Binding molecules against hepatitis B virus core antigen HBcAg and uses thereof

Abstract

The present application relates to the technical field of antibody drugs, and particularly relates to a binding molecule against hepatitis B virus core antigen HBcAg and application thereof. The binding molecule provided by the present application comprises at least one immunoglobulin single variable domain, and the amino acid sequences of CDR1, CDR2 and CDR3 in the immunoglobulin single variable domain are respectively shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. The binding molecule against hepatitis B virus core antigen provided by the present application can reduce the levels of HBcAg, HBeAg, HBsAg and HBV DNA, and can also reduce the assembly of infectious hepatitis B virus particles, block the secretion of infectious hepatitis B virus complete particles, inhibit the infection and amplification of hepatitis B virus, and has a significant therapeutic effect on hepatitis B.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody drugs, and in particular to a binding molecule against hepatitis B virus core antigen HBcAg and uses thereof. Background Art

[0002] Hepatitis B virus (HBV) infection is a serious global public health issue. Statistics show that over 2 billion people worldwide have been infected with HBV, of whom approximately 350 million suffer from chronic HBV infection (CHB). Due to the extremely high infection rate of HBV, liver cancer, which develops in patients with CHB, ranks second among all cancer mortality rates in my country.

[0003] The HBV genome consists of a small circular DNA structure containing 3.2 kb base pairs (3182 to 3221 base pairs, with slight variations in genome length among different HBV subtypes), making it one of the smallest known eukaryotic DNA viruses. The HBV genome has four open reading frames (ORFs): the S-ORF, the P-ORF, the C-ORF, and the X-ORF. The S-ORF encodes three envelope proteins: SHBs (S), MHBs (preS2+S), and LHBs (preS1+preS2+S). The C-ORF comprises the pre-C region (preC) and the C gene region. The pre-C region is a secreted protein (HBeAg) that plays an important role in the development of immune tolerance to chronic infection, while the C gene region forms the viral capsid and encapsidates the viral genome. This region is the most conserved in the HBV genome and serves as a target epitope for immune attack. The gene product of the X-ORF, the HBx protein, has the function of transactivating enhancers and promoters for transcription and is a multifunctional trans-regulatory factor that is crucially involved in viral infection, transcription, replication, apoptosis, and the development of HCC. The P-ORF is the longest ORF in the HBV genome and is responsible for transcribing DNA polymerase. Furthermore, the various open reading frames of HBV highly overlap, resulting in a very compact structure.

[0004] The HBV genome contains 4 genes, which transcribe 4 different mRNAs and can encode the synthesis of 9 different viral proteins, including the structural proteins that assemble the virus's outer membrane protein (HBsAg) and nucleocapsid (HBcAg); 3 P proteins (terminal protein, DNAp and RNase H) and HBx protein that regulates viral replication function. Another HBeAg protein encoded by the pc / C gene is also a functional protein.

[0005] The HBV outer membrane protein (HBsAg), also known as the Australia antigen, was first discovered by Blumberg in 1965 and is widely used to quantify serum HBsAg levels to predict the level of HBV infection in patients. The HBV outer membrane protein is encoded by the S-ORF and includes three outer membrane components: L-HBsAg, M-HBsAg, and S-HBsAg. These three proteins have their own start codons but share the S-HBsAg stop codon.

[0006] The HBV core protein, encoded by the C-ORF, exists in two forms: HBcAg, a structural component of the viral nucleocapsid; and HBeAg, a soluble, secreted form. The HBV C gene has two related ORFs, one of which is divided into the pre-C (Pre-C) and C regions by two start codons, but shares a common stop codon. Pre-C / P mRNA synthesizes the p21 nucleocapsid protein HBcAg, while pre-C-mRNA produces the p25 pre-C protein, which ultimately becomes HBeAg after a series of processing. HBcAg can spontaneously assemble into a capsid structure in various cell lines, such as bacteria, yeast, and mammalian cells, in the absence of other HBV-related proteins or components, making it suitable for use as a vector for genetically engineered vaccines.

[0007] Currently, the primary strategy for combating HBV is preventive vaccines. However, effective antiviral drugs are still needed for patients with chronic hepatitis B. Currently approved drugs for the clinical treatment of chronic hepatitis B include interferons and nucleoside analogs. Although these two classes of drugs have shown positive effects in inhibiting viral replication and delaying disease progression, they still face challenges such as long treatment cycles, low response rates, severe side effects (interferons), and potential drug resistance (nucleoside analogs). Summary of the Invention

[0008] In view of this, the present invention provides a binding molecule against hepatitis B virus core antigen HBcAg and its use. The binding molecule can specifically react with HBcAg and can significantly reduce the HBV virus content in cell models and mouse models, and has a significant effect in treating hepatitis B.

[0009] To solve the above technical problems, the first aspect of the present invention provides a binding molecule against hepatitis B virus core antigen HBcAg, wherein the binding molecule comprises at least one immunoglobulin single variable domain, and the amino acid sequences of CDR1, CDR2 and CDR3 in the immunoglobulin single variable domain are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0010] The present invention immunizes alpacas with HBcAg antigen, collects and separates peripheral blood mononuclear cells (PBMCs) from the immunized alpacas, sorts HBcAg-positive B cells, extracts total RNA and obtains cDNA, obtains an antibody library through phage display technology, and further uses HBcAg to screen and obtain binding molecules that can specifically react with HBcAg. The binding molecules can specifically react with HBcAg and can effectively reduce the content of HBV virus in cell models and mouse models, and have a significant effect in treating hepatitis B.

[0011] Specifically, the sequence of SEQ ID NO: 1 is SGSSFSTVA, the sequence of SEQ ID NO: 2 is QITSIGVT, and the sequence of SEQ ID NO: 3 is CKLYTTYGPPY.

[0012] In conjunction with the first aspect, the binding molecule comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NO: 4 to SEQ ID NO: 10.

[0013] In conjunction with the first aspect, the binding molecule comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 4 to SEQ ID NO: 10.

[0014] In conjunction with the first aspect, the binding molecule comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NO: 4 to SEQ ID NO: 10.

[0015] In combination with the first aspect, the amino acid sequence of the binding molecule is any one of SEQ ID NO: 4 to SEQ ID NO: 10.

[0016] In particular, the sequence of SEQ ID NO: 4 is QVQLVESGGGLVQPGGSLRLSCSASGSSFSTVAMAWYRQAAGQQRELVAQITSIGVTHYADSVKGRFTISRDNAKNTVDLHMNSLKPEDTAVYYCKLYTTYGPPYWGQGTQVTVSS; the sequence of SEQ ID NO: 5 is QLQLVESGGGLVQPGGSLRLSCSASGSSFSTVAMAWFRQAPGKGRELGVAQITSIGVTNYADSVKGRFTVSRDDAKNTVYLQMNSLRAEDTAVYYCKLYTTY GPPYWGQGTTVTVSA; the sequence of SEQ ID NO: 6 is QVQLVESGGGLVQPGGSLRLSCSASGSSFSTVAMAWFRQAPGKGRELGVAQITSIGVTNYADSVKGRFTVSRDDAKNTVYLQMNSLRAEDTAVYYCKLYTTY GPPYWGQGTTVTVSA; the sequence of SEQ ID NO: 7 is QLQLVESGGGLVQPGGSLRLSCSASGSSFSTVAMAWFRQAPGKGRELGVAQITSIGVTNYADSVKGRFTVSRDDAKNTVYLQMNSLRAEDTAVYYCKLYTTY GPPYWGQGTTVTVSS; the sequence of SEQ ID NO: 8 is QLQLVESGGGLVQPGGSLRLSCSASGSSFSTVAMAWFRQAPGKGRELGVAQITSIGVTNAADSVKGRFTVSRDDAKNTVYLQMNSLRAEDTAVYYCKLYTTY GPPYWGQGTTVTVSA; the sequence of SEQ ID NO: 9 is QVQLVESGGGLVQPGGSLRLSCSASGSSFSTVAMAWFRQAPGKGRELGVAQITSIGVTNYADSVKGRFTVSRDDAKNTVYLQMNSLRAEDTAVYYCKLYTTY GPPYWGQGTTVTVSS; the sequence of SEQ ID NO: 10 is QLQLVESGGGLVQPGGSLRLSCSASGSSFSTVAMAWFRQAPGKGRELGVAQITSIGVTNYADSVKGRFTVSRDAAKNTVYLQMNSLRAEDTAVYYCKLYTTY GPPYWGQGTTVTVSS.

[0017] In connection with the first aspect, the binding molecule is an antibody against the hepatitis B virus core antigen HBcAg.

[0018] In combination with the first aspect, the binding molecule is a nanobody against hepatitis B virus core antigen HBcAg.

[0019] The second aspect of the present invention provides a fusion protein comprising the above-mentioned binding molecule and an Fc fragment, wherein the binding molecule is fused with the Fc fragment of an immunoglobulin.

[0020] The third aspect of the present invention provides a multispecific antibody comprising the above-mentioned binding molecule and other antibody fragments.

[0021] The fourth aspect of the present invention provides a use of the above-mentioned binding molecule against hepatitis B virus core antigen HBcAg in the preparation of diagnostic and / or therapeutic products for hepatitis B virus.

[0022] The beneficial effects of the present invention are: the anti-hepatitis B virus core antigen HBcAg binding molecules provided by the present invention can reduce HBcAg levels, HBeAg levels, HBsAg levels and HBV DNA levels, and can also reduce the assembly of infectious hepatitis B virus particles, block the secretion of complete infectious hepatitis B virus particles, inhibit the transmission and amplification of hepatitis B virus, and have a significant therapeutic effect on hepatitis B. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram of PBMC separation in an embodiment of the present invention;

[0024] Figure 2 Graphs showing the analysis of HBcAg-FITC-positive B cells in PBMCs in an embodiment of the present invention, wherein Figure a is a negative control group without the addition of HBcAg-FITC, and Figure b is a sample group with the addition of HBcAg-FITC;

[0025] Figure 3 This is an electrophoresis diagram of the PCR amplification product in the embodiment of the present invention;

[0026] Figure 4 This is a Coomassie blue staining image of the antibody SDS-PAGE in the embodiment of the present invention;

[0027] Figure 5 This is a diagram showing the ELISA test of HBcAg antibodies and HBcAg protein reactivity in an embodiment of the present invention;

[0028] Figure 6 The changes in serum HBsAg levels in transgenic mice in the anti-HBcAg group (Anti-HBcAg) and the control group (Control) at different times in Experimental Example 1 of the present invention;

[0029] Figure 7The changes in serum HBeAg levels in transgenic mice in the anti-HBcAg group (Anti-HBcAg) and the control group (Control) at different times in Experimental Example 1 of the present invention;

[0030] Figure 8 The changes in HBV DNA levels in transgenic mice in the anti-HBcAg group (Anti-HBcAg) and the control group (Control) at different times in Experimental Example 1 of the present invention;

[0031] Figure 9 This is the change in HBcAg levels in the liver of mice in the anti-HBcAg group (Anti-HBcAg) and the control group (Control) in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Example 1

[0034] (1) Preparation of HBcAg antigen:

[0035] The immunogen used in this experiment was the hepatitis B core antigen (HBcAg) in alpacas. The protein was expressed using an E. coli prokaryotic expression system. The cells were disrupted and centrifuged, and then purified using a nickel column. The purified protein was quantitatively analyzed using a 12% polyacrylamide gel. The purified protein was then aliquoted, freeze-dried, and stored at -80°C to avoid protein degradation caused by repeated freeze-thaw cycles.

[0036] (2) Alpaca immunization and blood collection operations are as follows:

[0037] Alpacas were immunized four times with HBcAg. The injection site was the cervical lymph nodes of the alpaca. Half of the sample was injected into each of the left and right lymph nodes of the alpaca's neck. During the operation, care should be taken to keep the protein and adjuvant at low temperature throughout the process to avoid protein degradation caused by repeated freezing and thawing. The initial immunization dose was 300 μg mixed with complete Freund's adjuvant. The booster immunization at week 2 was 200 μg HBcAg mixed with incomplete Freund's adjuvant. The booster immunization at week 6 was 200 μg HBcAg mixed with incomplete Freund's adjuvant. The booster immunization at week 10 was 200 μg HBcAg mixed with incomplete Freund's adjuvant. Before each immunization, 10 mL of blood was collected, anticoagulated with EDTA, and plasma and PBMC were separated.

[0038] After the fourth immunization, 40 mL of blood was collected from the alpaca's jugular vein. This experiment used 10 mL anticoagulant tubes. If the blood sample needs to remain for more than 2 hours before the next step, the volume of blood sample placed in each tube should not exceed 5 mL. If the blood sample is to be placed in the next step within 2 hours, the volume of blood sample placed in each tube should not exceed 8 mL. Each time blood is drawn into the anticoagulant tube, it should be immediately inverted several times to mix thoroughly to prevent uneven mixing and clotting.

[0039] (III) Isolation of peripheral blood mononuclear cells (PBMCs) from alpacas:

[0040] Fresh blood samples should be stored at 18-20°C. Peripheral blood mononuclear cell isolation should be completed as soon as possible within 2-6 hours (preferably within 2 hours) after blood collection. The steps for peripheral blood mononuclear cell isolation are as follows:

[0041] 1) Take out the Histopaque-1077 peripheral blood mononuclear cell separation medium from the 4°C refrigerator one day in advance and allow it to cool to room temperature.

[0042] 2) Transfer 5 mL of room temperature Histopaque-1077 peripheral blood mononuclear cell separation medium to a 15 mL centrifuge tube. Carefully and slowly add 8 mL of whole blood to the top of the Histopaque-1077 peripheral blood mononuclear cell separation medium.

[0043] 3) Set the centrifugal force to 400g and centrifuge at 20℃ for 30 minutes. The sample after centrifugation is divided into four layers, such as Figure 1 As shown, from top to bottom, are serum, peripheral blood mononuclear cells, dextran in peripheral blood mononuclear cell separation solution, and red blood cells. Carefully aspirate the mononuclear cells from the second, turbid layer, avoiding the lower layer. Transfer the aspirated mononuclear cells to a new 15mL centrifuge tube and aliquot the upper serum into a 1.5mL centrifuge tube for long-term storage at -80°C.

[0044] 4) Add 10 mL of room temperature isotonic phosphate buffered saline (PBS), gently invert to mix, and centrifuge at 250 g for 10 minutes at 20°C.

[0045] 5) Remove the supernatant and retain the pellet. Add 5 mL of room temperature PBS to resuspend the cells. Mix gently and centrifuge at 250 g for 10 minutes at 20°C.

[0046] 6) Remove the supernatant and retain the pellet. Remove as much PBS as possible, as this will result in a cloudy precipitate in the lysate in subsequent steps.

[0047] 7) Add freezing solution to suspend the cells and freeze in liquid nitrogen for later use.

[0048] (IV) Isolation of HBcAg-positive B cells from alpaca peripheral blood mononuclear cells (PBMCs):

[0049] The isolated alpaca peripheral blood mononuclear cells (PBMCs) were reacted with FITC-labeled HBcAg (HBcAg-FITC) and Anti-CD19-PE antibodies, and HBcAg-positive B cells were sorted by flow cytometry. Figure 2 The HBcAg-positive B cells obtained by sorting were used as library cells, and positive antibodies were further obtained using phage display technology.

[0050] (V) Library construction:

[0051] 1. Extraction of total RNA

[0052] A. Collect 50 mL of peripheral blood from alpacas and separate peripheral blood mononuclear cells using peripheral blood mononuclear cell separation solution.

[0053] B. Total RNA was extracted using Trizol.

[0054] C. UV spectrophotometric analysis of RNA 6711 revealed OD260 / 280 = 1.99 and OD260 / 230 = 1.43, indicating no significant RNA degradation and acceptable purity. The total RNA concentration was 809.3 ng / μL. Agarose gel electrophoresis revealed two bands, 28S and 18S.

[0055] 2.RT-PCR

[0056] The reverse transcription system is as follows:

[0057] Step 1

[0058]

[0059] After mixing, heat at 65°C for 5 min and then quickly put in ice bath;

[0060] Step 2

[0061]

[0062] After mixing, reverse transcription was performed under the following conditions: 42°C, 30 min; 50°C, 15 min; 70°C, 15 min.

[0063] 3. VHH gene amplification (nested PCR)

[0064] The VHH gene was amplified using nested PCR as follows:

[0065] Step 1

[0066]

[0067] Reaction conditions:

[0068]

[0069] The PCR product was purified and concentrated using a DNA purification kit, and then a ~750 bp band was recovered using a DNA product gel recovery kit and quantified using an UV spectrophotometer;

[0070] Step 2

[0071]

[0072] Reaction conditions:

[0073]

[0074] The PCR products were recovered using a DNA product gel recovery kit and quantified using an ultraviolet spectrophotometer.

[0075] Nested PCR was used to amplify the VHH gene, and the target gene VHH of about 500 bp was finally obtained. VHH was recovered using a PCR product gel recovery kit.

[0076] (6) Library transformation:

[0077] The target gene VHH and vector pHEN1 were double-digested with SfiI and Not1. Figure 2 As shown in Figure 1, the digested VHH and pHEN1 were ligated using T4 DNA ligase and transformed into TG1 electroporation competent cells to construct a VHH gene library. A total of 15 transformations were performed, and the mixture was evenly spread on six 150 mm Ø culture dishes. At the same time, 0.1 μL, 0.01 μL, 0.001 μL, and 0.0001 μL of the mixed transformation solution were each evenly spread on a 90 mm Ø culture dish for library capacity calculation. As shown in Table 1, the calculated library capacity was 1.395 × 10 8 cfu.

[0078] Table 1 Library capacity calculation (based on the plate with 30-300 colonies)

[0079]

[0080] 48 colonies were randomly selected from the culture dish used to calculate the library capacity, and colony PCR was performed to measure the target gene insertion rate of the library. The library insertion rate was 100%, and the actual library capacity was 1.395×10 8cfu. Another 36 colonies were randomly selected for amplification and sequencing, sequence diversity was analyzed, DNA sequences were translated into amino acid sequences, sequence alignment and functional partitioning were performed, and the results were as follows: Figure 3 shown.

[0081] (VII) Library rescue:

[0082] 10-100 times the library volume of living cells were taken from the above gene library for inoculation and culture. After culture to the logarithmic phase, M13K07 phage was used for rescue. After rescue culture, phages were collected by centrifugation and purified with PEG-NaCl to obtain a phage display library with a titer of 3.4×10 13 cfu / mL. Can be directly used for subsequent affinity screening of specific phages.

[0083] (VIII) Screening of specific binding molecules:

[0084] Avidin magnetic beads were used for screening. The expressed and purified HBcAg was labeled with biotin, mixed with the labeled antigen and the phage library, and then the avidin magnetic beads were added. Since the selected phage were amplified after the first round of screening, theoretically, phage carrying the correct Flag antibody were enriched. After the second round of screening, more positive results should be obtained than in the first round. The phage eluted from the second round of screening were then amplified, rescued by the addition of helper phage, and then the third round of screening was continued. After three rounds of screening, high-titer phage were obtained.

[0085] ELISA experiments were used to further select different monoclonal clones from the phage obtained by magnetic bead screening for properties. The antigen was coated on an enzyme-linked plate and incubated with a nanobody solution to allow it to bind to the antigen. Subsequently, a mouse anti-M13 p III monoclonal antibody was used to bind to the VHH-p III fusion protein in the nanobody, and a goat anti-mouse antibody labeled with alkaline phosphatase (AP) was used as a secondary antibody, and pNPP was used as a substrate for color development.

[0086] (IX) Sequencing the screened binding molecules: The bacterial solution corresponding to the wells with more obvious color reaction in the ELISA experiment was sent for testing. The Sanger sequencing service was provided by Guangzhou Aiji Biotechnology Co., Ltd., and the sequences of the binding molecules were measured as SEQ ID NO: 4 to SEQ ID NO: 10.

[0087] Among them, the amino acid sequences of the three CDR regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0088] Test Example 1

[0089] The seven binding molecules obtained above (including the sequences SEQ ID NO: 4 to SEQ ID NO: 10, respectively) were constructed in the pET25b prokaryotic expression plasmid, and then transformed into B21 competent bacteria. The grown single clones were cultured at 37°C and 200 rpm overnight (about 12 to 20 hours). The activated bacterial solution was inoculated into a 1L triangular flask, and 400 μL of the corresponding resistance was added according to 400 mL / bottle LB medium and a 1:1000 inoculation ratio, i.e. 400 μL. The culture was cultured at 37°C and 200 rpm for 3 to 6 hours. When the OD600 reached 0.6 to 1, IPTG induction was performed, and 1M IPTG was added at a ratio of 1:5000 to obtain a final concentration of 0.2 μM.

[0090] The cells were expressed at 18°C ​​and 200 rpm for 16 to 24 hours. After expression, the cells were collected and disrupted by ultrasound. The supernatant after centrifugation was then subjected to affinity chromatography on a Ni medium and eluted with 100 to 500 mM imidazole. The purified antibodies were subjected to SDS-PAGE electrophoresis and then stained with Coomassie Brilliant Blue. Figure 4 As shown, numbers 1 to 7 represent 7 binding molecules (including sequences SEQ ID NO: 4 to SEQ ID NO: 10, respectively).

[0091] Depend on Figure 4 It can be seen that the seven binding molecules obtained were all successfully expressed and purified, with molecular weights ranging from 15 to 20 kD and purities greater than 90%.

[0092] Test Example 2

[0093] Detect the reactivity of the purified HBcAg binding molecules with HBcAg protein, specifically:

[0094] HBcAg protein was coated in a 96-well plate and then reacted with different concentrations of HBcAg antibody at 37°C for 60 minutes. After washing the plate three times, Anti-VHH-HRP secondary antibody was added and reacted at 37°C for 30 minutes. After washing the plate three times, luminescent solution was added and the chemiluminescence intensity was detected. The results are as follows: Figure 5 As shown, numbers 1# to 7# represent 7 binding molecules (including sequences SEQ ID NO: 4 to SEQ ID NO: 10, respectively).

[0095] Depend on Figure 5 It can be seen that except for the 6# binding molecule (including the sequence SEQ ID NO: 9), the other 6 binding molecules have high affinity reaction activity with HBcAg.

[0096] Test Example 3

[0097] The anti-HBcAg binding molecule (SEQ ID NO: 4) obtained by screening in Example 1 was injected into the tail vein of HBV transgenic mice to form an anti-HBcAg group (Anti-HBcAg). Mice injected with anti-GFP nanobody were used as a control group (Control). The serum HBsAg and HBeAg levels of mice and the levels of HBcAg and HBV replication in the liver were measured. The results were as follows: Figures 6-9 shown.

[0098] Depend on Figure 6 It can be seen that compared with the control group, the anti-HBcAg binding molecule treatment group significantly reduced the serum HBsAg level of mice; Figure 7 It can be seen that compared with the control group, the anti-HBcAg binding molecule treatment group significantly reduced the serum HBeAg level of mice; Figure 8 It can be seen that compared with the control group, the anti-HBcAg binding molecule treatment group significantly reduced the HBV DNA level in the serum of HBV transgenic mice; Figure 9 It can be seen that compared with the control group, the anti-HBcAg binding molecule treatment group significantly reduced the HBcAg level in the mouse liver.

[0099] The above is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with this technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A nanobody against hepatitis B virus core antigen HBcAg, characterized in that: The nanobody comprises at least one immunoglobulin single variable domain, and the amino acid sequences of CDR1, CDR2 and CDR3 in the immunoglobulin single variable domain are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

2. The Nanobody according to claim 1, wherein The Nanobody comprises an amino acid sequence that is at least 80% identical to any one of the amino acid sequences of SEQ ID NO: 4 to SEQ ID NO:

10.

3. The Nanobody according to claim 2, wherein The Nanobody comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NO: 4 to SEQ ID NO:

10.

4. The Nanobody according to claim 3, wherein The Nanobody comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NO: 4 to SEQ ID NO:

10.

5. The Nanobody according to claim 4, wherein The amino acid sequence of the nanobody is any one of SEQ ID NO: 4 to SEQ ID NO:

10.

6. A fusion protein, characterized in that Composed of the Nanobody according to any one of claims 1 to 5 and an Fc fragment.

7. Use of the nanobody against hepatitis B virus core antigen HBcAg according to claim 1 in the preparation of a diagnostic and / or therapeutic product for hepatitis B virus.

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