μg / mL), it has no significant toxic effect on the proliferation of SKBR3 cells and no significant decrease in cell viability, indicating that the reagent itself has low cytotoxicity and will not interfere with subsequent internalization efficiency detection.

[0065] Example 4: Application of HK-D4-MMAE in the detection of trastuzumab internalization efficiency 1. Cell plating: SKBR3 cells were digested, collected, and counted, at a density of 4 × 10⁶ cells / cells. 3 Seeds were placed in columns 3-11 of a 96-well plate at 90 μL / well; 90 μL of blank cell culture medium was added to column 2 as a blank control; the wells were sealed with 200 μL of blank culture medium or PBS. A detailed plating diagram is shown below. Figure 5 As shown, the plate was placed in a 37℃, 6% CO2 incubator and incubated overnight for 16 hours.

[0066] 2. Complex preparation: The following day, HK-D4-MMAE was diluted to a stock solution concentration of 2.5 μg / mL in DMEM medium free of serum and antibiotics, serving as the antibody diluent. The test antibody, trastuzumab, was diluted to 15 μg / mL using this diluent, followed by 10-fold serial dilutions. The diluted samples were gently mixed and incubated at room temperature for 20 min to form the trastuzumab-HK-D4-MMAE complex.

[0067] 3. Drug Addition: Remove the cultured 96-well plate and add 10 μL of the complex at different dilutions to the wells in columns 3-10. Since 2.5 μg / mL HK-D4-MMAE was used as the antibody diluent during complex preparation, the working concentration of HK-D4-MMAE in each well is 0.25 μg / mL. The concentration of trastuzumab starts from 1.5 μg / mL and is serially diluted 10-fold. 10 μL of 2.5 μg / mL HK-D4-MMAE is added to column 2 as a blank control (without cells) to achieve a working concentration of 0.25 μg / mL in the plate. 10 μL of 2.5 μg / mL HK-D4-MMAE is added to column 11 as a positive control (containing cells, without the test antibody) to achieve a working concentration of 0.25 μg / mL in the plate. A schematic diagram of the drug addition layout is shown below. Figure 7 As shown.

[0068] 4. Culture and detection: The 96-well plates with added reagent were placed in an incubator at 37℃ and 6% CO2 for 4 days. 10 μL of CCK8 reagent was added to each well, and the plates were incubated at 37℃ for 1.5 h. The absorbance at OD450 was measured using a microplate reader.

[0069] The formula for calculating cell viability is: Cell viability (%) = (As - Ab) / (Ac - Ab) × 100% Where: As: absorbance of experimental group (including cells, culture medium, CCK-8 and test antibody + HK-D4-MMAE). Ac: Absorbance of the positive control group (containing cells, culture medium, CCK-8 and HK-D4-MMAE, but excluding the antibody to be tested); Ab: Absorbance of blank control group (containing culture medium, CCK-8 and HK-D4-MMAE, but excluding cells and test antibody).

[0070] 5. Results: like Figure 8As shown, cell proliferation inhibition curves were plotted and IC50 values ​​were calculated using trastuzumab alone and trastuzumab-MMAE (self-made, prepared as before, DAR=3.2) as references. The IC50 value of trastuzumab acting in combination with HK-D4-MMAE was 0.007564 μg / mL (approximately 0.0319 nM), and the IC50 value of trastuzumab-MMAE was 0.009062 μg / mL (approximately 0.0604 nM). Considering the toxin loading, the two are not significantly different and are quite comparable. This indicates that the HK-D4-MMAE provided by this invention can relatively accurately predict the internalization effect of the test antibody after being loaded as an ADC.

[0071] The above-disclosed embodiments are merely a few specific examples of the present invention. However, the embodiments of the present invention are not limited thereto, and any variations that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A nanobody-conjugated toxin reagent for detecting antibody endocytosis and internalization efficiency, characterized in that, The reagent is formed by linking nanobodies with toxin drugs via linkers; The amino acid sequence of the nanobody is shown in SEQ ID NO:1, and the amino acid sequence of its complementarity-determining region (CDR) is shown below: CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:

4.

2. The nanobody-conjugated toxin reagent as described in claim 1, characterized in that, In the complementarity-determining region (CDR), at least one CDR has an amino acid sequence that is the sequence shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, or a sequence that has at least 80% sequence identity with the sequence shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

3. The nanobody-conjugated toxin reagent as described in claim 1, characterized in that, The nanobody is selected from at least one of monovalent nanobody, multivalent nanobody, multispecific antibody and fusion nanobody.

4. The nanobody-conjugated toxin reagent as described in claim 1, characterized in that, The toxin drug is selected from at least one of microtubule inhibitors, DNA synthesis inhibitors, and cell-killing toxins. The microtubule inhibitor is selected from at least one of MMAE, MMAF, DM1, DM4, tubulosicin, cryptomycin, and antimitotic EG5 inhibitor. The DNA synthesis inhibitor is selected from at least one of Ducamycin, pyrrolobenzodiazepines, pyrrolobenzodiazepines, indolechlorobenzodiazepines, enediyne derivatives, and topoisomerase inhibitors; The cytotoxic toxins are selected from at least one of apoptosis inducers, tylanlustatin and its analogues, amatoxins, nicotinamide phosphoribosyltransferase inhibitors, diphtheria toxins and their derivatives, and carbamycin.

5. The nanobody-conjugated toxin reagent as described in claim 1, characterized in that, The molar ratio of the nanobody to the toxin drug is 1:(1-10).

6. The nanobody-conjugated toxin reagent as described in claim 1, characterized in that, The connector is selected from either a cuttable connector or a non-cuttable connector; The cleavable linker is selected from at least one of chemically cleavable linkers and enzyme-cleavable linkers; The incisional linker is selected from at least one of MCC, SMCC, MC, pyrophosphate diester group, PEG, or negatively charged sulfonate group.

7. A method for preparing a nano-antibody-conjugated toxin reagent for detecting antibody endocytosis efficiency, used to prepare the nano-antibody-conjugated toxin reagent according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Synthesize the gene encoding the above-mentioned nanobody, construct a recombinant plasmid, transform it into an expression host cell for fermentation expression, and purify to obtain the nanobody; S2. Coupling nanobodies with toxic drugs via a chemical reaction; S3. Purify the conjugated product, identify it by HPLC and mass spectrometry, detect the drug-antibody ratio, and obtain the nanobody-conjugated toxin reagent.

8. The application of the nanobody-conjugated toxin reagent as described in any one of claims 1-6 in the detection of antibody endocytosis internalization efficiency.

9. The application as described in claim 8, characterized in that, The application is implemented through the detection method of the following steps: S1. Prepare a solution of nano-antibody-conjugated toxin reagent using cell culture medium as an antibody diluent; S2. The antibody to be tested is added to the antibody diluent for serial dilution and incubated at room temperature to form the antibody-nanobody conjugate toxin reagent complex. S3. Seed the target cells into cell culture plates and culture overnight. Add different dilutions of the complex to each well, set up a blank control group and a positive control group, and continue culturing. S4. Add cell proliferation and toxicity detection reagents, incubate, and measure the absorbance at OD450 to calculate cell viability. S5. Plot the proliferation inhibition curve based on cell viability, calculate the IC50 value, and quantify the endocytosis and internalization efficiency of the antibody to be tested.

10. The application as described in claim 9, characterized in that, In step S1, the concentration of the solution is 0.1–1 μg / mL; In step S2, the gradient dilution is a 5-10 fold gradient dilution; In step S3, the overnight culture time is 16-18 hours; The blank control group consisted of only cell culture medium and nanobody-conjugated toxin reagent, without the addition of target cells; The positive control group consisted of target cells, cell culture medium, and nanobody-conjugated toxin reagent, but no antibody to be tested. The continued culture period is 3 to 7 days.

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