An anti-ANGPTL3-FLD nanobody and its application

Abstract

The present invention belongs to the fields of biotechnology and antibody engineering pharmaceuticals, and specifically relates to an anti-ANGPTL3-FLD nanobody and its application. The three prepared anti-ANGPTL3-FLD nanobodies are VHH128, VHH252, and VHH376, and the fusion proteins are 128-Fc, 252-Fc, and 376-Fc. The three nanobody fusion proteins VHH-Fc all have good thermal stability and colloidal stability, and similar particle sizes; the three nanobody fusion proteins have good affinity with the antigen hANGPTL3-His. The KD value of 128-Fc is 1.59E-10 M, the KD value of 252-Fc is 3.82E-10 M, and the KD value of 376-Fc is 1.65E-10 M3; the purity of the prepared nanobody fusion protein is as high as 100%.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and antibody engineering pharmaceuticals, and specifically relates to an anti-ANGPTL3-FLD nanobody and its applications. Background Art

[0002] Nanobodies (VHHs) are single-domain antibodies composed of the variable regions (VHHs) of heavy-chain antibodies naturally present in the sera of camelids (such as camels and alpacas). The uniqueness of this kind of antibody lies in that it only contains a heavy-chain variable region without a light chain, which makes them have a smaller molecular weight and higher tissue penetrability than traditional antibodies (S. Muyldermans. “Nanobodies: natural single-domain antibodies.” Annual Review of Biochemistry (2013).; Wang Landong, Feng Dongxiao, Zhang Shumin. Research progress of nanobodies [J]. Letters in Biotechnology, 2016, 27(03): 453-458.). Due to characteristics such as their small molecular weight, high stability, and low immunogenicity, nanobodies have significant advantages in fields such as cancer treatment (Zhou Huihui, Chen Qu, Yang Xiaomei, etc. Research progress of nanobodies in cancer treatment [J]. Life Sciences, 2022, 34(04): 468-476.), prevention, diagnosis, and treatment of infectious diseases (Mei Yaxian, Wang Yue, Luo Wenxin. Application of nanobodies in the prevention, diagnosis, and treatment of infectious diseases [J]. China Biotechnology, 2020, 40(10): 24-34.), and immunoassay (Mei Yaxian, Wang Yue, Luo Wenxin. Application of nanobodies in the prevention, diagnosis, and treatment of infectious diseases [J]. China Biotechnology, 2020, 40(10): 24-34.); moreover, they have high expression levels, good water solubility, strong stability, are easy to produce and preserve, and are suitable for the development of engineered antibodies.

[0003] Nanobody fusion proteins are obtained by genetically engineering techniques to fuse nanobodies with other functional proteins or fragments to enhance their functions and application scopes. Since the molecular weight of a nanobody itself is only about 15 kD, it will be rapidly cleared by filtering through the glomerular barrier during in vivo metabolism, and because it does not have an Fc segment and cannot produce cytotoxic effects such as ADCC / CDC like traditional antibodies, the VHH antibody is often fused with the Fc segment for expression. While increasing the half-life of the VHH-Fc fusion protein in vivo to extend the protein's action time, ADCC and CDC activities are added as appropriate depending on the specific situation.

[0004] Angiopoietin-like protein 3 (ANGPTL3) is a secreted glycoprotein, whose structure includes an amino-terminal coiled-coil domain (CCD), a secretion signal peptide, a short linker peptide, and a carboxyl-terminal globular fibrinogen-like domain (FLD). Studies have shown that elevated levels of ANGPTL3 are associated with the occurrence of various diseases, including coronary heart disease, diabetes, hepatocellular carcinoma, breast cancer, etc. Especially in patients with coronary heart disease, the elevated plasma ANGPTL-3 level is positively correlated with the severity of the disease (Gao Kele. Plasma angiopoietin-like protein 3 level and its clinical significance in patients with coronary heart disease [J]. Guangxi Medical Journal, 2021, 43(1): 31-34, 51.). In addition, ANGPTL3 is also closely related to the invasion and metastasis of hepatocellular carcinoma, and its expression intensity in tumor tissues is significantly higher than that in the peritumoral tissues (Song Qitong, Shan Yunfeng, Zhou Mengtao, et al. Study on the expression of angiopoietin-like protein 3 and its relationship with the invasion and growth of hepatocellular carcinoma [J]. Chinese Journal of Hepatobiliary Surgery, 2006, 12(7): 450-452.). As an important secreted protein, ANGPTL3 plays an important role in the pathogenesis of various diseases such as lipid metabolism regulation, cardiovascular diseases, diabetes and its complications. Therefore, in-depth study of the function of ANGPTL3 and its role in diseases is of great significance for the development of new treatment strategies. Summary of the Invention

[0005] Based on the advantages of nanobodies such as the ability to recognize special epitopes, low immunogenicity, good permeability, and easy modification, the present invention constructs an anti-ANGPTL3-FLD nanobody with high affinity, and thus completes the present invention.

[0006] In a first aspect, the present invention provides an anti-ANGPTL3-FLD nanobody, wherein the variable heavy chain region of the nanobody is VHH128, VHH252 or VHH376, and the amino acid sequence of VHH128 is as shown in SEQ ID NO.1; the amino acid sequence of VHH252 is as shown in SEQ ID NO.2; the amino acid sequence of VHH376 is as shown in SEQ ID NO.3.

[0007] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-ANGPTL3-FLD nanobody as described in the first aspect of the present invention.

[0008] In a third aspect, the present invention provides an antibody conjugate, which includes the anti-ANGPTL3-FLD nanobody as described in the first aspect of the present invention.

[0009] In a fourth aspect, the present invention provides a composition, which contains the anti-ANGPTL3-FLD nanobody as described in the first aspect of the present invention or a pharmaceutically acceptable carrier thereof.

[0010] Fifth aspect, the present invention provides an application of the anti-ANGPTL3-FLD nanobody as described in the first aspect of the present invention, or the antibody conjugate as described in the third aspect of the present invention, or the composition as described in the fourth aspect of the present invention in the preparation of a drug for preventing or treating tumors, kidney diseases or metabolic-related diseases.

[0011] Sixth aspect, the present invention provides an application in a reagent for detecting ANGPTL3-FLD protein, wherein the reagent contains the anti-ANGPTL3-FLD nanobody as described in the first aspect of the present invention.

[0012] Seventh aspect, the present invention provides a kit for detecting ANGPTL3-FLD protein, wherein the kit includes an instruction manual and a detection reagent, and the detection reagent is the reagent as described in the sixth aspect of the present invention.

[0013] Beneficial effects

[0014] Both the anti-ANGPTL3-FLD nanobody and its fusion protein prepared by the present invention have good thermal stability and colloidal stability, and similar particle sizes; the prepared nanobody has the advantages of being able to recognize special epitopes, low immunogenicity, good permeability and easy modification; the purity of the prepared nanobody fusion protein is as high as 100%, and it has good affinity with the antigen. Description of the drawings

[0015] Figure 1 SDS-PAGE results, concentration and total amount of recombinant expressed antigen.

[0016] Figure 2 Detection results of serological titers before and after alpaca immunization.

[0017] Figure 3 Agarose gel electrophoresis results of total RNA of PBMC after the third and fourth immunizations. M: DNA marker; 1: RNA after the third immunization; 2: RNA after the fourth immunization.

[0018] Figure 4 Agarose gel electrophoresis results of the first-round amplification products of nested PCR.

[0019] Figure 5 Agarose gel electrophoresis results of the second-round amplification products of nested PCR.

[0020] Figure 6 Results of plate culture for determination of library transformants.

[0021] Figure 7 Agarose gel electrophoresis results of colony PCR.

[0022] Figure 8 Results of amino acid alignment sequence by diversity sequencing of library construction.

[0023] Figure 9 Evolutionary tree of the results of library construction diversity sequencing analysis.

[0024] Figure 10 Results of affinity panning of recombinant antigen hANGPTL3-FLD-His.

[0025] Figure 11 Results of phage supernatant ELISA detection.

[0026] Figure 12 SDS-PAGE results of recombinant expression of VHH-His.

[0027] Figure 13 ELISA detection results of recombinant expression of VHH-His.

[0028] Figure 14 SPR detection results of recombinant expressed candidate VHH-His.

[0029] Figure 15 Schematic diagram of the construction of nanobody fusion protein VHH-Fc.

[0030] Figure 16 SDS-PAGE results of nanobody fusion protein VHH-Fc.

[0031] Figure 17 SEC-HPLC results of nanobody fusion protein VHH-Fc.

[0032] Figure 18 Results of UNcle instrument detection of nanobody fusion protein VHH-Fc.

[0033] Figure 19 SPR affinity detection results of nanobody fusion protein VHH-Fc. Specific embodiments

[0034] The specific embodiments of the present invention will be further described below. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0035] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all commercially available through conventional commercial channels unless otherwise specified.

[0036] Example 1 Recombinant expression of antigen

[0037] 1.1 Test method

[0038] Considering the differences in the in vivo half-life and structure of different forms of antigens, two antigens, hANGPTL3-FLD-Fc and hANGPTL3-FLD-His, were designed and synthesized. The former was mainly used for animal immunization, and the latter was mainly used for antibody screening. After ligating the above gene fragments with the pTT5 vector and transferring them into cells for expression, the purified protein was verified by SDS-PAGE for subsequent experiments.

[0039] 1.2 Test results

[0040] After recombinant expression and purification of the antigens, 12 mg of hANGPTL3-FLD-Fc with a concentration of 10 mg / ml and 20.4 mg of hANGPTL3-FLD-His with a concentration of 17 mg / ml were obtained. The molecular weights of the two antigens under reducing and non-reducing conditions were consistent with expectations on SDS-PAGE and the protein purity was good (as Figure 1 shown).

[0041] Example 2 Alpaca immunization

[0042] 2.1 Test materials

[0043] Test animals: Adult healthy alpacas;

[0044] Recombinant expression antigen: hANGPTL3-FLD-Fc.

[0045] 2.2 Test methods

[0046] Multiple rounds of alpaca immunization were carried out. Negative sera before immunization were collected as controls, and sera after the second, third, and fourth immunizations were used for evaluating the immunization effect. The specific process is as follows:

[0047] Day 0: 10 mL of blood was taken and reserved as a negative serum control. The hANGPTL3-FLD-Fc antigen was mixed with CFA and injected;

[0048] Day 21: The hANGPTL3-FLD-Fc antigen was mixed with IFA and injected;

[0049] Day 28: Blood was collected to detect the titer;

[0050] Day 42: The hANGPTL3-FLD-Fc antigen was mixed with IFA and injected;

[0051] Day 49: Blood was collected to detect the titer, and peripheral blood lymphocytes were isolated;

[0052] Day 63: The hANGPTL3-FLD-Fc antigen was mixed with IFA and injected;

[0053] Day 70: Blood was collected to detect the titer, and peripheral blood lymphocytes were isolated.

[0054] 2.3 Detection of immunological titer by ELISA method

[0055] Dilute the recombinant antigen hANGPTL3-FLD-His and coat it overnight;

[0056] Discard the coating solution, wash, add skim milk and block;

[0057] Wash, add diluted serum and incubate;

[0058] Wash, add diluted Goat anti-Alpaca IgG(H+L)HRP and incubate;

[0059] Wash the plate, add TMB chromogenic solution for color development, incubate, add stop solution and detect the optical density.

[0060] 2.4 Test results

[0061] The pre-immune serum was used as a negative control, and the sera after the second, third, and fourth immunizations were serially diluted. ELISA was performed using the recombinant expressed antigen hANGPTL3-FLD-His coated overnight. An OD450 > 0.2 after dilution of the serum at 1:32K - 1:64K was considered a positive result. The results showed that the OD450 of the serum after the third immunization at 1:32K dilution was 0.2646, and the OD450 of the serum after the fourth immunization at 1:32K dilution was 0.6134, and the OD450 at 1:64K dilution was 0.3548, indicating that the sera after the third and fourth immunizations both reached positive serological titers and could be used for subsequent experiments (such as Figure 2 shown).

[0062] Example 3 RNA extraction and reverse transcription products

[0063] 3.1 RNA extraction and reverse transcription products

[0064] Collect peripheral blood after the third and fourth immunizations, isolate peripheral blood mononuclear cells (PBMC), extract total RNA, verify the purity by electrophoresis, and perform reverse transcription; then mix the reverse transcription products cDNA of the third and fourth immunizations in equal proportions and dilute, perform PCR amplification, electrophoresis, gel extract the VHH fragment of about 750 bp and then amplify, perform electrophoresis, and purify after gel extraction to obtain the target VHH fragment.

[0065] Digest the vector pComb3XSS and the target fragment VHH with SfiI respectively, recover, ligate, construct a plasmid for electroporation, resuscitate, and collect the resuscitation products.

[0066] After gradient dilution, it was spread on plates to determine the number of library transformants. The rest was centrifuged, the supernatant was discarded, and the pellet was resuspended, spread, and the number of library transformants was inferred from the number of clones on the plates. Clones were picked to determine the insertion rate, sequenced, and the sequencing primer was pComb3XSS-F. The sequencing results were analyzed using Sequencher Scanner v1.0 and DNASTAR software to obtain the amino acid alignment sequence and phylogenetic tree of library diversity.

[0067] 3.2 Test Results

[0068] After collecting peripheral blood, separating PBMCs and extracting RNA, agarose gel electrophoresis was performed. The results showed that the RNA bands were clear and the purity was good (as Figure 3 shown); the above RNA was reverse transcribed. The cDNA of the third and fourth immunizations was mixed in equal proportions and diluted, and then PCR first-round amplification was performed. The amplified product was subjected to agarose gel electrophoresis. The results showed that obvious bands were present at around 750 bp and 1000 bp, corresponding to the VHH and VH fragments respectively (as Figure 4 shown). The VHH fragment around 750 bp was recovered by gel extraction and subjected to second-round amplification. The agarose gel electrophoresis results showed that the VHH band was bright and clear (as Figure 5 shown). After gel extraction, it was purified using a DNA product purification kit to obtain the target VHH fragment.

[0069] Example 4 Phage Library Packaging

[0070] 4.1 Test Method

[0071] The bacterial library was inoculated into 2YT + A + G medium with an initial OD600 = 0.1 - 0.2 and cultured until OD600 > 0.8;

[0072] Helper phage M13KO7 was added and mixed evenly;

[0073] After centrifugation, the supernatant was discarded, and the pellet was resuspended in the medium and incubated overnight;

[0074] Centrifuged, the supernatant was collected, the precipitate was discarded, centrifuged again, and the supernatant was collected;

[0075] PEG8000 / NaCl was added, mixed evenly, and placed on ice bath;

[0076] After centrifugation, the supernatant was discarded, the precipitate was resuspended, and PEG8000 / NaCl was added for secondary precipitation;

[0077] After centrifugation, the supernatant was discarded, the precipitate was resuspended, glycerol was added to a final concentration of 50%, mixed evenly, aliquoted, and stored;

[0078] The library phage was serially diluted, aspirated and mixed evenly with TG1 bacterial solution, spread on Amp-resistant plates, cultured, and the phage library titer was calculated based on the number of clones on the titer plates.

[0079] 4.2 Test Results

[0080] Take the electrotransformation and resuscitation products, dilute them serially to 10 -3 and 10 -4 coat them on plates, and count 240 clones on the in-library transformant assay plates the next day (as Figure 6 shown), then the library capacity can be deduced to be 2.40×10 9 (240×10 4 ×10).

[0081] Select 48 clones from the in-library transformant titration plates, perform colony PCR on them, and then perform agarose gel electrophoresis. The results show that the vector fragments of all clones are clearly visible, and the insertion rate is 100% (as Figure 7 shown).

[0082] After the diversity sequencing analysis of the above 48 clones, the amino acid alignment sequence results and phylogenetic tree indicate that the diversity of the library is good (as Figure 8 and Figure 9 shown). In summary, the next step of screening can be carried out.

[0083] Example 5 Affinity Panning of Phage Library

[0084] 5.1 Affinity Panning Test Method

[0085] Dilute the recombinant antigen hANGPTL3-FLD-His to a final concentration of 5 μg / mL, add it to the enzyme-labeled wells, coat 8 wells for each target molecule, and coat overnight;

[0086] Discard the coating solution, wash, add 3% OVA-PBS blocking solution, and block;

[0087] Wash, add the phage library, and incubate;

[0088] Aspirate the unbound phages, and wash;

[0089] Add the elution solution, incubate, elute the specifically bound phages, transfer this elution solution to a centrifuge tube, and neutralize it with a buffer;

[0090] Dilute serially, measure the titer, calculate the panning recovery rate, mix the remaining eluates, and perform amplification and purification for the next round of affinity panning. Change the panning conditions, and the panning conditions for each round are shown in Table 1.

[0091] Table 1. Panning Conditions

[0092]

[0093] 5.2 Amplification of the Library after Panning

[0094] Mix the panning eluate with the E. coli TG1 culture in the early logarithmic growth phase, add glucose, culture, add M13K07 phage and 4 μl Amp+, add medium, and culture;

[0095] Aliquot the culture, centrifuge, resuspend the pellet, and culture;

[0096] After centrifugation, aspirate the supernatant, add PEG-NaCl, and mix;

[0097] After centrifugation, discard the supernatant, resuspend the pellet, add PEG / NaCl, and mix;

[0098] After centrifugation, discard the supernatant, suspend the pellet in PBS, which is the amplified product. Determine the titer for panning or analysis.

[0099] 5.3 Test Results

[0100] A total of 2 rounds of affinity panning were actually carried out (as Figure 10 shown):

[0101] In the first round, the antigen was coated at 5 μg / ml, the input library amount was 2×10 11 cfu, the recovery amount was 9.68×10 5 cfu, and the recovery rate was 4.84×10 -6 ;

[0102] In the second round, the antigen was coated at 2 μg / ml, the input library amount was 1x10 11 cfu, the recovery amount was 1.62×10 7 cfu, and the recovery rate was 1.62×10 -4 , and the enrichment factor was 33.47.

[0103] Example 6 Identification and Analysis of Specific Phage Clones

[0104] 6.1 Rescue of Phagemid

[0105] Select clones, inoculate, and culture;

[0106] Take the above culture, add M13K07 phage, and culture;

[0107] Add 2×YT-AK, and culture;

[0108] Centrifuge and take the supernatant for monoclonal ELISA identification.

[0109] 6.2 Identification of Positive Phage Clones

[0110] A. Test Method

[0111] Dilute the recombinant antigen hANGPTL3-FLD-His to a final concentration of 2 μg / mL, add it to the enzyme-labeled wells, and coat overnight;

[0112] Discard the coating solution, wash, add skim milk, and block;

[0113] Wash, add the supernatant of the phage culture broth and skim milk, and incubate;

[0114] Wash, add horseradish peroxidase-labeled anti-M13 antibody;

[0115] Wash the plate, add TMB chromogenic solution for color development, add the stop solution, and measure the optical density at 450 nm.

[0116] B. Test Results

[0117] Select 480 clones from the second-round titer determination plate, take the phage supernatant after expanded culture for ELISA determination, and the results show that a total of 432 positive clones are screened (as Figure 11 shown), 396 monoclonal antibodies are successfully sequenced, including a total of 92 full-length specific sequences and 53 CDR3 specific sequences.

[0118] 6.3 Sequencing and Analysis of Positive Phage Clones

[0119] The positive phage clones obtained by the above screening were sent for sequencing, and the sequencing results were analyzed using Sequencher Scannerv1.0 and DNASTAR software to obtain the library diversity amino acid alignment sequences and phylogenetic trees.

[0120] Further summarize the full-length specific sequences, especially pay attention to the CDR3 specific sequences. After sequence specificity analysis using DNASTAR software, combined with phage ELISA data, select sequences to construct VHH-His proteins, and detect their affinity with antigens by ELISA and SPR methods after recombinant expression to determine candidate sequences.

[0121] 6.4 Screening of Nanobody Candidate Sequences

[0122] Combined with the sequence specificity and phage supernatant ELISA detection data, 11 His-tagged nanobodies VHH-His were selected and constructed. Except for antibody No. 6 showing abnormally low expression, the remaining 10 antibodies could be normally expressed and had good purity (as Figure 12 shown). ELISA and SPR methods were used to detect the affinity between VHH-His and human ANGPTL3 molecules, and 3 high-affinity candidate antibodies were obtained (as Figure 13 and Figure 14 shown), and the candidate sequences are as follows:

[0123] VHH128

[0124] QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYTIGWFRQAPGKEREGISCINSRGSGTNYA

[0125] DSVAGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADVADIGGPTCPLYSDFLDYWGQ

[0126] GTQVTVSS

[0127] VHH252

[0128] EVQVVESGGGLVQPGGSLRLSCAASGFTLDNYYIVWLRQAPGKEREGVSCISRDGGTTKY

[0129] ADSATGRFTISRDNAKDTVYLQMNSLKPEDTGVYYCAAVFSTLQYICFITVRNADYWGQ

[0130] GTQVIVSS

[0131] VHH376

[0132] QLQLVESGGGLVQPGGSLRLSCTASGFTLDYYAIAWFRQAPGKEREGISCISSRGGSTNYA

[0133] DSVRGRFTISRDNAKNMVYLQMNRLKPEDTAIYYCAADVGALSGCVFGPGYEYDYWGQ

[0134] GTQVTVSS

[0135] Example 7 Construction and Characterization of Nanobody Fusion Proteins

[0136] 7.1 Construction of Nanobody Fusion Proteins

[0137] The human IgG1-Fc sequence was ligated to the C-terminus of the candidate antibody sequence. After gene synthesis of the fusion protein sequence, it was ligated to the pcDNA3.4 vector. Protein expression was performed using HEK293F cells. After centrifugation of the cell supernatant, it was purified by Protein A affinity chromatography and ultrafiltration to obtain the target protein.

[0138] The human IgG1-Fc sequence was respectively ligated to the C-terminus of the above 3 candidate antibody sequences. After gene synthesis, digestion sites XbaI and EcoRV were used to achieve ligation with the vector pcDNA3.4. The constructed plasmids were used for subsequent transfection (such asFigure 15 as shown). 7.2 SDS-PAGE was used to detect the molecular weight of the antibody

[0139] Prepare a Tris-glycine gel and perform electrophoresis under reducing and non-reducing conditions. Set the voltage to 80 V for concentration and 120 V for separation. After electrophoresis, take out the gel, stain it with Coomassie Brilliant Blue, and then rinse it with decolorizing solution overnight. Take an image and determine the molecular weight according to the position of the band.

[0140] After the above plasmid was transfected into HEK293F cells for protein expression, it was purified by affinity chromatography on a Protein A column and then verified by SDS-PAGE. The results showed that the three nanobody fusion proteins VHH-Fc showed clear protein bands under both reducing and non-reducing conditions, with the molecular weight meeting the expectations and good purity (as Figure 16 shown).

[0141] 7.3 SEC-HPLC was used to detect the purity of the antibody

[0142] After diluting the purified protein, inject it into the chromatographic column and detect it under ultraviolet irradiation to judge the purity of the antibody.

[0143] SEC-HPLC was performed on the above three nanobody fusion proteins VHH-Fc to detect the protein purity. The results showed that the protein purity was all very good, reaching 100% (as Figure 17 shown).

[0144] 7.4 DLS was used to detect the stability, particle size and dispersity coefficient of the antibody

[0145] Use a protein stability analyzer to obtain the polydispersity, particle size and particle size distribution of the sample, and explore the unfolding and aggregation characteristics of the protein during the heating process, so as to determine the Tm and Tagg values to reveal the thermal stability and colloidal stability of the antibody.

[0146] Use the UNcle instrument to further characterize the above three nanobody fusion proteins VHH-Fc. The results showed that the Tm value of 128-Fc was 61.62 °C, the Tagg value was 67.02 °C, the particle size was 7.46 nm, and the PDI was 0.226; the Tm value of 252-Fc was 61.09 °C, the Tagg value was 79.59 °C, the particle size was 8.06 nm, and the PDI was 0.053; the Tm value of 376-Fc was 60.41 °C, the Tagg value was 60.49 °C, the particle size was 7.46 nm, and the PDI was 0.141 (as Figure 18 shown).

[0147] The above results indicate that all three nanobody fusion proteins VHH-Fc have good thermal stability and colloidal stability, and similar particle sizes, but there are significant differences in the polydispersity coefficients, suggesting that the existence forms of different proteins in solution may be different. 7.5 SPR detection of the affinity between antibody and antigen

[0148] Surface plasmon resonance (SPR) analysis was used. The human IgG capture antibody was pre-fixed on the CM5 chip, and the antibody was captured on the chip by adjusting the capture time. Subsequently, the antigen hANGPTL3 was flowed through the chip for binding and dissociation, and the affinity was calculated based on the binding and dissociation parameters generated by the system.

[0149] The SPR experimental method was used to detect the affinity between the above three nanobody fusion proteins VHH-Fc and the antigen hANGPTL3-His by a Biacore T200 instrument. The results showed that the KD value of 128-Fc was 1.59E-10 M, the KD value of 252-Fc was 3.82E-10 M, and the KD value of 376-Fc was 1.65E-10 M, all showing good and similar affinities (as Figure 19 shown).

Claims

1. An anti-ANGPTL3-FLD nanobody, wherein the heavy chain variable region of the nanobody is VHH128, VHH252 or VHH376, wherein: The amino acid sequence of VHH128 is shown in SEQ ID NO.1; the amino acid sequence of VHH252 is shown in SEQ ID NO.2; and the amino acid sequence of VHH376 is shown in SEQ ID NO.

3.

2. A nucleic acid molecule encoding the anti-ANGPTL3-FLD nanobody according to claim 1.

3. An antibody conjugate, comprising the anti-ANGPTL3-FLD nanobody according to claim 1.

4. A composition comprising the anti-ANGPTL3-FLD nanobody according to claim 1 and a pharmaceutically acceptable carrier.

5. Use of an anti-ANGPTL3-FLD nanobody in the preparation of a reagent for detecting ANGPTL3-FLD protein, wherein the anti-ANGPTL3-FLD nanobody is as described in claim 1.

6. A kit for detecting ANGPTL3-FLD protein, the kit comprising instructions and a detection reagent, wherein the detection reagent is the reagent according to claim 5.

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