Anti-idiotypic antibodies to aflibercept and uses thereof

Abstract

The application discloses an anti-idiotype antibody of aflibercept and application thereof. The anti-idiotype antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1-3 with amino acid sequences as shown in SEQ ID NOs: 3-5 respectively; and the light chain variable region comprises LCDR1-3 with amino acid sequences as shown in SEQ ID NOs: 6-8 respectively. The anti-idiotype antibody of the application is a blocking type antibody, has high binding force and high specificity for aflibercept, can be used for detecting the content of aflibercept in a sample, plays an important role in pharmacokinetics and pharmacodynamics analysis of the aflibercept, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of antibody drugs, specifically relating to an anti-idiotype antibody against aflibercept and its application. Background Technology

[0002] Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a key regulator of angiogenesis. VEGF activates a series of signaling pathways by binding to tyrosine kinase receptors (VEGFR1 and VEGFR2) expressed on the surface of vascular endothelial cells, promoting endothelial cell proliferation, budding, migration, and tubular formation. In normal physiological processes, VEGF plays an important role in embryonic development, wound healing, and tissue repair. However, in the tumor microenvironment, VEGF enhances blood supply to tumor cells and diseased tissues, providing essential nutrients for tumor growth and metastasis. Therefore, VEGF and its related pathways have become key targets in anticancer therapy and immunotherapy for diseases such as ADM.

[0003] Afibercept is a homodimeric glycoprotein formed by the fusion of the extracellular domains of human vascular endothelial growth factor receptor (VEGFR) (i.e., the VEGFR1 Ig2 and VEGFR2 Ig3 regions) with the Fc domain of human IgG1. It can efficiently bind to VEGF-A, VEGFRB, and placental growth factor PIGF, acting as a decoy antibody to block the interaction of these factors with their endogenous receptors, inhibiting angiogenesis and vascular permeability. Afibercept is now widely used in ophthalmic diseases such as wet age-related macular degeneration (wAMD), diabetic macular edema (DME), and macular edema caused by retinal vein occlusion.

[0004] Anti-idiotype antibodies are antibodies that target specific epitopes (idiotypes) on the variable region of an antibody molecule and can affect the antibody's ability to recognize antigens. Idiotype antibodies are important tools in the development of antibody drugs because they specifically bind to epitopes of specific antibodies. They are mainly used for: 1) detecting the levels of corresponding antibody drugs in the blood, playing an important role in the pharmacokinetics and pharmacodynamics evaluation of antibody drugs; 2) serving as positive controls for anti-drug antibodies (ADAs) of corresponding antibody drugs to assess the immunogenicity of antibody drugs; and 3) blocking drug binding to the target, serving as positive controls for anti-drug neutralizing antibodies of antibody drugs to evaluate the analysis of anti-drug neutralizing antibodies of antibody drugs.

[0005] Therefore, developing unique antibodies with high binding affinity and high specificity against aflibercept is of great importance for preclinical studies, clinical studies, and immunogenicity assessments of its analogues and derivatives. Summary of the Invention

[0006] To address the technical problem of the lack of highly binding and highly specific idiotype antibodies against aflibercept in the existing technology, this invention provides an anti-idiotype antibody against aflibercept and its application.

[0007] Specifically, the present invention provides the following technical solutions to solve the above-mentioned technical problems.

[0008] A first aspect of the present invention provides an anti-idiotype antibody or antigen-binding fragment of aflibercept, said anti-idiotype antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region;

[0009] The heavy chain variable region includes heavy chain complementarity-determining regions HCDR1-3 as shown in SEQ ID NO: 3-5, respectively; the light chain variable region includes light chain complementarity-determining regions LCDR1-3 as shown in SEQ ID NO: 6-8, respectively.

[0010] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; and / or, the light chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2.

[0011] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2.

[0012] In some embodiments, the anti-idiotype antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; and / or, the anti-idiotype antibody or its antigen-binding fragment is selected from full-length antibodies, Fab, Fab', F(ab')2, Fv, and scFv.

[0013] In some embodiments, the anti-idiotype antibody comprises a heavy chain constant region and / or a light chain constant region.

[0014] In some implementations, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3 or IgG4.

[0015] In some implementations, the light chain constant region is the light chain constant region of the κ chain or the λ chain.

[0016] In some implementations, the heavy chain constant region includes CH1, CH2 and CH3 or CH2 and CH3.

[0017] A second aspect of the invention provides an isolated nucleic acid that encodes an anti-idiotype antibody or an antigen-binding fragment thereof as described in the first aspect of the invention.

[0018] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the second aspect of the present invention.

[0019] A fourth aspect of the present invention provides a transformant, wherein the transformant is a host cell containing a nucleic acid as described in the second aspect of the present invention or a recombinant expression vector as described in the third aspect of the present invention, or expressing an antibody or antigen-binding fragment as described in the first aspect of the present invention; the transformant is not an animal variety or a plant cell variety.

[0020] A fifth aspect of the present invention provides a kit comprising the anti-idiotype antibody or its antigen-binding fragment as described in the first aspect of the present invention, the nucleic acid as described in the second aspect, the recombinant expression vector as described in the third aspect, or the transformant as described in the fourth aspect.

[0021] A sixth aspect of the present invention provides a method for detecting aflibercept or a derivative thereof, the method comprising contacting a sample to be tested with an anti-idiotype antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention or a kit as described in the fifth aspect of the present invention.

[0022] In some implementations, the method is for non-diagnostic or non-therapeutic purposes.

[0023] The seventh aspect of the present invention provides the use of the anti-idiotype antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the kit as described in the fifth aspect of the present invention, in the preparation of the following reagents:

[0024] (1) The reagent is used to detect the content or concentration of aflibercept or its derivatives;

[0025] (2) The reagent is used to detect the bioactivity of aflibercept or its derivatives;

[0026] (3) The reagent is used to evaluate the immunogenicity of aflibercept or its derivatives;

[0027] (4) The reagent is used to detect antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) of aflibercept or its derivatives.

[0028] In this invention, aflibercept is a fusion protein comprising the following amino acid sequence:

[0029] SDTGPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSP SHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 9).

[0030] In some embodiments, the aflibercept derivative comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, and having the same biological activity or immunogenicity as aflibercept.

[0031] In some embodiments, the aflibercept derivative comprises an amino acid sequence with 1-3 substituted / deleted / added amino acids compared to the amino acid sequence shown in SEQ ID NO: 9.

[0032] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0033] The reagents and raw materials used in this invention are all commercially available.

[0034] The positive and progressive effects of this invention are as follows:

[0035] 1. The anti-idiotype antibody of this application has high binding affinity and high specificity for aflibercept, and can be used to detect the content of aflibercept in samples, thus playing an important role in the pharmacokinetic and pharmacodynamic analysis of aflibercept.

[0036] 2. The anti-idiotype antibody of this application has good performance and can be used as a positive control for immunogenicity ADA analysis, playing an important role in immunogenicity assessment.

[0037] 3. The anti-idiotype antibody of this application is a target-blocking anti-idiotype antibody, which can be used as a positive control for neutralizing antibody analysis of aflibercept. Attached Figure Description

[0038] Figure 1 This is the drug quantification curve of aflibercept blood concentration.

[0039] Figure 2 This is a curve analysis of the anti-idiotype antibody ADA.

[0040] Figure 3 This is a dose-response diagram of neutralization for idiotype antibodies. Detailed Implementation

[0041] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0042] Example 1: Preparation of anti-idiotype antibodies

[0043] 1) Animal immunization and antibody preparation

[0044] Pepsin (Sigma, catalog number P6887) was used to digest aflibercept protein (manufactured by Maiwei (Shanghai) Biotechnology Co., Ltd.), removing the Fc region of the protein to prepare the F(ab)2 sample. The obtained F(ab)2 sample was used as an immunogen to immunize five Balb / c mice, with each mouse immunized three times, each cycle lasting three weeks. The serum of the immunized mice should specifically bind to the corresponding immunogen, with an antiserum titer of 1:10000 or higher. Spleen cells from 1-2 mice with the highest titers among the immunized mice were fused with SP2 / 0 myeloma cells. The hybridoma cells were expanded and cultured, and the supernatant was used to detect the titer and antibody subtype by ELISA. Cells that were positive for aflibercept and negative for irrelevant human IgG were selected for cloning. To ensure the positive rate of the hybridoma cell line and the stable production of antibodies, 3-4 cloning processes were performed, with each cloning interval of 1-2 weeks. After 3-4 cloning processes yielded 100% positive clones, the cells were expanded and cultured, then inoculated into mice to prepare ascites fluid, obtaining anti-idiotype antibodies. During the cloning process, the specificity of the hybridoma supernatant was detected using an indirect ELISA method, specifically, the mouse monoclonal antibody in the supernatant bound only to the humanized aflibercept monoclonal antibody and not to irrelevant human IgG. Finally, a unique monoclonal antibody was obtained, and the results are shown in Table 1.

[0045] Table 1. Specificity identification results of hybridoma supernatant

[0046]

[0047] 2) Target competitive and non-competitive testing

[0048] An antibody produced by hybridoma cells was tested and found to meet the following conditions:

[0049] 1) Non-blocking antibody: Detected by ELISA, it recognizes aflibercept but not human IgG, and cannot block the binding of aflibercept to VEGF (ELISA plate coated with antigen, aflibercept incubated with the prepared anti-idiotype antibody and then added to the ELISA plate, detected by anti-human IgG secondary antibody, the signal value was not reduced compared with the group without anti-idiotype antibody incubation (buffer group));

[0050] 2) Target blocking antibody: Detected by ELISA, it recognizes aflibercept but not the control human IgG, and can block the binding of aflibercept to VEGF (ELISA plate coated with antigen, aflibercept incubated with the prepared anti-idiotype antibody and then added to the ELISA plate, detected by anti-human IgG secondary antibody, the signal value is significantly reduced compared with the group without anti-idiotype antibody incubation (PBS).

[0051] The results are shown in Table 2. The target blocking ability of the anti-idiotype antibody was determined by comparing the difference in OD values ​​between the antibody-added group and the antibody-unadded group. 16E8G10 is a target blocking antibody.

[0052] Table 2. Results of Target Competitive and Non-Competitive Tests

[0053]

[0054] 3) Sequencing of anti-idiotype antibodies

[0055] Experimental Procedure

[0056] Hybridoma cells were cultured on a large scale, and total RNA was extracted from the cells. cDNA was obtained by reverse transcription. The heavy and light chain variable regions of the antibody were amplified by PCR, sequenced, and subjected to bioinformatics analysis to remove non-functional antibody genes. The antibody was named 16E8G10 according to its hybridoma cell clone of origin. The amino acid sequences of its heavy chain variable region VH, light chain variable region VL, and CDR variable region are shown in Table 3.

[0057] Table 3. Sequencing results (CDRs are defined according to Kabat numbering rules)

[0058]

[0059] The aforementioned antibodies can be isolated monoclonal antibodies through conventional genetic engineering. For example, the gene for synthesizing the aforementioned antibodies can be used to construct a recombinant vector, which can then be transfected into 293 cells for recombinant expression, followed by purification.

[0060] Example 2: Application of 16E8G10

[0061] Application of aflibercept idiotype antibodies in non-clinical and clinical drug analysis

[0062] I. Application of anti-idiotype antibodies in non-clinical pharmacokinetics

[0063] Using the enzyme-linked immunosorbent assay (ELISA) method, taking the pharmacokinetic analysis of rhesus monkey serum as an example, the application of the anti-idiotype antibody in the present invention in non-clinical pharmacokinetic analysis was investigated using an idiotype antibody as a capture reagent and an anti-human Fc antibody as a detection reagent.

[0064] Test methods

[0065] Coating and blocking: Dilute the unique antibody (16E8G10) to 1 μg / mL with coating buffer and incubate overnight at 2-8°C; blot dry the ELISA plate and rinse the ELISA plate 3 times with washing buffer; blot dry the ELISA plate, add 3% BSA / PBST, and incubate at room temperature for 2 hours; blot dry the blocked ELISA plate and rinse the ELISA plate 3 times with washing buffer.

[0066] Standard curve and quality control dilution: A standard curve was prepared by diluting afliberceptumab with 100% pooled monkey serum (PMS) at concentrations of 25000 ng / mL, 8333.33 ng / mL, 2777.78 ng / mL, 925.93 ng / mL, 308.64 ng / mL, 102.88 ng / mL, 34.294 ng / mL, and 11.431 ng / mL. Five quality control concentrations were also prepared using 100% pooled monkey serum: 2000 ng / mL (ULOQ, upper limit of quantification), 1500 ng / mL (HQC, high-concentration control), 500 ng / mL (MQC, medium-concentration control), 250 ng / mL (LQC, low-concentration control), and 100 ng / mL (LLOQ, lower limit of quantification).

[0067] Diluting and adding the test samples: The monkey serum samples, standard curve samples and quality control samples were pre-diluted 50 times with 3% BSA / PBST and then added to the ELISA plate at a rate of 100 μL / well. The plates were set up with replicates and incubated at room temperature for 1 hour.

[0068] Add the detection secondary antibody: After incubation, pat the ELISA plate dry and rinse the ELISA plate 3 times with washing buffer; dilute the goat anti-mouse IgG HRP-labeled secondary antibody (Sino biological, catalog number 10702-MM01T-H) 10000 times with 3% BSA / PBST and add it to the ELISA plate, then incubate at room temperature for 1 hour.

[0069] Color development: After incubation, pat the ELISA plate dry and rinse the ELISA plate 3 times with washing buffer; add the TMB substrate (KPL, catalog number 5120-0047) that has been equilibrated to room temperature to the ELISA plate and incubate at room temperature in the dark for about 10 minutes; add 1 M sulfuric acid to terminate the reaction.

[0070] Reading the plate: After the colorimetric reaction is terminated, use 650nm as the reference wavelength and read the OD value at a wavelength of 450nm.

[0071] Concentration calculation: The concentration of the standard curve is fitted with the OD value using four parameters, and the drug concentration of the sample to be tested is calculated using the fitted equation.

[0072] This method was used to detect the concentration of aflibercept in monkey serum, and the standard curve is shown below. Figure 1 Antigen-blocking antibodies can be used as detection antibodies in pharmacokinetic analysis, exhibiting high method detection sensitivity and signal response values. The accuracy and precision of the method were investigated using this antibody, and the results are shown in Table 4. This antibody can accurately quantify the drug concentration in rhesus monkey serum.

[0073] Table 4. Accuracy and precision of methods for quantifying aflibercept blood concentrations

[0074]

[0075] II. Application of anti-idiotype antibodies in the detection of drug immunogenicity (ADA)

[0076] Immune responses to therapeutic protein drugs can affect their pharmacokinetics, pharmacodynamics, safety, and efficacy. Most adverse drug reactions are caused by humoral immune responses, and the detection of anti-drug antibodies (ADAs) is a primary method for assessing drug immunogenicity. This example primarily examines the application of anti-idiotype antibodies in the detection of immunogenicity ADAs.

[0077] Test Procedure

[0078] Coating and blocking: Dilute aflibercept to 1 μg / mL with CBS coating buffer and incubate overnight at 2-8°C; blot dry the ELISA plate and wash the ELISA plate 3 times with washing buffer; add 3% BSA / PBST to block at room temperature for 2 hours and then wash the plate 3 times.

[0079] Prepare the test sample: Dilute the anti-idiotype antibody (16E8G10) with 10% mixed monkey serum (10% PMS) to 80 μg / mL as the starting concentration, and perform serial dilutions of 3-5 times for a total of 7 points, with 10% PMS as the 0 point.

[0080] Sample addition: Aflibercept was labeled with EZ-LINK NHS-LC-LC-Biotin (Thermo, catalog number 21343) according to the manufacturer's instructions to obtain aflibercept-Biotin, which was then diluted to 2000 ng / mL with 0.5% BSA / PBST. The diluted anti-idiotype antibody sample and aflibercept-Biotin were added to ELISA plates at 100 μL / well, with replicates, and incubated at room temperature for 1 h.

[0081] Add SA-HRP: After incubation, blot the ELISA plate dry and rinse it three times with washing buffer. Dilute SA-HRP (Thermo, catalog number 21126) with 0.5% BSA / PBST at a ratio of 1:5000 and add it to the ELISA plate. Incubate at room temperature for 1 hour.

[0082] Color development: After incubation, blot the ELISA plate dry and rinse it three times with washing buffer. Add the pre-equilibrated TMB substrate (Seracare, catalog number 5120-0077) to the ELISA plate and incubate at room temperature in the dark for about 10 minutes. Stop the reaction by adding 1 M sulfuric acid.

[0083] Reading the plate: After the colorimetric reaction is terminated, use 650nm as the reference wavelength and read the OD value at a wavelength of 450nm.

[0084] Test results

[0085] The results are as follows Figure 2 As shown, the 16E8G10 anti-idiotype antibody exhibits good performance, with a good dose-response relationship between the signal value and antibody concentration, and can be used as a positive control for ADA analysis.

[0086] III. Application of anti-idiotype antibodies in drug neutralization and antibody detection

[0087] Neutralizing activity refers to the ability of an anti-drug antibody to inhibit the biological activity of a drug. Neutralizing antibodies can block the binding of a product to its target or interfering receptor / ligand, thus interfering with the drug's in vivo activity. This example evaluates the neutralizing activity of anti-idiotype antibodies using ligand-binding-based analytical methods.

[0088] Test Procedure

[0089] Sample acid hydrolysis: The acid-hydrolyzed serum sample and alkaline neutralizing reagent were added to a pre-coated and sealed ELISA microplate and incubated at room temperature with shaking to remove VEGF interference;

[0090] Secondary acid hydrolysis of supernatant: Take the supernatant and perform secondary acid hydrolysis;

[0091] Sample incubation: On the preparation plate, the acid-digested serum sample, alkaline neutralizing reagent and the detection reagent Ru-aflibercept were incubated at room temperature with shaking. Then the mixture was added to a pre-coated target antigen rhVEGF165 (Beijing Yiqiao Shenzhou Technology Co., Ltd.; catalog number: 11066-HNAH) and sealed MSD microplate and incubated at room temperature with shaking.

[0092] Reading: After washing the plate, add MSD Read Buffer T(2x) (MSD, part number: R92TC-1, is MSD Read Buffer T(4x), dilute before use), and read the instrument signal on the MESO QUICKPLEX SQ120;

[0093] If the sample to be tested does not contain neutralizing active antibodies, the rh VEGF165 in the system can fully bind to the detection reagent Ru-9MW0813, resulting in a high instrument response value (ECLU) on the electrochemiluminescence detector and a low inhibition rate for the negative control sample. If the sample to be tested contains neutralizing antibodies or quality control positive control antibodies, its ECLU value will be low, and its inhibition rate will be high.

[0094] The results are as follows Figure 3As shown in Table 5, 16E8G10 is a target-blocking anti-idiotype antibody that can block the binding of aflibercept to rhVEGF in a concentration-dependent manner. Therefore, the target-blocking antibody 16E8G10 can be used as a positive control in aflibercept neutralizing antibody assay.

[0095] Table 5. Analysis data of neutralizing antibodies against idiotype antibodies

[0096]

[0097] In summary, the unique monoclonal antibody that specifically recognizes anti-aflibercept in this application has good homogeneity and strong binding affinity, and can be widely used in the field of drug development, mainly for immunogenicity analysis and pharmacokinetic analysis of antibody drugs.

Claims

1. An anti-idiotype antibody against aflibercept or its antigen-binding fragment, characterized in that, The anti-idiotype antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes heavy chain complementarity-determining regions HCDR1-3 as shown in SEQ ID NO: 3-5, respectively; the light chain variable region includes light chain complementarity-determining regions LCDR1-3 as shown in SEQ ID NO: 6-8, respectively.

2. The anti-idiotype antibody or its antigen-binding fragment as described in claim 1, characterized in that, (i) The heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; and / or, the light chain variable region comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; or, (ii) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:

2.

3. The anti-idiotype antibody or its antigen-binding fragment as described in claim 2, characterized in that, The anti-idiotype antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody; and / or, the anti-idiotype antibody or its antigen-binding fragment is selected from full-length antibodies, Fab, Fab', F(ab')2, Fv, and scFv.

4. The anti-idiotype antibody or its antigen-binding fragment as described in claim 3, characterized in that, The anti-idiotype antibody comprises a heavy chain constant region and / or a light chain constant region.

5. The anti-idiotype antibody or its antigen-binding fragment as described in claim 4, characterized in that, The heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4; and / or, the light chain constant region is the light chain constant region of the κ chain or λ chain; And / or, the heavy chain constant region includes CH1, CH2 and CH3 or CH2 and CH3.

6. An isolated nucleic acid, characterized in that, The nucleic acid encodes an anti-idiotype antibody or its antigen-binding fragment as described in any one of claims 1-5.

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 6.

8. A transformant, characterized in that, The transformant is an engineered host cell containing the nucleic acid as described in claim 6 or the recombinant expression vector as described in claim 7, or expressing an antibody or antigen-binding fragment as described in any one of claims 1-5; the transformant is not an animal variety or a plant cell variety.

9. A reagent kit, characterized in that, The kit contains any one of the anti-idiotype antibodies or antigen-binding fragments thereof as claimed in any one of claims 1-5.

10. A reagent kit, characterized in that, The kit comprises the nucleic acid as described in claim 6, the recombinant expression vector as described in claim 7, or the transformant as described in claim 8.

11. A method for detecting aflibercept, characterized in that, The method comprises contacting the anti-idiotype antibody or its antigen-binding fragment as described in any one of claims 1-5 or the kit as described in claim 9 with the sample to be tested; the method is not for diagnostic or therapeutic purposes.

12. The use of the anti-idiotype antibody or its antigen-binding fragment as described in any one of claims 1-5, or the kit as described in claim 9, in the preparation of the following reagents: (1) The reagent is used to detect the content or concentration of aflibercept; (2) The reagent is used to detect the bioactivity of aflibercept; (3) The reagents are used to assess the immunogenicity of aflibercept; (4) The reagent is used to detect antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated phagocytosis or complement-dependent cytotoxicity of aflibercept.

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