Anti-angptl3-FLD nanobodies and use thereof

Abstract

Provided are anti-ANGPTL3-FLD nanobodies and the use thereof. The three prepared anti-ANGPTL3-FLD nanobodies are VHH128, VHH252 and VHH376 respectively; fusion proteins are 128-Fc, 252-Fc and 376-Fc, and the three nanobody fusion proteins VHH-Fc all have good thermal stability and colloidal stability, and have similar particle sizes. The three nano-fusion proteins have good affinity with the antigen hANGPTL3-His, the KD value of 128-Fc being 1.59E-10M, the KD value of 252-Fc being 3.82E-10M, and the KD value of 376-Fc being 1.65E-10M. The purities of the prepared nanobody fusion proteins are up to 100%.

Description

Anti-angptl3-fld nanobody and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and antibody engineering pharmaceuticals, and specifically relates to an anti-ANGPTL3-FLD nanobody and application thereof. BACKGROUND

[0002] Nanobody (VHH) is a single-domain antibody composed of the variable region (VHH) of heavy chain antibody naturally existing in the serum of Camelidae (such as camels and llamas). The unique feature of this antibody is that it only contains a heavy chain variable region without a light chain, which makes them have a smaller molecular weight and higher tissue penetration 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]. Biotechnology Communications, 2016, 27(03): 453-458.). Nanobodies have significant advantages in cancer treatment (Zhou Huihui, Chen Qu, Yang Xiaomei, et al. Research progress of nanobodies in cancer treatment [J]. Life Science, 2022, 34(04): 468-476.), prevention, diagnosis and treatment of infectious diseases (Mei Yaxian, Wang Yue, Luo Wuxin. Application of nanobodies in prevention, diagnosis and treatment of infectious diseases [J]. Chinese Journal of Bioengineering, 2020, 40(10): 24-34.) and immunoassay (Mei Yaxian, Wang Yue, Luo Wuxin. Application of nanobodies in prevention, diagnosis and treatment of infectious diseases [J]. Chinese Journal of Bioengineering, 2020, 40(10): 24-34.) due to their small molecular weight, high stability and low immunogenicity; and, they have high expression, good water solubility, strong stability, easy production and preservation, and are suitable for engineering antibody development.

[0003] Nanobody fusion protein is a fusion of nanobody with other functional proteins or fragments through genetic engineering technology to enhance its function and application range. Since the molecular weight of nanobody itself is only about 15kD, it will be quickly cleared by filtering through the glomerular barrier during in vivo metabolism, and it cannot produce ADCC / CDC and other cytotoxic effects like traditional antibodies due to the absence of Fc segment, therefore, VHH antibody is often fused with Fc segment for expression to increase the half-life of VHH-Fc fusion protein in vivo to prolong the protein action time, while adding ADCC and CDC activity according to specific circumstances.

[0004] Angiopoietin-like protein 3 (ANGPTL3) is a secreted glycoprotein, whose structure includes an amino-terminal coiled-coil domain (CCD), a secretory signal peptide, a short connecting peptide, and a carboxy-terminal 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. In particular, in patients with coronary heart disease, elevated plasma levels of ANGPTL-3 are positively correlated with the severity of the disease (Gao KL. Plasma levels of angiopoietin-like protein 3 and its clinical significance in patients with coronary heart disease [J]. Guangxi Medicine, 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 tissue is significantly higher than that in peritumoral tissue (Song QT, Shan YF, Zhou MT, et al. Expression of angiopoietin-like protein 3 and its relationship with 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 diseases such as lipid metabolism regulation, cardiovascular disease, 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.

[0005] SUMMARY

[0006] Based on the advantages of nanobodies, such as recognition of special epitopes, low immunogenicity, good permeability, and easy modification, an anti-ANGPTL3-FLD nanobody is constructed, which has high affinity, and based on this, the present application is completed.

[0007] In a first aspect, the present application provides an anti-ANGPTL3-FLD nanobody, wherein the variable region of the heavy chain of the nanobody is VHH128, VHH252 or VHH376, wherein the amino acid sequence of VHH128 is shown as SEQ ID NO. 1; the amino acid sequence of VHH252 is shown as SEQ ID NO. 2; and the amino acid sequence of VHH376 is shown as SEQ ID NO. 3.

[0008] In a second aspect, the present application provides a nucleic acid molecule encoding the anti-ANGPTL3-FLD nanobody according to the first aspect of the present application.

[0009] In a third aspect, the present application provides an antibody conjugate comprising the anti-ANGPTL3-FLD nanobody according to the first aspect of the present application.

[0010] In a fourth aspect, the present application provides a composition comprising the anti-ANGPTL3-FLD nanobody according to the first aspect of the present application or a pharmaceutically acceptable carrier thereof.

[0011] In a fifth aspect, the present application provides use of the anti-ANGPTL3-FLD nanobody according to the first aspect of the present application, or the antibody conjugate according to the third aspect of the present application, or the composition according to the fourth aspect of the present application in the preparation of a drug for preventing or treating tumors, kidney diseases or metabolism-related diseases.

[0012] In a sixth aspect, the present application provides use of a reagent for detecting ANGPTL3-FLD protein, wherein the reagent comprises the anti-ANGPTL3-FLD nanobody according to the first aspect of the present application.

[0013] In a seventh aspect, the present application provides a kit for detecting ANGPTL3-FLD protein, wherein the kit comprises an instruction and a detection reagent, and the detection reagent is the reagent according to the sixth aspect of the present application. Advantages

[0014] The anti-ANGPTL3-FLD nanobody and the fusion protein thereof prepared by the present application have good thermal stability and colloidal stability, and similar particle sizes; the prepared nanobody can recognize special epitopes, has low immunogenicity, good permeability and is easy to modify; the prepared nanobody fusion protein has a purity of 100%, and has good affinity with antigens. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a result of SDS-PAGE and concentration and total amount of a recombinant antigen.

[0016] Figure 2 is a result of serological titer detection before and after immunization of a llama.

[0017] Figure 3 is a result of agarose gel electrophoresis of total RNA of PBMC after three immunizations and four immunizations.

[0018] Figure 4 is a result of agarose gel electrophoresis of a first round of amplification products of nested PCR.

[0019] Figure 5 is a result of agarose gel electrophoresis of a second round of amplification products of nested PCR.

[0020] Figure 6 is a result of plate culture of library transformants.

[0021] Figure 7 is a result of agarose gel electrophoresis of colony PCR.

[0022] Figure 8 is a result of amino acid alignment sequence of sequencing of library diversity.

[0023] Figure 9 is a result of phylogenetic tree of analysis of sequencing of library diversity.

[0024] Figure 10 is a result of affinity selection of a recombinant antigen hANGPTL3-FLD-His.

[0025] Figure 11. The results of phage supernatant ELISA detection.

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

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

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

[0029] Figure 15. The schematic diagram of nanobody fusion protein VHH-Fc construction.

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

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

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

[0033] Figure 19. The results of SPR affinity detection of nanobody fusion protein VHH-Fc. DETAILED DESCRIPTION

[0034] The specific embodiments of the present application are described below. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. 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. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0036] Example 1. Recombinant expression of antigen

[0037] 1.1 Test method

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

[0039] 1.2 Test results

[0040] After the antigen was purified by recombinant expression, 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 weight of the two antigens in the reduced and non-reduced states on SDS-PAGE was consistent with the expected value and the protein purity was good (as shown in Figure 1).

[0041] Example 2 Alpaca immunization

[0042] 2.1 Test materials

[0043] Test animals: adult healthy alpacas (Alpaca);

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

[0045] 2.2 Test method

[0046] Multiple rounds of alpaca immunization were performed, and pre-immune negative serum was taken as a control, and post-immune serum was used for immune effect evaluation, and the specific process is as follows:

[0047] Day 0: Take blood 10 mL, leave as negative serum control, mix hANGPTL3-FLD-Fc antigen with CFA and inject;

[0048] Day 21, mix hANGPTL3-FLD-Fc antigen with IFA and inject;

[0049] Day 28, take blood to test titer;

[0050] Day 42, mix hANGPTL3-FLD-Fc antigen with IFA and inject;

[0051] Day 49, take blood to test titer, and isolate peripheral blood lymphocytes;

[0052] Day 63, mix hANGPTL3-FLD-Fc antigen with IFA and inject;

[0053] Day 70, take blood to test titer, and isolate peripheral blood lymphocytes.

[0054] 2.3 ELISA method for detecting immunological titer

[0055] Dilute recombinant antigen hANGPTL3-FLD-His, coat 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, incubate;

[0059] Wash, add TMB color developing solution, incubate, add stop solution, detect optical density.

[0060] 2.4 Test results

[0061] The pre-immune serum was taken as a negative control, and the secondary, tertiary, and fourth immune sera were gradient diluted and subjected to ELISA detection using the recombinant expression antigen hANGPTL3-FLD-His antigen coated overnight. The serum was diluted to 1:32K-1:64K, and OD450>0.2 was considered a positive result. The results showed that the OD450 of the tertiary immune serum diluted to 1:32K was 0.2646, and the OD450 of the fourth immune serum diluted to 1:32K was 0.6134, and the OD450 of the fourth immune serum diluted to 1:64K was 0.3548, indicating that the tertiary and fourth immune sera reached a positive serological titer and could be used for subsequent experiments (as shown in FIG. 2).

[0062] Example 3 RNA extraction and reverse transcription product

[0063] 3.1 RNA extraction and reverse transcription product

[0064] Peripheral blood was collected after the third and fourth immunizations, peripheral blood mononuclear cells (PBMCs) were separated, total RNA was extracted, purity was verified by electrophoresis, and reverse transcription was performed. The cDNA of the third and fourth immunization reverse transcription products was then mixed at an equal ratio, diluted, PCR amplified, electrophoresed, and the 750bp VHH fragments were recovered and amplified. The purified VHH fragments were obtained by electrophoresis and gel recovery.

[0065] The vector pComb3XSS and the target fragment VHH were recovered after being digested with SfiI enzyme, ligated, and constructed into a plasmid for electroporation transformation, recovery, and collection of the recovery product.

[0066] The dilution was coated on the plate to determine the number of library transformants. The remaining supernatant was resuspended, coated, and the number of library transformants was inferred from the number of colonies on the plate. The insertion rate was determined by picking colonies, sequencing, and sequencing primer pComb3XSS-F. The library diversity amino acid alignment sequence and phylogenetic tree were obtained by analyzing the sequencing results using Sequencher Scanner v1.0 and DNASTAR software.

[0067] 3.2 Test results

[0068] After collecting peripheral blood, separating PBMC, extracting RNA, and performing agarose gel electrophoresis, the results show that the RNA band is clear and has good purity (as shown in Figure 3); the above-mentioned RNA is subjected to reverse transcription, and the cDNA of the three and four reverse transcription products is mixed at the same proportion and then diluted, and subjected to one round of PCR amplification, and the product after amplification is subjected to agarose gel electrophoresis, and the results show that there are obvious bands of about 750 bp and 1000 bp, respectively, for VHH and VH fragments (as shown in Figure 4), the VHH band of about 750 bp is recovered from the gel and subjected to two rounds of amplification, and the results of agarose gel electrophoresis show that the VHH band is bright and clear (as shown in Figure 5), and the target VHH fragment is obtained by using a DNA product purification kit after gel recovery.

[0069] Example 4 Packaging of phage library

[0070] 4.1 Test method

[0071] The bacterial library is inoculated into 2YT+A+G medium, the initial OD600 is 0.1-0.2, and the culture is cultured to OD600 is more than 0.8;

[0072] Add helper phage M13KO7 and mix well;

[0073] After centrifugation, the supernatant is discarded, the medium is resuspended, and it is cultured overnight;

[0074] Centrifugation, collection of supernatant, discard of precipitate, centrifugation, collection of supernatant;

[0075] Add PEG8000 / NaCl, mix well, and ice bath;

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

[0077] After centrifugation, the supernatant is discarded, the precipitate is resuspended, glycerol is added to a final concentration of 50%, and after mixing, it is aliquoted and stored;

[0078] Take the library phage gradient dilution, suck and mix with TG1 bacterial liquid, coat Amp resistant plate, culture, and calculate the phage library titer by the number of clones on the titer plate.

[0079] 4.2 Test results

[0080] Take the electroporation recovery product, gradient dilute to 10 -3 and 10 -4 Coat on the plate, count 240 clones on the library transfectant assay plate every other day (as shown in Figure 6), and then deduce that the library capacity is 2.40×10 9 (240×10 4 ×10).

[0081] 48 clones were picked from the library transformation plate and subjected to colony PCR, and then the PCR products were subjected to agarose gel electrophoresis. The results showed that the vector fragments of all the clones were clearly visible, and the insertion rate was 100% (as shown in Figure 7).

[0082] After the diversity sequencing analysis of the above 48 clones, the amino acid alignment sequence results and the phylogenetic tree showed that the diversity of the library was good (as shown in Figures 8 and 9), and the next step of screening could be performed.

[0083] Example 5 Affinity panning of the phage library

[0084] 5.1 Test method of affinity panning

[0085] The recombinant antigen hANGPTL3-FLD-His was diluted to a final concentration of 5 μg / mL, and was added to the enzyme-labeled hole. Each target molecule coated 8 holes, and the coating was performed overnight;

[0086] The coating solution was discarded, and the washing was performed. Then, 3% OVA-PBS blocking solution was added for blocking;

[0087] The washing was performed, and the phage library was added for incubation;

[0088] The unbound phage was aspirated, and the washing was performed;

[0089] The eluent was added for incubation, and the specifically bound phage was eluted. The eluent was transferred to a centrifuge tube, and was neutralized with a buffer;

[0090] Gradient dilution was performed, the titer was determined, the panning recovery rate was calculated, the remaining eluent was mixed for amplification and purification, and was used for the next round of affinity panning. The panning conditions were changed, and the conditions of each round of panning are shown in Table 1.

[0091] Table 1. Panning conditions

[0092] 5.2 Amplification of the library after panning

[0093] The panning eluent was mixed with the E. coli TG1 culture in the early logarithmic growth phase, glucose was added, and the culture was performed. Then, M13K07 phage and 4 ul Amp+ were added, and the medium was added for culture;

[0094] The culture was dispensed, centrifuged, resuspended, and cultured.

[0095] After centrifugation, the supernatant was aspirated, and PEG-NaCl was added for mixing;

[0096] After centrifugation, the supernatant was discarded, the precipitate was resuspended, PEG / NaCl was added, and the mixture was mixed;

[0097] After centrifugation, the supernatant was discarded, the precipitate was suspended in PBS, and the amplification product was obtained. The titer was determined, and was used for panning or analysis.

[0098] 5.3 Test results

[0099] Two rounds of panning were actually performed (as shown in Figure 10):

[0100] The first round was coated with 5 μg / ml of antigen, and the library input was 2 x 10 11 cfu, and the recovery was 9.68 x 10 5 cfu, and the recovery was 4.84 x 10 -6 ;

[0101] The second round was coated with 2 μg / ml of antigen, and the library input was 1 x 10 11 cfu, and the recovery was 1.62 x 10 7 cfu, and the recovery was 1.62 x 10 -4 , and the enrichment was 33.47.

[0102] Example 6 Identification and analysis of specific phage clones

[0103] 6.1 Rescue of phagemids

[0104] The clones were selected, inoculated, and cultured;

[0105] The above culture was taken, M13K07 phage was added, and the culture was taken;

[0106] 2 x YT-AK was added, and the culture was taken;

[0107] The supernatant was taken by centrifugation, and used for identification of monoclonal ELISA.

[0108] 6.2 Identification of positive phage clones

[0109] A. Test method

[0110] The recombinant antigen hANGPTL3-FLD-His was diluted to a final concentration of 2 μg / mL, added to the enzyme-labeled hole, and coated overnight;

[0111] The coating solution was discarded, washed, skim milk was added, and blocked;

[0112] Washed, added phage culture supernatant and skim milk, and incubated;

[0113] Washed, added horseradish peroxidase-labeled anti-M13 antibody;

[0114] Washed the plate, added TMB color developing solution, developed color, added stop solution, and measured optical density at 450 nm.

[0115] B. Test results

[0116] From the second round of titer determination plate, 480 clones were selected, after expansion culture, the phage supernatant was taken for ELISA determination, the results showed that 432 positive clones were screened (as shown in Figure 11), 396 single clones were successfully sequenced, and 92 full-length specific sequences and 53 CDR3 specific sequences were included.

[0117] 6.3 Sequencing and analysis of positive phage clones

[0118] The positive phage clones obtained by the above screening were sent for sequencing, and the sequencing results were analyzed by Sequencher Scanner v1.0 and DNASTAR software to obtain library diversity amino acid alignment sequences and phylogenetic tree.

[0119] Further summarize the full-length specific sequence, pay special attention to the CDR3 specific sequence, after sequence specificity analysis by DNASTAR software, combined with phage ELISA data, select sequences to construct VHH-His protein, after recombinant expression, use ELISA and SPR method to detect its affinity with antigen to determine the candidate sequence.

[0120] 6.4 Nanobody candidate sequence screening

[0121] Combined with the specificity of the sequence and the ELISA detection data of the phage supernatant, 11 His-tagged nanobody VHH-His were selected and constructed, except for No. 6 antibody which showed abnormally low expression, the remaining 10 antibodies could be normally expressed and had good purity (as shown in Figure 12), 3 high-affinity candidate antibodies were obtained by using ELISA and SPR method to detect the affinity of VHH-His and human ANGPTL3 molecule (as shown in Figures 13 and 14), the candidate sequences are as follows:

[0122] Example 7 Nanobody fusion protein construction and characterization

[0123] 7.1 Nanobody fusion protein construction

[0124] The human IgG1-Fc sequence was connected to the C-terminal of the candidate antibody sequence, the fusion protein sequence was synthesized by gene and connected to the pcDNA3.4 vector, the protein was expressed by using HEK293F cells, and the supernatant was centrifuged and purified by Protein A affinity chromatography column, and the ultrafiltration was obtained. The target protein.

[0125] The human IgG1-Fc sequence was connected to the C-terminal of the above three candidate antibody sequences, and the gene was synthesized by using enzyme digestion sites XbaI and EcoRV to realize the connection with the vector pcDNA3.4, and the constructed plasmid was used for subsequent transfection (as shown in Figure 15).

[0126] 7.2 SDS-PAGE detection of the molecular weight of the antibody

[0127] Tris-glycine gel was configured, and electrophoresis was performed under reducing and non-reducing conditions. The voltage was set to 80 V for concentration and 120 V for separation. After electrophoresis, the gel was taken out, stained with Coomassie brilliant blue, and then rinsed overnight with a destaining solution. Imaging was performed, and the molecular weight was determined according to the position of the band.

[0128] After the above-mentioned plasmid was transfected into HEK293F cells for protein expression and purified by Protein A column affinity chromatography, SDS-PAGE was performed for verification. The results showed that the three nanobody fusion proteins VHH-Fc exhibited clear protein bands under reducing and non-reducing conditions, with a molecular weight consistent with the expected value and good purity (as shown in Figure 16).

[0129] 7.3 SEC-HPLC detection of the purity of the antibody

[0130] After diluting the purified protein, it was injected into the chromatographic column, and the purity of the antibody was determined under ultraviolet irradiation.

[0131] The above-mentioned three nanobody fusion proteins VHH-Fc were subjected to SEC-HPLC detection of protein purity, and the results showed that the protein purity was very good, reaching 100% (as shown in Figure 17).

[0132] 7.4 DLS detection of the stability, particle size, and polydispersity index of the antibody

[0133] A protein stability analyzer was used to obtain the polydispersity, particle size, and particle size distribution of the sample, to explore the unfolding and aggregation characteristics of the protein during heating, and to determine the Tm and Tagg values to reveal the thermal stability and colloidal stability of the antibody.

[0134] The above-mentioned three nanobody fusion proteins VHH-Fc were further characterized using UNcle instruments, and the results showed that the Tm value of 128-Fc was 61.62℃, the Tagg value was 67.02℃, the particle size was 7.46 nm, and the PDI was 0.226; the Tm value of 252-Fc was 61.09℃, the Tagg value was 79.59℃, the particle size was 8.06 nm, and the PDI was 0.053; the Tm value of 376-Fc was 60.41℃, the Tagg value was 60.49℃, the particle size was 7.46 nm, and the PDI was 0.141 (as shown in Figure 18).

[0135] The above-mentioned results showed that the three nanobody fusion proteins VHH-Fc all had good thermal stability and colloidal stability, and the particle sizes were similar, but the polydispersity coefficients were quite different, suggesting that the forms of different proteins in solution might be different.

[0136] 7.5 SPR detection of the affinity of the antibody to the antigen

[0137] Using surface plasmon resonance (SPR) analysis, human IgG capture antibody was pre-fixed on a CM5 chip, and the antibody was captured on the chip by adjusting the capture time, and then the antigen hANGPTL3 was flowed through the chip for binding, dissociation, and the affinity was calculated by the binding and dissociation parameters generated by the system.

[0138] The affinities of the above three nanobody fusion proteins VHH-Fc and the antigen hANGPTL3-His were detected by the Biacore T200 instrument using the SPR experimental method, and the results showed that the KD value of 128-Fc was 1.59E-10M, the KD value of 252-Fc was 3.82E-10M, and the KD value of 376-Fc was 1.65E-10M, all showing good and similar affinities (as shown in Figure 19).

Claims

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

3.

2. A nucleic acid molecule encoding the anti-ANGPTL3-FLD Nanobody of claim 1.

3. An antibody conjugate comprising the anti-ANGPTL3-FLD Nanobody of claim 1.

4. A composition comprising the anti-ANGPTL3-FLD Nanobody of claim 1 or a pharmaceutically acceptable carrier thereof.

5. Use of the anti-ANGPTL3-FLD Nanobody of claim 1 or the antibody conjugate of claim 3 or the composition of claim 4 in the preparation of a medicament for preventing or treating a tumor, a kidney disease or a metabolism-related disease.

6. Use of an agent for detecting an ANGPTL3-FLD protein, the agent comprising the anti-ANGPTL3-FLD Nanobody of claim 1.

7. A kit for detecting an ANGPTL3-FLD protein, the kit comprising an instruction and a detection agent, the detection agent being the agent of claim 6.

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