Epitope peptides and antibodies for the treatment of HBV infection and related diseases

Abstract

The present invention relates to the field of molecular virology and immunology, in particular to the field of treating Hepatitis B virus (HBV) infection. In particular, the present invention relates to an epitope peptide (or a variant thereof) capable of treating Hepatitis B virus infection, a recombinant protein comprising the epitope peptide (or a variant thereof) and a carrier protein, and an antibody (e.g., nanobody) against the epitope peptide. The epitope peptide and antibody of the present invention can be used to prevent and / or treat HBV infection or a disease associated with HBV infection (e.g., Hepatitis B), to reduce serum levels of HBV DNA and / or HBsAg in a subject (e.g., a human), or to activate a subject (e.g., a chronic HBV-infected person or a chronic Hepatitis B patient) against HBV humoral immune response.

Description

[Technical field]

[0001] The present invention relates to the field of molecular virology and immunology, in particular to the field of treatment of Hepatitis B virus (HBV) infection. In particular, the present invention relates to an epitope peptide (or a variant thereof) that can be used for the treatment of Hepatitis B virus infection, a recombinant protein comprising such an epitope peptide (or a variant thereof) and a carrier protein, and an antibody (e.g., nanobody) targeting such an epitope peptide. The epitope peptide and antibody of the present invention can be used to prevent and / or treat HBV infection or a disease associated with HBV infection (e.g., Hepatitis B), neutralize the toxicity of HBV in a subject (e.g., a human), reduce serum levels of HBV DNA and / or HBsAg in a subject, or activate a humoral immune response against HBV in a subject (e.g., a chronic HBV-infected person or a chronic Hepatitis B patient). [Background technology]

[0002] HBV is a DNA virus that can cause a range of liver diseases of varying severity, and chronic hepatitis B (CHB) infection carries a significant risk of developing severe liver disease, including liver cirrhosis (LC) and primary hepatocellular carcinoma (HCC). HBV represents a significant public health burden worldwide.

[0003] The ideal therapeutic endpoint for CHB patients is to achieve HBsAg serological negativity (HBsAg disappearance). However, due to the high HBsAg titers in patients, immune tolerance occurs and it is difficult to achieve the desired endpoint with existing drug (interferon or nucleoside / nucleotide analogue) based therapies. Therefore, the development of innovative therapeutic agents and treatments for patients with chronic HBV infection is urgent and necessary.

[0004] The development of new drugs for the treatment of chronic HBV infection based on immunological methods is one of the important research directions in this field, which may include active immunotherapy (corresponding drug forms including vaccines, etc.) and passive immunotherapy (corresponding drug forms including antibodies, etc.). Active immunotherapy refers to stimulating the body of a patient with chronic HBV infection by administering a therapeutic vaccine (including protein vaccines, polypeptide vaccines, nucleic acid vaccines, etc.) to actively generate a cellular immune response (such as CTL effect) and / or a humoral immune response (such as antibodies) against HBV, thereby achieving the purpose of inhibiting or eliminating HBV. Passive immunotherapy (taking antibodies as an example) refers to passively administering an antibody with therapeutic properties to a patient with HBV infection, using antibody-mediated virus neutralization to prevent new HBV infection of liver cells, or using antibody-mediated immune clearance to remove the virus and infected liver cells from the body, thereby achieving a therapeutic effect. Summary of the Invention [Problem to be solved by the invention]

[0005] However, it is known in the art that patients with chronic hepatitis B often develop immune exhaustion (tolerance) to HBV due to high levels of HBsAg in the body, leading to prolonged infection. In order to functionally cure CHB, it is important to remove serum HBsAg and restore or activate HBV-specific immunity. [Means for solving the problem]

[0006] Although there are multiple B cell response (antibody response) epitopes on various proteins of the HBV virus, not all antibodies against any epitope can be used to treat HBV infection (especially chronic HBV infection). Therefore, in order to develop an immunotherapeutic drug / method that can effectively treat HBV infection, it is important to identify a target (epitope) that can induce an antibody response that can effectively eliminate the virus and virus-infected cells in the body, and obtain an antibody against that target (epitope). The present invention identifies such a target (epitope) and provides an antibody, particularly a nanobody, against such an epitope peptide / epitope. Nanobodies have the advantages of both monoclonal antibodies and small molecule drugs. Nanobodies can pass the blood-brain barrier, can be easily designed, developed and stably produced, and are less difficult and costly to manufacture. It is expected that immunotherapy via the epitope peptide or nanobody of the present invention will be a new strategy for CHB patients to fight persistent infection with the HBV virus.

[0007] Epitope peptide In one aspect, the present invention provides an isolated epitope peptide or variant thereof, wherein the epitope peptide or variant thereof comprises an epitope located within amino acid residues 157 to 174 of HBsAg protein, the epitope comprising at least amino acid residues 163 to 165 of HBsAg protein, and wherein the variant differs from the epitope peptide from which it is derived by mutation (e.g., substitution, addition or deletion) of one or a few (e.g., 1, 2, 3, 4 or 5) amino acid residues, and retains the biological function of the epitope peptide from which it is derived.

[0008] The biological functions of the epitope peptides of the present invention include, but are not limited to, one or more selected from the following: 1) specifically binds to a nanobody (e.g., 125s) comprising CDR1 to CDR3 set forth in SEQ ID NO:1 to SEQ ID NO:3; 2) reducing serum levels of HBV DNA and / or HBsAg in a subject (optionally after fusing the epitope peptide to a carrier protein); 3) inducing an antibody response capable of effectively eliminating HBV and HBV-infected cells in a subject and / or inducing an immune response (e.g., a humoral immune response) against HBV (optionally after fusing the epitope peptide to a carrier protein); and 4) Preventing and / or treating HBV infection or a disease associated with HBV infection (e.g., hepatitis B) in a subject.

[0009] In certain embodiments, the subject in any one of 2) to 4) may be an HBV-uninfected subject or an HBV-infected subject, such as a subject with chronic HBV infection. In certain embodiments, the epitope peptide of the present invention has a biological function as described in any one of 1) to 4) after being fused to a carrier protein.

[0010] In certain embodiments, the epitope located within amino acid residues 157 to 174 of the HBsAg protein includes at least amino acid residues 163 to 165, as well as amino acid residues 158, 160, and 171 of the HBsAg protein. In certain embodiments, the epitope further includes amino acid residue 157.

[0011] In certain embodiments, the epitope located within amino acid residues 157 to 174 of the HBsAg protein includes at least amino acid residues 163 to 166 of the HBsAg protein (eg, as set forth in SEQ ID NO: 37).

[0012] In certain embodiments, the epitope located within amino acid residues 157 to 174 of the HBsAg protein includes at least amino acid residues 163 to 166 of the HBsAg protein (e.g., as set forth in SEQ ID NO: 37), as well as amino acid residues 158, 160, and 171. In certain embodiments, the epitope further includes amino acid residue 157.

[0013] In certain embodiments, the epitope located within amino acid residues 157 to 174 of the HBsAg protein is a conformational epitope consisting of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 non-consecutive amino acid residues within amino acid residues 157 to 174 of the HBsAg protein and including at least amino acid residues 163 to 165 of the HBsAg protein.

[0014] In certain embodiments, the conformational epitope comprises at least amino acid residues 163 to 165, as well as amino acid residues 158, 160, and 171 of the HBsAg protein. In certain embodiments, the conformational epitope further comprises amino acid residue 157.

[0015] In certain embodiments, the conformational epitope comprises at least amino acid residues 163 to 166 of the HBsAg protein (eg, as set forth in SEQ ID NO:37).

[0016] In certain embodiments, the conformational epitope includes at least amino acid residues 163 to 166 (e.g., as set forth in SEQ ID NO: 37) of the HBsAg protein, as well as amino acid residues 158, 160, and 171. In certain embodiments, the conformational epitope further includes amino acid residue 157.

[0017] In certain embodiments, the epitope peptide consists of at least 5 (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20) and / or at most 80 (e.g., at most 75, at most 70, at most 65, at most 60, at most 55, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20) consecutive amino acid residues and comprises an epitope.

[0018] In certain embodiments, the epitope peptides are 5 to 80 (e.g., 5 to 75, 5 to 70, 5 to 65, 5 to 60, 5 to 55, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 74, 75, or 80 consecutive amino acid residues and contains an epitope.

[0019] In certain embodiments, the epitope peptide comprises at least amino acid residues 157 to 174 of the HBsAg protein. In certain embodiments, amino acid residues 157 to 174 of the HBsAg protein are as set forth in any one of SEQ ID NO: 19, SEQ ID NO: 38 to SEQ ID NO: 44.

[0020] In certain embodiments, the variant does not contain a mutation at a position corresponding to amino acid position 163 to amino acid position 165 of the HBsAg protein.

[0021] In certain embodiments, the variant does not contain a mutation at a position corresponding to amino acid position 163 to amino acid position 166 of the HBsAg protein.

[0022] In certain embodiments, the variant does not contain a mutation at a position corresponding to amino acid position 162 to amino acid position 167 of the HBsAg protein.

[0023] In certain embodiments, the variant does not further comprise a mutation at a position corresponding to amino acid position 157 of the HBsAg protein.

[0024] In certain embodiments, the variant does not further comprise a mutation at a position corresponding to amino acid position 171 of the HBsAg protein.

[0025] In certain embodiments, the variant does not further comprise a mutation at one or more (eg, one, two or three) positions corresponding to amino acid position 158, amino acid position 160 of the HBsAg protein.

[0026] In certain embodiments, the variant comprises a mutation at one or more (e.g., one, two, three, four or five) positions corresponding to amino acid position 161, amino acid position 162, amino acid position 167, amino acid position 168, amino acid position 169, amino acid position 170, amino acid position 172, amino acid position 173, amino acid position 174 of the HBsAg protein.

[0027] In certain embodiments, the mutations are amino acid substitutions, such as conservative substitutions.

[0028] In certain embodiments, the amino acid substitution comprises substituting the amino acid at that position with alanine.

[0029] In certain embodiments, the above amino acid positions refer to positions in the sequence set forth in SEQ ID NO: 36. It is well known to those skilled in the art that in order to obtain positions in the variants corresponding to the above amino acid positions, the amino acid positions can be determined by optimal alignment of the sequences (i.e., when the sequences are aligned so as to obtain the highest percentage identity).

[0030] In certain embodiments, the epitope peptide or variant thereof has at least the structure: AX1X2X3X4X5WEWAX6X7X8X9SX 10 X 11 X 12 (SEQ ID NO:51), wherein X1 (corresponding to amino acid position 158 of the HBsAg protein) is F or L; X2 (corresponding to amino acid position 159 of the HBsAg protein) is A or G; X3 (corresponding to amino acid position 160 of the HBsAg protein) is K or R; X4 (corresponding to amino acid position 161 of the HBsAg protein) is any naturally occurring amino acid (e.g., Y, F, or A); X5 (corresponding to amino acid position 162 of the HBsAg protein) is any naturally occurring amino acid (e.g., L or A); X6 (corresponding to amino acid position 167 of the HBsAg protein) is any naturally occurring amino acid (e.g., S or A); X7 (corresponding to amino acid position 168 of the HBsAg protein) is any naturally occurring amino acid (e.g., V or A); X8 (corresponding to amino acid position 169 of the HBsAg protein) is any naturally occurring amino acid (e.g., R, H, or A); X9 (corresponding to amino acid position 170 of the HBsAg protein) is any naturally occurring amino acid (e.g., F or A); X 10 (corresponding to amino acid position 172 of the HBsAg protein) is any naturally occurring amino acid (e.g., W or A); X 11(corresponding to amino acid position 173 of the HBsAg protein) is any naturally occurring amino acid (e.g., L or A); X 12 (corresponding to amino acid position 174 of the HBsAg protein) is any naturally occurring amino acid (e.g., S or A);

[0031] In certain embodiments, X5 is L, X6 is S, and X1-X4 and X7-X 12 is as defined above.

[0032] In certain embodiments, X5 is L, X6 is S, X9 is F, and X 10 is W and X 11 is L and X 12 is S, and X1 to X4 and X7 to X8 are as defined above.

[0033] In certain embodiments, the epitope peptide or a variant thereof comprises at least an amino acid sequence selected from the sequences set forth in any one of SEQ ID NO:19, and SEQ ID NO:38 to SEQ ID NO:44.

[0034] In certain embodiments, the HBsAg protein comprises the sequence set forth in SEQ ID NO: 36, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity thereto.

[0035] In certain embodiments, the epitope peptide or a variant thereof comprises or consists of a sequence set forth in any one of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 to SEQ ID NO: 35, and SEQ ID NO: 38 to SEQ ID NO: 44.

[0036] Recombinant Proteins The epitope peptides of the present invention or variants thereof can be fused to carrier proteins to enhance the immunogenicity of the epitope peptides or variants thereof and / or to maintain the conformational epitopes contained in the epitope peptides or variants thereof, thereby allowing them to be recognized by the body's immune system and induce an immune response in the body.

[0037] Therefore, in another aspect, the present invention provides a recombinant protein comprising the above-mentioned isolated epitope peptide or variant thereof and a carrier protein, wherein the recombinant protein is not a naturally occurring protein or a fragment thereof. In the recombinant protein, the epitope peptide or variant thereof can be linked to the N-terminus or C-terminus of the carrier protein, inserted inside the carrier protein, or replace a part of the amino acid sequence of the carrier protein, depending on the specific carrier protein used. In addition, optionally, the epitope peptide or variant thereof is linked to the carrier protein via a linker (e.g., a rigid linker or a flexible linker, such as a peptide linker containing one or more glycines and / or one or more serines). The recombinant protein of the present invention is not limited by its production method. For example, the recombinant protein can be produced by genetic engineering (recombinant technology) or chemical synthesis.

[0038] In certain embodiments, the biological functions of the recombinant protein include one or more selected from, but are not limited to: 1) specifically binding to a nanobody (e.g., 125s) comprising CDR1-CDR3 as set forth in SEQ ID NO:1-SEQ ID NO:3; 2) reducing serum levels of HBV DNA and / or HBsAg in a subject; 3) inducing an antibody response capable of effectively eliminating HBV and HBV-infected cells in a subject and / or inducing an immune response (e.g., a humoral immune response) against HBV; and 4) preventing and / or treating HBV infection or a disease associated with HBV infection (e.g., hepatitis B) in a subject.

[0039] In certain embodiments, the carrier protein is selected from an immunoglobulin Fc domain, such as an IgG Fc domain (eg, an IgG1, IgG2, IgG3, or IgG4 Fc domain).

[0040] In certain embodiments, the epitope peptide or variant thereof is linked to the N-terminus or C-terminus (e.g., the N-terminus) of the immunoglobulin Fc domain, optionally via a linker (e.g., a rigid or flexible linker, such as a peptide linker comprising one or more glycines and / or one or more serines). In certain embodiments, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO:14.

[0041] In certain embodiments, the carrier protein is selected from proteins capable of self-assembling to form nanoparticles, thereby enabling the recombinant protein to form antigen nanoparticles. Antigen nanoparticles are collections of polypeptides presenting multiple copies of an antigen (e.g., an epitope peptide of the invention or a variant thereof), which may result in multiple binding sites (affinity), leading to improved antigen stability and immunogenicity. The creation of self-assembled protein nanoparticles and their use in vaccines are well known to those skilled in the art. As non-limiting examples, self-assembled nanoparticles can be based on ferritin, encapsulin, or lumazine synthase (LS).

[0042] In certain embodiments, the protein capable of self-assembling to form nanoparticles is ferritin, such as mouse ferritin.

[0043] In certain embodiments, the epitope peptide or variant thereof is linked via a peptide bond or an isopeptide bond to the N-terminus or C-terminus (e.g., the N-terminus) of a protein capable of self-assembling to form a nanoparticle.

[0044] In certain embodiments, the epitope peptide or variant thereof is linked via an isopeptide bond to the N-terminus or C-terminus (eg, the N-terminus) of a protein capable of self-assembling to form a nanoparticle.

[0045] In certain embodiments, the isopeptide bond is formed by a protein-protein bond pair.

[0046] In certain embodiments, the protein-protein binding pair consists of a first member (e.g., a first peptide tag) and a second member (e.g., a second peptide tag), where the first member and the second member are linked via an isopeptide bond, and the first member is linked to the N-terminus or C-terminus of an epitope peptide or a variant thereof, optionally via a first linker (e.g., a rigid linker or a flexible linker, such as a peptide linker comprising one or more glycines and / or one or more serines) to form a first protein, and the second member is linked to the N-terminus or C-terminus (e.g., the N-terminus) of a protein capable of self-assembling to form a nanoparticle, optionally via a second linker (e.g., a rigid linker or a flexible linker, such as a peptide linker comprising one or more glycines and / or one or more serine peptide linkers) to form a second protein.

[0047] In certain embodiments, the first protein and / or the second protein may further comprise a protein tag (eg, at its N-terminus or C-terminus).

[0048] In certain embodiments, the protein-protein binding pair is selected from: (i) a SpyTag:SpyCatcher pair; (ii) a SpyTag:KTag pair; (iii) a Isopeptag:pilin-C pair; (iv) a SnoopTag or SnoopTagJr:SnoopCatcher pair; (v) a SpyTag002:SpyCatcher002 pair; (vi) a RrgATag, RrgATag2 or DogTag:RrgACatcher pair; (vii) a IsopepTag N:Pilin N pair; (viii) a PsCsTag:PsCsCatcher pair.

[0049] In certain embodiments, the protein-protein binding pair is a SpyTag:SpyCatcher pair, where, for example, SpyTag comprises the sequence set forth in SEQ ID NO:46 and, for example, SpyCatcher comprises the sequence set forth in SEQ ID NO:47 or 48.

[0050] In certain embodiments, the recombinant protein of the invention is formed by linking a first protein and a second protein via an isopeptide bond, the first protein comprises an epitope peptide or variant thereof and a first member of a SpyTag:SpyCatcher pair, the first member being linked to the epitope peptide or variant thereof (e.g., linked to the N-terminus or C-terminus of the epitope peptide or variant thereof) optionally via a first linker; The second protein comprises a protein capable of self-assembling to form a nanoparticle and a second member of a SpyTag:SpyCatcher pair, the second member being linked to the protein capable of self-assembling to form a nanoparticle (e.g., linked to the N-terminus of the protein capable of self-assembling to form a nanoparticle) optionally via a second linker.

[0051] In certain embodiments, the first protein comprises an epitope peptide or a variant thereof and SpyCatcher or an active fragment thereof (e.g., an N-terminal truncation), and the second protein comprises a protein capable of self-assembling to form nanoparticles and a SpyTag.

[0052] In certain embodiments, the first protein comprises the sequence set forth in SEQ ID NO:49 and the second protein comprises the sequence set forth in SEQ ID NO:50.

[0053] Production of epitope peptides and recombinant proteins In another aspect, the present invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the epitopic peptide of the present invention or a variant thereof, or the recombinant protein of the present invention.

[0054] In certain embodiments, the isolated nucleic acid molecule encodes an epitopic peptide of the invention or a variant thereof.

[0055] In certain embodiments, the isolated nucleic acid molecule encodes a recombinant protein of the present invention.

[0056] In certain embodiments, the recombinant protein encoded by the isolated nucleic acid molecule is formed by linking a first protein and a second protein via an isopeptide bond, where the first protein comprises an epitope peptide or a variant thereof and a first member of a protein-protein binding pair, and the second protein comprises a protein capable of self-assembly to form a nanoparticle and a second member of the protein-protein binding pair. In such embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first protein and a second nucleotide sequence encoding the second protein, where the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules.

[0057] In another aspect, the present invention also provides a vector comprising the isolated nucleic acid molecule described above. The vector of the present invention can be a cloning vector or an expression vector. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a cosmid, etc. In certain embodiments, the vector is capable of expressing the epitope peptide of the present invention or a variant thereof, or the recombinant protein of the present invention in a subject (e.g., a mammal such as a human).

[0058] In certain embodiments, the vector encodes an epitopic peptide of the invention or a variant thereof.

[0059] In certain embodiments, the vector encodes a recombinant protein of the invention.

[0060] In certain embodiments, the recombinant protein encoded by the vector is formed by linking a first protein and a second protein via an isopeptide bond, where the first protein comprises an epitope peptide or a variant thereof and a first member of a protein-protein binding pair, and the second protein comprises a protein capable of self-assembly to form a nanoparticle and a second member of the protein-protein binding pair. In such embodiments, the vector comprises a first nucleotide sequence encoding the first protein and a second nucleotide sequence encoding the second protein, where the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors.

[0061] In another aspect, the present invention further provides a host cell comprising the isolated nucleic acid molecule or vector of the present invention. Such host cells include, but are not limited to, prokaryotic cells, such as bacterial cells (e.g., E. coli cells), and eukaryotic cells, such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). The cells of the present invention may be cell lines, such as HEK293 cells. In certain embodiments, the host cell is a microorganism, such as a bacterial cell or a fungal cell.

[0062] In another aspect, the present invention further provides a method for producing the epitope peptide or variant thereof, or recombinant protein of the present invention, comprising culturing a host cell of the present invention under suitable conditions and recovering the epitope peptide or variant thereof, or recombinant protein of the present invention from the cell culture.

[0063] Multimers In another aspect, the present invention provides a multimer comprising a plurality of monomers, each monomer being independently selected from a recombinant protein of the present invention.

[0064] In certain embodiments, the multimer is a dimer, trimer, or tetramer.

[0065] In certain embodiments, the monomers are identical to each other.

[0066] In certain embodiments, the monomers are identical to each other and are selected from recombinant proteins comprising an epitopic peptide of the invention or a variant thereof and an immunoglobulin Fc domain as a carrier protein.

[0067] In certain embodiments, the biological functions of the multimer include one or more selected from, but are not limited to, 1) specifically binding to a nanobody (e.g., 125s) comprising CDR1-CDR3 as set forth in SEQ ID NO:1-SEQ ID NO:3; 2) reducing serum levels of HBV DNA and / or HBsAg in a subject; 3) inducing an antibody response capable of effectively eliminating HBV and HBV-infected cells in a subject, and / or inducing an immune response (e.g., a humoral immune response) against HBV; and 4) preventing and / or treating HBV infection or a disease associated with HBV infection (e.g., hepatitis B) in a subject.

[0068] particle In another aspect, the present invention provides particles displaying the isolated epitope peptide of the present invention or a variant thereof on the surface thereof.

[0069] In certain embodiments, the particle is a nanoparticle, a virus-like particle (VLP) or a core-like particle (CLP).

[0070] In certain embodiments, the particles comprise or consist of a recombinant protein or multimer of the invention.

[0071] In certain embodiments, the particle is a nanoparticle formed by a recombinant protein comprising an epitopic peptide of the invention or a variant thereof and a protein capable of self-assembling to form nanoparticles as a carrier protein.

[0072] In certain embodiments, the biological functions of the particles include one or more selected from, but are not limited to, 1) specifically binding to a nanobody (e.g., 125s) comprising CDR1 to CDR3 set forth in SEQ ID NO:1 to SEQ ID NO:3; 2) reducing serum levels of HBV DNA and / or HBsAg in a subject; 3) inducing an antibody response capable of effectively eliminating HBV and HBV-infected cells in a subject, and / or inducing an immune response (e.g., a humoral immune response) against HBV; and 4) preventing and / or treating HBV infection or a disease associated with HBV infection (e.g., hepatitis B) in a subject.

[0073] Use of epitope peptides In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned epitopic peptide or a variant thereof, recombinant protein, isolated nucleic acid molecule, vector, host cell, multimer, or particle.

[0074] In certain embodiments, the pharmaceutical composition is a vaccine.

[0075] In certain embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier and / or excipient (e.g., an adjuvant). In certain embodiments, the pharma- ceutically acceptable carrier and / or excipient comprises an adjuvant. The adjuvant co-administered or included in the pharmaceutical composition of the present invention is preferably a potentially safe, well-tolerated and effective adjuvant in the human body. Such adjuvants are well known to those skilled in the art.

[0076] In certain embodiments, the pharmaceutical composition is a protein vaccine comprising the above epitope peptide or variants thereof, recombinant protein, multimer, or particle. In certain embodiments, the protein vaccine comprises one or more of the epitope peptides or variants thereof of the present invention, which may be separate or in tandem, modified or unmodified, and may or may not be bound to other proteins.

[0077] In certain embodiments, the pharmaceutical composition is a nucleic acid vaccine comprising the isolated nucleic acid molecule or vector described above. In certain embodiments, the nucleic acid vaccine comprises DNA or RNA. In such embodiments, the DNA or RNA may be naked or may be encapsulated in a shell that has a delivery and / or protection function. In some embodiments, the shell may be that of an adenovirus, an adeno-associated virus, a lentivirus, a retrovirus, etc., or other materials that can be synthesized by chemical methods and perform similar functions.

[0078] The epitope peptide or variant thereof, recombinant protein, multimer, particle, or pharmaceutical composition can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, liquids, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form varies depending on the intended mode of administration and therapeutic application. The epitope peptide or variant thereof, recombinant protein, multimer, particle, or pharmaceutical composition is desirably sterile and stable under the conditions of manufacture and storage. One of the preferred dosage forms is an injection. Such an injection may be a sterile injection solution. In addition, the sterile injection solution may be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) for ease of storage and use. Such sterile, lyophilized powder can be dispersed in a suitable carrier, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., 0.01% polysorbate 20), a pH buffer (e.g., phosphate buffer), Ringer's solution, and any combination thereof, before use.

[0079] The epitope peptide or its variant, recombinant protein, multimer, particle, or pharmaceutical composition can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal. However, for many therapeutic applications, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or mode of administration will vary depending on the intended purpose.

[0080] The epitope peptide or its variant, recombinant protein, multimer, particle, or pharmaceutical composition is preferably administered in an effective amount. The appropriate amount can be determined according to the specific disease or condition to be treated or prevented, its severity, the age of the subject, and other personal characteristics of the specific subject (e.g., the overall health status of the subject and the status of the subject's immune system). The determination of the effective amount is also guided by studies in animal models followed by human clinical trials, and by administration regimens that significantly reduce the occurrence or severity of symptoms or conditions of the target disease in the subject.

[0081] In another aspect, the present invention provides the use of the above-mentioned epitope peptide or variant thereof, recombinant protein, isolated nucleic acid molecule, vector, host cell, multimer, particle or pharmaceutical composition in the manufacture of a preparation, the preparation being used to reduce serum levels of HBV DNA and / or HBsAg in a subject (e.g., a human), to induce an immune response (e.g., a humoral immune response) against HBV in a subject (e.g., a human), and / or to prevent and / or treat HBV infection or a disease associated with HBV infection (e.g., hepatitis B) in a subject (e.g., a human).

[0082] In certain embodiments, the preparation is a vaccine.

[0083] In certain embodiments, the preparation is a protein vaccine. In certain embodiments, the invention provides the use of the above epitope peptides or variants, recombinant proteins, multimers, or particles for the manufacture of a protein vaccine, which is used to reduce serum levels of HBV DNA and / or HBsAg in a subject (e.g., a human), to induce an immune response (e.g., a humoral immune response) against HBV in a subject (e.g., a human), and / or to prevent and / or treat HBV infection or a disease associated with HBV infection (e.g., Hepatitis B) in a subject (e.g., a human).

[0084] In certain embodiments, the preparation is a nucleic acid vaccine. In certain embodiments, the invention provides for the use of a nucleic acid molecule or vector as described above for the manufacture of a nucleic acid vaccine, which is used to reduce serum levels of HBV DNA and / or HBsAg in a subject (e.g., a human), to induce an immune response (e.g., a humoral immune response) against HBV in a subject (e.g., a human), and / or to prevent and / or treat HBV infection or a disease associated with HBV infection (e.g., Hepatitis B) in a subject (e.g., a human).

[0085] In certain embodiments, the subject is a non-HBV infected subject. In certain embodiments, the subject is a subject infected with HBV, such as a subject with chronic HBV infection or a subject with chronic hepatitis B.

[0086] In another aspect, the present invention provides a method for reducing serum levels of HBV DNA and / or HBsAg in a subject (e.g., human), inducing an immune response against HBV in a subject (e.g., human), and / or preventing and / or treating HBV infection or a disease associated with HBV infection (e.g., hepatitis B) in a subject (e.g., human), comprising administering to a subject in need thereof an effective amount of the above-mentioned epitopic peptide or variant thereof, recombinant protein, isolated nucleic acid molecule, vector, host cell, multimer, particle, or pharmaceutical composition. In certain embodiments, the subject is an HBV-uninfected subject. In certain embodiments, the subject is an HBV-infected subject, such as a subject with chronic HBV infection or a subject with chronic hepatitis B.

[0087] For prophylactic applications, the epitopic peptides or variants thereof, recombinant proteins, isolated nucleic acid molecules, vectors, host cells, multimers, particles, or pharmaceutical compositions described above may be used before any symptoms appear, e.g., before infection. Prophylactic administration is used after exposure or suspected exposure to the virus, or after the occurrence of actual infection, to prevent or ameliorate any subsequent infection, and to reduce the severity, duration, or extent of symptoms of anticipated infection and / or associated diseases. In some embodiments, the subject being treated may be a subject at risk of suffering from HBV infection, e.g., due to exposure or potential exposure to HBV.

[0088] For therapeutic applications, the epitopic peptide or variant thereof, recombinant protein, isolated nucleic acid molecule, vector, host cell, multimer, particle, or pharmaceutical composition may be administered at or after the onset of a disease or infection symptom, for example, provided after the onset of a symptom of HBV infection or after diagnosis of HBV infection, and in some embodiments, the subject to be treated may be a subject infected with HBV, such as a subject with chronic HBV infection or a chronic hepatitis B patient.

[0089] In certain embodiments, a protein vaccine comprising the above-mentioned epitopic peptide or variants thereof, recombinant protein, multimer, or particle is administered to the subject.

[0090] In certain embodiments, a nucleic acid vaccine comprising the isolated nucleic acid molecule or vector described above is administered to the subject.

[0091] Nanobodies and their derivatives In another aspect, the invention provides a Nanobody or an antigen-binding fragment thereof capable of specifically binding to HBsAg. Nanobodies generally consist of four framework regions (FR) and three complementarity determining regions (CDRs), termed FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and an antigen-binding fragment comprises at least a portion of the Nanobody, which portion is sufficient to confer to the fragment the ability to specifically bind to HBsAg. In some embodiments, the Nanobody of the invention may be truncated at the N-terminus or C-terminus to comprise only a portion of FR1 and / or FR4 or to lack one or two of the framework regions, so long as it substantially retains its antigen-binding ability and specificity.

[0092] In certain embodiments, the Nanobody or an antigen-binding fragment thereof is capable of specifically binding to said epitopic peptide or a variant thereof or an epitope contained therein, recombinant protein, multimer, or particle.

[0093] In certain embodiments, the Nanobody, or antigen-binding fragment thereof, comprises a CDR1 having the sequence set forth in SEQ ID NO: 1 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. one, two or three amino acid substitutions, deletions or additions), a CDR2 having the sequence set forth in SEQ ID NO: 2 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. one, two or three amino acid substitutions, deletions or additions), and a CDR3 having the sequence set forth in SEQ ID NO: 3 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. one, two or three amino acid substitutions, deletions or additions).

[0094] In certain embodiments, the Nanobody or antigen-binding fragment thereof further comprises FR1 having the sequence set forth in SEQ ID NO: 4 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. substitutions, deletions or additions of one, two or three amino acids); FR2 having the sequence set forth in SEQ ID NO: 5 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. substitutions, deletions or additions of one, two or three amino acids); FR3 having the sequence set forth in SEQ ID NO: 6 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. substitutions, deletions or additions of one, two or three amino acids); and FR4 having the sequence set forth in SEQ ID NO: 7 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. substitutions, deletions or additions of one, two or three amino acids).

[0095] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 8, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity thereto, or a sequence having one or several amino acid substitutions, deletions or additions thereto (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto. In certain embodiments, the substitutions are conservative substitutions.

[0096] In certain embodiments, the Nanobody or antigen-binding fragment thereof is humanized, hi certain embodiments, the Nanobody or antigen-binding fragment thereof is a humanized VHH, i.e. a VHH in which one or more framework regions have been substantially replaced with human framework regions.

[0097] In certain embodiments, the Nanobody, or antigen-binding fragment thereof, further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene), where the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations from human residues to camelid residues.

[0098] In certain embodiments, the Nanobody or antigen-binding fragment thereof is selected from the group consisting of IGHV3-23 * 04 FR1 to FR3 derived from germline gene sequence and IGHJ5 * and FR1-FR4 derived from the germline gene sequence, wherein FR1-FR4 optionally contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations from human residues to camelid residues.

[0099] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises FR1 having the sequence set forth in SEQ ID NO: 9 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. one, two or three amino acid substitutions, deletions or additions); FR2 having the sequence set forth in SEQ ID NO: 10 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. one, two or three amino acid substitutions, deletions or additions); FR3 having the sequence set forth in SEQ ID NO: 11 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. one, two or three amino acid substitutions, deletions or additions); and FR4 having the sequence set forth in SEQ ID NO: 12 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. one, two or three amino acid substitutions, deletions or additions).

[0100] In certain embodiments, the Nanobody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 13, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity thereto, or a sequence having one or several amino acid substitutions, deletions or additions thereto (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto. In certain embodiments, the substitutions are conservative substitutions.

[0101] In certain embodiments, a Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention is capable of specifically binding to HBsAg, neutralizing the toxicity of HBV, reducing serum levels of HBV DNA and / or HBsAg in a subject, and / or activating a humoral immune response against HBV in a subject (e.g. a subject with chronic HBV infection or a patient with chronic hepatitis B). In certain embodiments, HBV includes genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H, genotype I, genotype J. HBV genotypes can be found in the NCBI Taxonomy database under accession number txid10407.

[0102] A Nanobody or antigen-binding fragment thereof of the invention may be derivatized, e.g. linked to another molecule (e.g. another polypeptide or protein). In general, derivatization (e.g. labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to HBsAg. Thus, a Nanobody or antigen-binding fragment thereof of the invention is intended to include such derivatized forms. For example, a Nanobody or antigen-binding fragment thereof of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular groups, such as another antibody (e.g. to form a bispecific antibody), an additional functional peptide (e.g. an Fc domain), a detection reagent, a pharmaceutical reagent, and / or a protein or peptide that can mediate binding of the antibody or antigen-binding fragment to another molecule (e.g. an anti-biotin protein or a polyhistidine tag).

[0103] Thus, in another aspect, the present invention further provides a polypeptide construct comprising a Nanobody of the invention, or an antigen-binding fragment thereof, and an additional polypeptide, wherein the polypeptide construct is capable of specifically binding to HBsAg.

[0104] In certain embodiments, the polypeptide construct comprises a Nanobody of the invention, or an antigen-binding fragment thereof, and an immunoglobulin Fc domain.

[0105] In this context, the Fc domain, also referred to as the Fc region, refers to a portion of the heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc domain comprises a hinge, CH2 and CH3. When the Fc domain comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc domain can be any isotype of antibody heavy chain constant region. In some embodiments, the Fc domain is IgG1, IgG2, IgG3 or IgG4.

[0106] In certain embodiments, the Fc domain contained in the polypeptide construct of the invention is a native Fc region that comprises an amino acid sequence that corresponds to the amino acid sequence of an Fc region found in nature. For example, the Fc domain can be a human IgG1 Fc region with native sequence, a human IgG2 Fc region with native sequence, a human IgG3 Fc region with native sequence, or a human IgG4 Fc region with native sequence. The native Fc region can have an effector function. Exemplary "effector functions" include binding to Fc receptors, C1q binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. A functional change can be produced by replacing at least one amino acid residue of the native Fc region with a different residue or by chemical modification, for example, changing the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q), thereby altering (e.g., reducing or enhancing) the effector function.

[0107] Thus, in certain embodiments, the Fc domain comprised in a polypeptide construct of the invention may be a variant Fc region, which may comprise one or more (e.g. 1 to 10, e.g. 1 to 5) amino acid mutations compared to a native Fc region, or may be chemically modified to alter one or more of the following properties of an antibody of the invention: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function or complement function.

[0108] In certain embodiments, the immunoglobulin Fc domain is linked to the N-terminus and / or C-terminus of the Nanobody or antigen-binding fragment thereof, optionally via a peptide linker. In certain embodiments, the immunoglobulin Fc domain is linked to the C-terminus of the Nanobody or antigen-binding fragment thereof, optionally via a peptide linker.

[0109] In certain embodiments, the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1, IgG2, IgG3 or IgG4 Fc domain).

[0110] In certain embodiments, the immunoglobulin Fc domain is a human immunoglobulin Fc domain, such as a human IgG Fc domain (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc domain). In certain embodiments, the immunoglobulin Fc domain comprises a sequence as set forth in SEQ ID NO: 14, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity thereto, or a sequence having one or several amino acid substitutions, deletions or additions thereto (e.g., one, two, three, four or five amino acid substitutions, deletions or additions thereto). In certain embodiments, the substitutions are conservative substitutions.

[0111] In certain embodiments, the immunoglobulin Fc domain is a mouse immunoglobulin Fc domain, such as a mouse IgG Fc domain (e.g., a mouse IgG1, IgG2, IgG3 or IgG4 Fc domain). In certain embodiments, the immunoglobulin Fc domain comprises a sequence as set forth in SEQ ID NO: 15, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity thereto, or a sequence having one or several amino acid substitutions, deletions or additions thereto (e.g., one, two, three, four or five amino acid substitutions, deletions or additions thereto). In certain embodiments, the substitutions are conservative substitutions.

[0112] In another aspect, the present invention also provides a conjugate comprising a Nanobody or an antigen-binding fragment thereof, or a polypeptide construct of the invention and a conjugation moiety.

[0113] In certain embodiments, the conjugated moiety is a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material), or biotin. The detectable label of the present invention can be any material that is detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are well known in the art and examples include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 Examples of suitable labels include, but are not limited to, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750), luminescent materials (e.g., chemiluminescent materials such as acridinium esters), calorimetric labels such as magnetic beads (e.g., Dynabeads®), colloidal gold, or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels. In certain embodiments, such labels are suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, said detectable labels can be linked to the Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention via linkers of different lengths to reduce potential steric hindrance.

[0114] In certain embodiments, the conjugated moiety is a therapeutic agent.

[0115] Production of antibodies and their derivatives The antibody or polypeptide construct of the present invention can be produced by various methods known in the art, such as recombinant genetic engineering techniques. For example, the DNA molecule encoding the antibody or polypeptide construct of the present invention can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cell is then cultured under specific conditions to express the antibody or polypeptide construct of the present invention.

[0116] Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a Nanobody or antigen-binding fragment thereof, or a polypeptide construct of the invention. In certain embodiments, the isolated nucleic acid molecule encodes a Nanobody or antigen-binding fragment thereof, or a polypeptide construct of the invention.

[0117] In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule described above. In certain embodiments, the vector of the invention is, for example, a plasmid, cosmid, phage, etc.

[0118] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule or vector described above. Such host cells include, but are not limited to, prokaryotic cells, such as bacterial cells (e.g., E. coli cells), and eukaryotic cells, such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell of the present invention is a mammalian cell, such as HEK293. In certain embodiments, the host cell is a microorganism, such as a bacterial cell or a fungal cell.

[0119] In another aspect, the invention further provides a method for making a Nanobody or antigen-binding fragment thereof of the invention, comprising culturing a host cell as described above under conditions that allow protein expression, and recovering the Nanobody or antigen-binding fragment thereof, or the polypeptide construct from the culture of the cultured host cells.

[0120] Therapeutic uses of antibodies In another aspect, the present invention provides a pharmaceutical composition comprising a Nanobody or antigen-binding fragment thereof, or a polypeptide construct of the invention, and a pharma- ceutically acceptable carrier and / or excipient.

[0121] In certain embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention may be used in combination with an additional pharma- ceutical active agent. Thus, the pharmaceutical composition of the invention may further comprise an additional pharma- ceutical active agent, such as a drug for the prevention or treatment of HBV infection or a disease associated with HBV infection (e.g. hepatitis B), e.g. an interferon drug, such as interferon or pegylated interferon.

[0122] In another aspect, there is provided a use of a Nanobody or antigen-binding fragment thereof, or a polypeptide construct of the invention, or a pharmaceutical composition of the invention, for the manufacture of a medicament for preventing and / or treating HBV infection in a subject (e.g. a human), or a disease associated with HBV infection (e.g. hepatitis B), for neutralizing HBV toxicity in vitro or in a subject (e.g. a human), for reducing serum levels of HBV DNA and / or HBsAg HBV in a subject (e.g. a human), and / or for activating a humoral immune response against HBV in a subject (e.g. a subject with chronic HBV infection or a chronic hepatitis B patient).

[0123] In another aspect, the invention provides a method for preventing or treating HBV infection or a disease associated with HBV infection (e.g. hepatitis B) in a subject (e.g. a human), neutralizing HBV toxicity in vitro or in a subject (e.g. a human), reducing serum levels of HBV DNA and / or HBsAg HBV in a subject (e.g. a human), and / or activating a humoral immune response against HBV in a subject (e.g. a subject with chronic HBV infection or a chronic hepatitis B patient), comprising administering to a subject in need thereof an effective amount of a Nanobody or antigen-binding fragment thereof, or a polypeptide construct of the invention, or a pharmaceutical composition of the invention.

[0124] In any of the above aspects, the subject may be a subject not infected with HBV, or the subject may be a subject infected with HBV, such as a subject with chronic HBV infection or a chronic hepatitis B patient.

[0125] In any of the above aspects, the HBV for which the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or pharmaceutical composition of the invention relates includes, but is not limited to, genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H, genotype I, genotype J.

[0126] The method of administration of the Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention, or the pharmaceutical composition of the invention, may be any conventional route of administration, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intravesicular, inguinal, intravesical, topical (e.g. powders, ointments or drops), or nasal. The Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention, may be administered by various methods known in the art. However, for many therapeutic applications, the preferred route / mode of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular injection). The skilled artisan will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention is administered by intravenous infusion or injection.

[0127] The Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention, or pharmaceutical composition of the invention can be formulated into a variety of dosage forms, such as liquid, semi-solid and solid dosage forms, for example solutions (e.g. injectables), dispersions or suspensions, tablets, powders, granules, emulsions, pills, syrups, powders, liposomes, capsules and suppositories. The preferred dosage form will vary depending on the intended mode of administration and therapeutic application.

[0128] For example, one of the preferred dosage forms is an injection. Such an injection may be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: incorporating the required amount of the nanobody or antigen-binding fragment thereof, or polypeptide construct of the present invention, and optionally other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, isotonicity agents, preservatives, diluents, or any combination thereof) in a suitable solvent, followed by filtration for sterilization. Furthermore, the sterile injection solution may be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) for ease of storage and use. Such a sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0129] Furthermore, the Nanobodies or antigen-binding fragments thereof, or polypeptide constructs of the invention may be presented in unit dosage form in pharmaceutical compositions for ease of administration. Pharmaceutical compositions of the invention are preferably sterile and stable under the conditions of manufacture and storage.

[0130] The Nanobody or antigen-binding fragment thereof, or polypeptide construct thereof, or pharmaceutical composition provided by the invention can be used alone or in combination, or in combination with additional pharma- ceutical active agents (e.g. additional antiviral agents, e.g. interferon drugs such as interferon or pegylated interferon). In certain embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention is used in combination with an additional antiviral agent to prevent and / or treat diseases associated with Hepatitis B virus infection. The Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention can be administered simultaneously, separately or sequentially with such antiviral agent. Such antiviral agents include, but are not limited to, interferon drugs, ribavirin, adamantane, IL-2, L-12, etc.

[0131] The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a Nanobody or antigen-binding fragment thereof, or a polypeptide construct of the invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, hinder or delay the onset of a disease (e.g. HBV infection or a disease associated with HBV infection). A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. A therapeutically effective amount of a Nanobody or antigen-binding fragment thereof, or a polypeptide construct of the invention may vary depending on factors such as the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration, and other treatments administered at the same time.

[0132] Dosage regimens can be adjusted to achieve the best desired response (e.g., a therapeutic or prophylactic response). For example, a single administration can be administered or multiple administrations can be administered over a period of time, or the dose can be proportionally reduced or increased as dictated by the requirements of the therapeutic situation.

[0133] Antibody detection applications A Nanobody or antigen-binding fragment thereof, or a polypeptide construct or conjugate of the invention is capable of specifically binding to HBsAg and can therefore be used to detect the presence or level of HBsAg in a sample.

[0134] Thus, in another aspect, the invention provides a method for detecting the presence or level of HBsAg protein in a sample comprising using a Nanobody or antigen-binding fragment thereof, or a polypeptide construct or conjugate of the invention. In some embodiments, the Nanobody or antigen-binding fragment thereof, or a polypeptide construct thereof of the invention further comprises a detectable label. In other embodiments, the method further comprises using a second antibody carrying a detectable label to detect the Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention. The method can be used for diagnostic or non-diagnostic purposes (e.g. the sample is not a sample derived from a patient but is a cell sample).

[0135] In certain embodiments, the method comprises (1) contacting a sample with a Nanobody or antigen-binding fragment thereof, or a polypeptide construct or conjugate of the invention, and (2) detecting the formation of an antigen-antibody complex or detecting the amount of the complex, wherein the formation of the complex indicates the presence of HBsAg protein and / or HBV.

[0136] In another aspect, the invention provides a method for diagnosing whether a subject is infected with HBV comprising using a Nanobody or antigen-binding fragment thereof, or a polypeptide construct or conjugate of the invention to detect the presence of HBsAg protein in a sample derived from the subject. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention further comprises a detectable label. In other embodiments, the method further comprises using a second antibody carrying a detectable label to detect the Nanobody or antigen-binding fragment thereof, or polypeptide construct of the invention.

[0137] In another aspect, there is provided the use of a Nanobody or antigen-binding fragment thereof, or a polypeptide construct or conjugate of the invention in the manufacture of a detection reagent for detecting the presence or level of HBsAg protein in a sample or for diagnosing whether a subject is infected with HBV.

[0138] In any of the above aspects, the HBV for which the Nanobody or antigen-binding fragment thereof, or polypeptide construct or conjugate of the invention relates includes, but is not limited to, genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H, genotype I, genotype J.

[0139] Antibodies that recognize epitope peptides and uses thereof In another aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to an epitopic peptide or variant thereof, or an epitope contained therein, recombinant protein, multimer, or particle of the present invention.

[0140] In certain embodiments, the antibody or antigen-binding fragment thereof competes with the above-mentioned Nanobody provided by the invention (e.g. 125s) for binding to HBsAg protein. Competitive binding refers to the ability to inhibit the binding of a Nanobody provided by the invention (e.g. 125s) to HBsAg protein by at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% or preferably at least 99%. Competitive binding can be determined by competitive binding assays.

[0141] Competitive binding assays are well known to those skilled in the art. Competitive binding assays are immunological assays that detect and quantify unknown substances by their ability to inhibit the binding of a labeled known antigen to its specific antibody, also called competitive inhibition assays. An exemplary method includes first pre-coating an antigen onto a microplate, then adding serial dilutions of an unlabeled antibody to be tested and a specific concentration of a labeled known monoclonal antibody (e.g., a nanobody provided by the present invention) together to the pre-coated microplate, incubating, and then, after washing, determining the amount of the known antibody bound to the microplate under different dilutions of the antibody to be tested. The greater the ability of the antibody to be tested to compete with the known antibody for binding to the antigen, the lower the ability of the known antibody to bind to the antigen, and the lower the amount of the known antibody bound to the microplate. For example, the antigen is pre-coated onto a 96-well microplate, and the ability of the monoclonal antibody to be tested to inhibit the labeled known monoclonal antibody is determined using a radioimmunoassay, an enzyme immunoassay such as ELISA, or a fluorescent immunoassay.

[0142] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from a Fab, Fab', F(ab')2, Fd, Fv, dAb, a complementarity determining region fragment, a single chain antibody (e.g., scFv), a nanobody, a humanized antibody, a chimeric antibody, or a bispecific or multispecific antibody.

[0143] In another aspect, the present invention provides a method for producing an antibody that specifically binds to an epitope peptide or variant thereof of the present invention, or an epitope contained therein, comprising using an epitope peptide or variant thereof, recombinant protein, multimer, particle, or polynucleotide encoding the epitope peptide or variant thereof, or recombinant protein described in the present invention.

[0144] In certain embodiments, the method includes immunizing an animal with an epitopic peptide or variant thereof, recombinant protein, multimer, particle, or polynucleotide, and screening for target antibodies from the immunized animal.

[0145] In certain embodiments, the method further comprises a humanization process.

[0146] In certain embodiments, the humanization process involves grafting the CDR sequences of an antibody made from an animal into the FR (framework) regions of a human antibody.

[0147] In certain embodiments, the humanization process involves the use of transgenic animals (e.g., transgenic mice) that are incapable of producing endogenous immunoglobulins and can, upon immunization, produce a fully human antibody repertoire.

[0148] In certain embodiments, the method comprises using a display technology. In certain embodiments, the display technology is selected from the group consisting of phage display, bacterial display, yeast display, or ribosome display. In certain embodiments, the library used in the display technology is designed to have sequences derived from human antibodies.

[0149] In certain embodiments, the method further comprises using the epitope peptide or variant thereof, recombinant protein, multimer, or particle of the invention to screen for antibodies that specifically bind to the target epitope.

[0150] In another aspect, the present invention further provides an isolated nucleic acid molecule, vector, or host cell comprising a nucleotide sequence encoding the above-mentioned antibody or antigen-binding fragment thereof.In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable carrier and / or excipient.

[0151] In another aspect, the present invention provides a method for preventing or treating HBV infection or a disease associated with HBV infection (e.g., hepatitis B) in a subject (e.g., a human), neutralizing the toxicity of HBV in a subject (e.g., a human), reducing serum levels of HBV DNA and / or HBsAg in a subject (e.g., a human), and / or activating a humoral immune response against HBV in a subject (e.g., a subject with chronic HBV infection or a chronic hepatitis B patient), comprising administering to a subject in need thereof an effective amount of the above-mentioned antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising same. The present invention further relates to the use of the above-mentioned antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising same, in the manufacture of a medicament for preventing and / or treating HBV infection in a subject (e.g., a human) or a disease associated with HBV infection (e.g., hepatitis B), neutralizing the toxicity of HBV in vitro or in a subject (e.g., a human), reducing serum levels of HBV DNA and / or HBsAg in a subject (e.g., a human), and / or activating a humoral immune response against HBV in a subject (e.g., a subject with chronic HBV infection or a chronic hepatitis B patient).

[0152] Definition of Terms In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the experimental procedures of virology, biochemistry and immunology used herein are routine procedures that are widely used in the corresponding fields.Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0153] As used herein, the term "HBsAg" refers to the major surface antigen protein of Hepatitis B virus (HBV), which is well known to those skilled in the art (see, e.g., NCBI GENBANK database accession number: ADC29595.1).

[0154] As used herein, when referring to the amino acid sequence of HBsAg, it is described using the sequence set forth in SEQ ID NO: 36. For example, the phrase "amino acid residues 157 to 174 of HBsAg" refers to amino acid residues 157 to 174 of the polypeptide set forth in SEQ ID NO: 36. However, it will be understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions such as different genotypes or subtypes of HBsAg) in the amino acid sequence of HBsAg may occur naturally or may be artificially introduced without affecting its biological function. Thus, in the present invention, the term "HBsAg" is intended to include all such sequences, including, for example, the sequence set forth in SEQ ID NO: 36 and natural or artificial variants thereof. Furthermore, when describing a sequence fragment of HBsAg, not only the sequence fragment of SEQ ID NO: 36 but also the corresponding sequence fragment of the natural or artificial variants thereof are included. For example, the expression "amino acid residues 157 to 174 of HBsAg" includes amino acid residues 157 to 174 of SEQ ID NO: 36 and the corresponding fragments of its (natural or artificial) variants. According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to a fragment that is located at an equivalent position in the sequences being compared when the sequences are optimally aligned, i.e. when the sequences are aligned so that the highest percentage identity is obtained.

[0155] As used herein, the term "epitope" refers to a portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, the epitope can be formed by contiguous amino acids or non-contiguous amino acids brought together by tertiary folding of the protein, and is called a linear epitope or a conformational epitope, respectively. Epitopes formed by contiguous amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. An epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.

[0156] As used herein, the term "epitope peptide" refers to a peptide segment on an antigen that can function as an epitope. In some cases, the epitope peptide alone is capable of specific recognition / binding by an antibody against that epitope. In other cases, it may be necessary to fuse the epitope peptide to a carrier protein so that the epitope peptide can be recognized by a specific antibody. As used herein, the term "carrier protein" refers to a protein that can act as a carrier for the epitope peptide, i.e., allows the insertion of the epitope peptide at a specific position (e.g., within the protein, at the N-terminus or C-terminus) so that the epitope peptide can be presented and recognized by the antibody or immune system. Optionally, the carrier protein can form multimers or self-assemble to form particles. Optionally, a linker can be used between the epitope peptide and the carrier protein to facilitate folding of each.

[0157] As used herein, the term "protein-protein binding pair" includes two proteins (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a covalent isopeptide bond under conditions that allow or promote isopeptide bond formation, where the term "cognate" refers to components that function together, i.e., react together to form an isopeptide bond. Thus, two proteins that react together efficiently to form an isopeptide bond under conditions that allow or promote isopeptide bond formation are sometimes referred to as a "complementary" pair of peptide linkers. Specific binding pairs that can interact to form covalent isopeptide bonds are reviewed in Veggiani G, Zakeri B, Howarth M. Superglue from bacteria: unbreakable bridges for protein nanotechnology. Trends Biotechnol. 2014;32(10):506-512. Non-limiting examples include SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, isopeptag:pilinC, SnoopTag:SnoopCatcher, etc.

[0158] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group, at least one of which is not derived from the protein backbone or is otherwise not part of the protein backbone. Isopeptide bonds can form within a single protein or can occur between two peptides or between a peptide and a protein. Thus, isopeptide bonds can form intramolecularly in a single protein or can form intermolecularly, i.e. between two peptide / protein molecules, for example between two peptide linkers. Typically, isopeptide bonds can occur between a lysine residue and an asparagine, aspartic acid, glutamine or glutamic acid residue or the terminal carboxyl group of a protein or peptide chain, or between the α-amino terminus of a protein or peptide chain and an asparagine, aspartic acid, glutamine or glutamic acid.

[0159] The SpyTag:SpyCatcher system is described in U.S. Patent No. 9,547,003 and Zakeri et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci US A. 2012;109(12):E690-E697, which are incorporated by reference in their entireties, and is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin-binding protein FbaB. Another specific binding pair derived from the CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase. See Fierer JO, Veggiani G, Howarth M. SpyLigase peptide-peptide ligation multimerizes affibodies to enhance magnetic cancer cell capture. Proc Natl Acad Sci US A. 2014;111(13):E1176-E1181, which is incorporated by reference in its entirety. SpyTag002: The SpyCatcher002 system is described in Keeble AH et al. Evolving Accelerated Amidation by SpyTag / SpyCatcher to Analyze Membrane Dynamics. Angew Chem Int Ed Engl. 2017;56(52):16521-16525, which is incorporated by reference in its entirety.

[0160] The SnoopTag:SnoopCatcher system is described in Veggiani G, Nakamura T, Brenner MD, et al. Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci US A. 2016; 113(5):1202-1207, which is incorporated by reference in its entirety. The D4 Ig-like domain of RrgA (which attaches to Streptococcus pneumoniae) is isolated to form SnoopTag and SnoopCatcher.

[0161] The isopeptag:pilin-C specific binding pair is derived from the major pilin protein Spy0128 of Streptococcus pyogenes. See Zakeri B, Howarth M. Spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting. J Am Chem Soc. 2010;132(13):4526-4527. (incorporated by reference in its entirety).

[0162] In this document, the term "antibody" is used in its broadest sense. "Antibody" includes basic four-chain antibodies, heavy-chain antibodies, nanobodies or single-domain antibodies, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, or multispecific antibodies, etc., as well as antibody fragments (e.g., antigen-binding fragments) having the required biological activity. A basic four-chain antibody generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) light chains and λ (lambda) light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into highly variable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each of the VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen binding site, respectively.The assignment of amino acids to each region or domain may be according to the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883. The term "antibody" is not limited to any particular method of making an antibody. Antibodies may be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM.

[0163] As used herein, the term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody that are involved in antigen binding. In a typical four-chain antibody, there are three CDRs, designated CDR1, CDR2, and CDR3, in each of the heavy and light chain variable regions. The exact boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al. al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, the CDRs defined by each numbering system can be readily identified by one of ordinary skill in the art. Furthermore, the correspondence between different numbering systems is well known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0164] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above.

[0165] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989) (incorporated herein by reference in its entirety for all purposes). Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

[0166] As used herein, the term "camelid antibody" refers to an antibody against an antigen produced by a camelid (including camels, alpacas and llamas (L. glama)) immunized or infected with the antigen. Among the antibodies produced by camelids, there is a "camelid heavy chain antibody (HCAb)" that lacks a light chain, and therefore, it is known to those skilled in the art that this antibody contains only one variable domain (VHH) of the heavy chain of HCAb and two conventional CH2 and CH3 regions, and that the cloned and expressed VHH region alone has good structural stability and antigen-binding activity. VHH is currently known to be the smallest unit capable of binding to an antigen.

[0167] As used herein, the term "nanobody", also known as "single domain antibody (sdAb)", is used interchangeably and refers to an antibody fragment that is composed of a single variable domain (e.g., heavy chain variable region) in an antibody, and is usually derived from the variable region of a heavy chain antibody (e.g., camelid antibody or shark antibody). Typically, nanobodies are composed of four framework regions and three complementarity determining regions, and have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies may be truncated at the N-terminus or C-terminus to include only a portion of FR1 and / or FR4, or to lack one or two of the framework regions, so long as they substantially maintain their antigen-binding ability and specificity. Nanobodies combine the advantages of monoclonal antibodies and small molecule drugs. Nanobodies can cross the blood-brain barrier and can be easily designed, developed and stably produced, reducing the difficulty and cost of production.

[0168] As used herein, the term "Fc domain" or "Fc region" refers to a portion of the heavy chain constant region, including CH2 and CH3. The Fc fragment of an antibody has many different functions, but is not involved in antigen binding. "Effector functions" mediated by the Fc region include Fc receptor binding, C1q binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In some embodiments, the Fc region includes a hinge, CH2, and CH3. When the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be any isotype of antibody heavy chain constant region, such as IgG1, IgG2, IgG3, or IgG4.

[0169] The Fc domain may comprise either a native Fc region or a variant Fc region. A native Fc region comprises an amino acid sequence that corresponds to the amino acid sequence of an Fc region found in nature, for example, a human Fc region having a native sequence includes a human IgG1 Fc region having a native sequence (non-A and A allotypes), a human IgG2 Fc region having a native sequence, a human IgG3 Fc region having a native sequence, a human IgG4 Fc region having a native sequence, and naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification. In some embodiments, a variant Fc region may have an altered effector function (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function) compared to a native Fc region.

[0170] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered and modified in amino acid sequence to increase sequence homology with human antibodies. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (receptor antibody). In certain embodiments, the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (receptor antibody). A humanized antibody generally retains the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, etc. In the present application, the donor antibody can be a camelid antibody with the desired properties (e.g., antigen specificity, affinity, reactivity, etc.). To generate a humanized antibody, the CDR regions of an immunized animal can be inserted into human framework sequences using methods known in the art. In the context of nanobodies, a humanized antibody may refer to a humanized VHH, i.e., a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some cases, certain framework regions (FR) of a human immunoglobulin are replaced with corresponding non-human residues. Furthermore, a humanized VHH may contain residues that are not found in either the original VHH or the human framework sequence, which are included to further improve and optimize the performance of the VHH or VHH-containing polypeptide.

[0171] As used herein, the term "humanization degree" refers to an index used to represent the number of non-human amino acid residues in a humanized antibody. The humanization degree of a humanized antibody can be calculated, for example, as follows: humanization degree = (number of amino acids in FR region - number of non-human amino acids retained in FR region) / number of amino acids in FR region x 100%.

[0172] As used herein, the term "germline antibody gene" or "germline antibody gene segment" refers to a sequence present in the genome of an organism that encodes an immunoglobulin that has not undergone a maturation process that may result in gene rearrangements and mutations for the expression of a particular immunoglobulin. In the present invention, the expression "heavy chain germline gene" refers to a germline antibody gene or gene fragment that encodes an immunoglobulin heavy chain, including a V gene (variable), a D gene (diversity), a J gene (joining), and a C gene (constant). Similarly, the expression "light chain germline gene" refers to a germline antibody gene or gene fragment that encodes an immunoglobulin light chain, including a V gene (variable), a J gene (joining), and a C gene (constant). In the present invention, the amino acid sequence encoded by a germline antibody gene or germline antibody gene fragment is also referred to as a "germline sequence". Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are known to those skilled in the art and can be obtained or retrieved from specialized databases (e.g., IMGT, unswag, NCBI, or VBASE2).

[0173] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. The strength or affinity of a specific binding interaction is determined by the equilibrium dissociation constant (K D In the present invention, "K D The term "equilibria" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0174] The specific binding properties between two molecules can be determined using methods known in the art. One approach involves measuring the rate at which the antigen-binding site / antigen complex is formed and dissociated. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentrations and the actual association and dissociation rates (see Malmqvist M, Nature, 1993, 361:186-187). The ratio kdis / kon is the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59:439-473). D The values ​​of k, k and k can be measured using any available method such as surface plasmon resonance (SPR) in a Biacore to measure dissociation constants, or bioluminescence interferometry or Kinexa to measure dissociation constants.

[0175] As used herein, a "neutralizing antibody" refers to an antibody or antigen-binding fragment thereof that can significantly reduce or completely inhibit the toxicity (e.g., ability to infect cells) of a target virus. Generally, a neutralizing antibody can recognize and bind to a target virus and prevent the target virus from invading / infecting a subject's cells. The antibody of the present invention is a neutralizing antibody.

[0176] However, in the present application, it should be understood that the virus neutralizing ability of an antibody is not entirely equivalent to the virus clearing ability of an antibody. As used herein, "neutralizing a virus" means neutralizing the toxicity of a target virus by inhibiting the target virus from entering / infecting the cells of a subject (i.e., significantly reducing or completely inhibiting the toxicity of the target virus). As used herein, "clearing a virus" means removing the target virus (whether or not it is infecting cells) from an organism, thereby changing the organism to a state before infection with the virus (e.g., the result of a serological test for the virus becomes negative). Thus, in general, a neutralizing antibody does not necessarily have a virus clearing ability. However, in the present application, the inventor surprisingly found that the antibody according to the present invention can not only neutralize HBV, but also clear the virus (i.e., clear HBV DNA and / or HBsAg in vivo and clear HBV and HBV-infected cells in vivo), and thus has important clinical value.

[0177] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, thereby allowing the genetic material elements carried by the vector to be expressed in the host cell. Vectors are known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Vectors can contain a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors can also contain an origin of replication site.

[0178] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell, such as E. coli or Bacillus subtilis, a fungal cell, such as a yeast cell or Aspergillus oryzae, an insect cell, such as S2 Drosophila cell or Sf9, or an animal cell, such as a fibroblast cell, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK293 cell, or a human cell.

[0179] As used herein, the term "identity" refers to the degree of correspondence between two polypeptides or two nucleic acids. If the two sequences being compared have the same monomer subunit of a base or amino acid at a particular site (e.g., two DNA molecules each have an adenine at a particular site, or two polypeptides each have a lysine at a particular site), then the two molecules are identical at that site. The percent identity between two sequences is a function of the number of identical sites shared by the two sequences over the total number of sites compared times 100. For example, if 6 out of 10 sites in two sequences are identical, then the two sequences have 60% identity. For example, the DNA sequences: CTGACT and CAGGTT have 50% identity (3 out of 6 sites are identical). Generally, the comparison of two sequences is performed to obtain the maximum identity. Such alignments can be performed using computer programs such as the Align program (DNAstar, Inc.), which is based on the method of Needleman, et al. (J. Mol. Biol. 48:443-453, 1970). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) as incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using a Blossum weighted residue table (GAP) and a GAP penalty of 12. The determination can be made using either a PAM62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6.

[0180] As used herein, the term "conservative substitution" refers to an amino acid substitution in an amino acid sequence that does not adversely affect or change the expected properties of a protein / polypeptide that contains the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions with amino acid residues that have similar side chains, e.g., amino acid residues that are physically or functionally similar to the corresponding amino acid residues (e.g., have similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to substitute a corresponding amino acid residue with another amino acid residue from the same side chain family. In addition, amino acid residues can be divided into categories defined by any physical and functional properties, such as alcohol-containing residues (S and T), aliphatic residues (I, L, V and M), cycloalkenyl-related residues (F, H, W and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K, N, Q, R, S and T), positively charged residues (H, K and R), small residues (A, C, D, G, N, P, S, T and V), very small residues (A, G and S), residues involved in corner formation (A, C, D, E, G, H, K, N, Q, R, S, P and T), flexible residues (Q, T, K, S, G, P, D, E and R).Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997)), which are incorporated herein by reference.

[0181] The twenty conventional amino acids in the present specification are described according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations that are well known in the art. For example, alanine can be represented by A or Ala.

[0182] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, osmotic pressure maintaining agents, absorption retarding agents, and preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic surfactants, anionic surfactants, or nonionic surfactants, such as Tween-80. Ionic strength enhancing agents include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin.

[0183] As used herein, the term "prevention / preventing" refers to a method performed to inhibit or delay the onset of a disease, disorder or condition in a subject, such as HBV infection or a disease associated with HBV infection. As used herein, the term "treatment / treating" refers to a method performed to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, and alleviation of symptoms (either partial or complete). In addition, "treatment" also refers to an increase in survival time compared to expected survival time (in the absence of treatment). In the present application, the antibodies of the present invention have the ability to neutralize HBV and can therefore be used to prevent / protect non-affected subjects or their cells from infection with HBV. In addition, the antibodies of the present invention have the ability to clear HBV (i.e., are capable of clearing HBV DNA and / or HBsAg from the body and clearing HBV and HBV-infected cells from the body) and therefore can be used to treat HBV infection or a disease associated with HBV infection in a subject.

[0184] As used herein, the term "subject" refers to a mammal, such as a primate mammal, for example a human.

[0185] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve an expected effect. For example, an amount effective for preventing a disease (such as HBV infection or a disease associated with HBV infection) refers to an amount effective for preventing, inhibiting or delaying the onset of the disease (such as HBV infection or a disease associated with HBV infection). An amount effective for treating a disease refers to an amount effective for curing or at least partially preventing the disease and its complications in a patient having the disease. Determining such an effective amount is within the capabilities of a person skilled in the art. For example, an amount effective for therapeutic use will vary depending on the severity of the disease to be treated, the overall state of the patient's immune system, the general condition of the patient, such as age, weight and sex, the method of administration of the drug, additional therapies used at the same time, etc. Effect of the Invention

[0186] Beneficial effects It is known in the art that patients with chronic hepatitis B often develop immune exhaustion (tolerance) to HBV due to high levels of HBsAg in the body, leading to prolonged infection. The epitope peptide or nanobody of the present invention can activate the regeneration of humoral immune response against HBV in a subject (e.g., a subject with chronic HBV infection or a patient with chronic hepatitis B), thereby reducing the serum level of HBV DNA and / or HBsAg in the subject and effectively eliminating HBV and HBV-infected cells in the body. Thus, the epitope peptide or nanobody of the present invention is suitable for the prevention and treatment of HBV infection and diseases associated with HBV infection (e.g., hepatitis B), and has a broad binding activity and a broad therapeutic activity against multiple genotypes of HBV. It is expected that immunotherapy via the epitope peptide or nanobody of the present invention will be a new strategy for CHB patients to fight persistent HBV infection. [Brief description of the drawings]

[0187] [Figure 1] Figure 1 shows the binding activity of 125s to HBsAg. The results showed that 125s has good binding activity to HBsAg. [Diagram 2] Figure 2 shows the neutralizing activity of 125s in a HepaRG cell model. In Figures 2A and 2B, "125s" on the left side refers to the experimental group to which the antibody to be tested was added, and "HBV" on the right side refers to the control group in which the antibody to be tested was replaced with PBS. The control group reflects the secretion levels of HbeAg (Figure 2A) or HBsAg (Figure 2B) in HepaAD38 cells not treated with neutralizing antibodies. Figure 2A shows that the HBeAg content in the cell supernatant decreases significantly with increasing 125s antibody concentration, and Figure 2B shows that the HBsAg content in the cell supernatant decreases significantly with increasing 125s antibody concentration, suggesting that 125s has neutralizing activity. [Diagram 3] 1 shows that 125s has binding activity to HBsAg particles of nine types of HBV genotypes (A to H, J), suggesting that 125s has a wide range of binding activity. [Figure 4] 4 shows the therapeutic effect of 125s in HBV-AAV mouse model. FIG. 4A shows that after a single injection treatment with 125s at a dose of 10 mg / kg in a mouse model of AAV-adw serotype, the serum HBsAg titer was less than 100 IU / ml and could be maintained for at least 7 days. FIG. 4B shows that after a single injection treatment with 125s at a dose of 10 mg / kg in a mouse model of AAV-ayw serotype, the serum HBsAg titer was less than 100 IU / ml and could be maintained for at least 7 days. The therapeutic effect in HBV-AAV mice suggests that 125s has long-term HBsAg inhibition and broad therapeutic activity. [Diagram 5] Figure 1 shows that after a single injection treatment of 125s at a dose of 10 mg / kg in an HBV transgenic mouse model, serum HBsAg titers rapidly dropped to less than 100 IU / ml and the HBsAg suppression state could be maintained for 7 days, which slowly returned to baseline levels from day 11 to day 14 after treatment. The therapeutic effect in HBV transgenic mice suggests that 125s has a long-term HBsAg inhibitory activity. [Figure 6]FIG. 1 shows binding of 125s to denatured HBsAg. [Figure 7] 7A shows the recognition epitope of 125s on HBsAg. Figure 7A shows a schematic diagram of a representation of the HBsAg extracellular segment fusion protein, which includes the extracellular segment of HBsAg (Surface, designated Sur), the N-terminal 56 amino acids (N56) of the HBsAg extracellular segment, the major hydrophilic region (MHR) of the HBsAg extracellular segment, the C-terminal 18 amino acids (Cter) of the HBsAg extracellular segment, the C-terminal 36 amino acids (Loop2-Cter) of the HBsAg extracellular segment, and the MHR and Cter portions of the HBsAg extracellular segment (MHR-Cter). Figure 7B shows the binding activity of 125s to six HBsAg extracellular segment fusion proteins (Sur, N56, MHR, Cter, Loop2-Cter, MHR-Cter), and the results showed that 125s had obvious binding reactivity to the complete extracellular segment of HBsAg (Sur) and the C-terminal 18 amino acids of the extracellular segment of HBsAg (Cter), suggesting that the major binding site of 125s is located at the C-terminus of HBsAg. Figure 7C shows the conservation analysis of the Cter region (aa157-aa174) of 10 HBV genotypes (A-J). Figure 7D shows the binding activity of 125s to HBsAg extracellular segment fusion proteins with alanine single point mutations at positions 158-174. The results showed that when single point mutations occurred at positions 158, 160, 163-165, and 171, the binding activity of 125s to HBsAg extracellular segment fusion protein was significantly reduced, suggesting that the major binding epitopes of 125s are associated with positions 158, 160, 163-165, and 171. [Figure 8] Figure 1 shows that the 125s humanized antibody (h125s-26) molecule has binding activity to HBsAg comparable to that of the parent 125s nanobody, demonstrating that the humanization modification of the 125s humanized antibody (h125s-26) molecule was successful. [Figure 9]FIG. 1 shows that HBsAg was rapidly cleared in mouse serum after a single injection of humanized nanobody molecule h125s-26, suggesting that the humanized 125s nanobody molecule h125s-26 has in vivo therapeutic activity. [Figure 10] FIG. 1 shows that Cter-Fc fusion proteins are immunogenic. [Figure 11] FIG. 1 shows that purified Spytag-mouse ferritin exhibited nanoparticle structures with uniform size and stable structure and morphology. [Figure 12] FIG. 1 shows that Spytag-mouse ferritin and Spycatcher-Cter could assemble to form particles. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0188] Sequence information The sequence descriptions for this application are shown in the table below.

[0189] [Table 1-1]

[0190] [Table 1-2]

[0191] [Table 1-3]

[0192] [Table 1-4] EXAMPLES

[0193] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. For specific techniques or conditions not specified in the examples, the techniques or conditions described in the literature of the field (for example, see J. Sambrook et al., "Molecular Cloning Experimental Guide", translated by Huang Peitang et al., third edition, Science Press) or the product instructions shall be followed. For reagents or equipment not specified by manufacturer, all are conventional products that can be purchased commercially.

[0194] Example 1: Generation of anti-HBsAg nanobodies 1.1 Preparation of immunogen The immunogen was recombinant Hepatitis B virus surface antigen major protein (HBsAg, genotype B, purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) expressed in CHO. The recombinant protein was diluted to 0.4mg / ml and mixed with an equal volume of Freund's adjuvant to completely emulsify. Freund's complete adjuvant was used for the primary immunization, and Freund's incomplete adjuvant was used for the subsequent booster immunizations.

[0195] 1.2 Construction and screening of nanobody phage libraries Healthy adult alpacas were immunized a total of four times with the above immunogens. After the immunization was completed, alpaca peripheral blood was extracted, total RNA was isolated, and VHH genes were obtained by reverse transcription and PCR amplification. The VHH genes were then cloned into the phage vector Pcgmt (stored in the laboratory) and transformed into ER2738 host cells (purchased from Lucigen) to construct a phage display library. The library was then screened using phage display technology, and 96 phage clones were randomly selected for ELISA identification, and the positive single clone 125s had good binding ability to HBsAg at the phage level. Furthermore, the sequence of 125s was determined using the method described in Kabat et al. The amino acid sequences of the complementarity determining regions CDR1 to CDR3 of nanobody 125s are set forth in SEQ ID NO:1 to SEQ ID NO:3, respectively, and the amino acid sequences of its framework regions FR1 to FR4 are set forth in SEQ ID NO:4 to SEQ ID NO:7, respectively. The amino acid sequence of the VHH domain of Nanobody 125s is set forth in SEQ ID NO:8.

[0196] 1.3 Eukaryotic expression of anti-HBsAg nanobodies The VHH domain of Nanobody 125s was linked to the N-terminus of a human IgG1 Fc fragment (SEQ ID NO: 14) or a mouse IgG2 Fc fragment (SEQ ID NO: 15) to obtain the Nanobody-Fc fusion proteins 125shIgG1 and 125smIgG2, respectively. Specifically, the VHH domain gene of Nanobody 125s was cloned into the ptt5hIgG1 vector (containing the human Fc region as set forth in SEQ ID NO: 14) or the 125smIgG2 vector (containing the mouse Fc region as set forth in SEQ ID NO: 15), after which the recombinant plasmid and the transfection reagent PEI were mixed 1:2 and transfected into HEK293F cells. The cells were cultured for 6 days at 37°C and 5% CO2 in a shaking incubator. The cell supernatant was collected, purified with Protein A, and the concentration was measured by UV spectroscopy, after which it was aliquoted into 1.5 mL tubes and stored at -80°C for further use.

[0197] Example 2: Binding activity of nanobody 125s to HBsAg 125shIgG1 was diluted with 20 mM PBS buffer and diluted 3-fold with a starting concentration of 1 μg / mL and a total gradient of 12. Humanized antibody 162 (detailed in China Patent Application Publication No. 201610879693.5) was used as a positive control, and an irrelevant antibody was used as a negative control. 100 μL of the diluted sample was added to each well of the HBsAg plate and reacted for 60 minutes in a 37°C incubator. After washing the ELISA plate five times with PBST washing solution, 100 μL of HRP-labeled goat anti-human (GAH) IgG reaction solution was added to each well and reacted for 30 minutes in a 37°C incubator. After the enzyme labeling reaction process was completed, the ELISA plate was washed five times with PBST washing solution, and 50 μL of TMB color reagent was added to each well and reacted for 15 minutes in a 37°C incubator. After the color reaction process was completed, 50 μL of stop solution was added to each well of the ELISA plate, and the OD450 / 630 value of each well was detected by a microplate reader. The results showed that 125shIgG1 had HBsAg specific binding activity (Figure 1).

[0198] Example 3: Determination of the neutralizing activity of Nanobody 125s HepaAD38 cells are cells generated in our laboratory that can controllably express HBV. When it was necessary to increase HepaAD38 cells without expressing HBV, tetracycline could be added to the culture medium to inhibit HBV transcription and replication. When it was necessary to express HBV, tetracycline-free medium could be used for the culture to initiate HBV transcription and replication. When the culture supernatant derived from HepaAD38 cells was contacted with differentiated HepaRG cells, it was determined that the culture supernatant contained HBV virus, indicating that the differentiated HepaRG cells were effectively infected. Using the HepaRG / HBV infection model described above, the ability of nanobody 125s to neutralize / block HBV infection was evaluated (HBV infectivity titer of 100 MOI).

[0199] The indicated concentrations of 125shIgG1 were pre-added to the virus solution used to infect cells, and then the virus solution was used to infect HepaRG cells. On the 7th day after infection, the cell culture supernatant was taken and its HBeAg and HBsAg levels (an important indicator of successful HBV infection) were determined. The detection method was as follows:

[0200] Anti-HBeAg (or anti-HBsAg) monoclonal antibody was diluted in 20 mM PB7.4 to reach a final concentration of 2 μg / ml. The diluted anti-HBeAg (or anti-HBsAg) monoclonal antibody was added to a 96-well microplate and incubated overnight at 4°C. The 96-well microplate was washed once with PBST and spun dry. Then, 200 μL of blocking solution was added to each well. 100 μl / well of the sample to be tested was added and incubated at 37°C for 60 minutes. The 96-well microplate was washed five times with PBST. Horseradish peroxidase-labeled anti-HBeAg (or anti-HBsAg) antibody was added and incubated at 37°C for 30 minutes. The reagents used in the above procedures were purchased from Beijing Wantai Biopharmaceutical Co., Ltd. The 96-microwell plate was washed five times with PBST. A chemiluminescence reagent was added to develop the color. Values ​​were read using a chemiluminescence microplate reader.

[0201] The experimental results are shown in Figures 2A and 2B, which demonstrated that 125s had good virus neutralizing activity.

[0202] Example 4: Determination of the binding activity of Nanobody 125s to HBsAg particles of nine HBV genotypes A total of nine types of HBsAg particles of HBV genotypes A to H and J produced in the laboratory were subjected to detection of their binding ability to Nanobody 125 by ELISA. The HBV genotypes can be found in the NCBI Taxonomy database under the accession number txid10407. The specific detection method was as follows.

[0203] HBsAg particles of nine genotypes were diluted to a final concentration of 2 μg / mL with 20 mM PBS buffer, added to the Elisa plate at 100 μL / well, and incubated in a 37 °C incubator for 60 minutes. After washing the Elisa plate once with PBST washing solution, 200 μL of blocking solution was added to each well and incubated at 37 °C for 2 hours. After washing the Elisa plate once with PBST washing solution, 100 μL / well of 125s nanobody diluted to a final concentration of 0.1 μg / mL was added and incubated in a 37 °C incubator for 1 hour. The Elisa plate was washed five times with PBST washing solution, and 100 μL of HRP-labeled goat anti-human IgG reaction solution was added to each well and incubated in a 37 °C incubator for 30 minutes. After the enzyme labeling reaction process was completed, the Elisa plate was washed five times with PBST washing solution, and 50 μL of TMB color-developing reagent was added to each well and reacted in a 37°C incubator for 15 minutes. After the color-developing reaction process was completed, 50 μL of stop solution was added to each well of the ELISA plate, and the OD450 / 630 value of each well was detected by a microplate reader. The results showed that 125shIgG1 had a wide range of HBsAg specific binding activity (Figure 3).

[0204] Example 5: Therapeutic efficacy of nanobody 125s in HBV-AAV mice of three serotypes 5.1 How to construct the AAV-HBV mouse model The HBV-AAV viruses included HBV-adw serotype (HBV-B genotype, purchased from Guangzhou Paizhen Biotechnology Co., Ltd.) and HBV-ayw serotype (HBV-D genotype, purchased from Guangzhou Paizhen Biotechnology Co., Ltd.). The above-mentioned viruses were delivered to C57BL / 6 mice (purchased from Shanghai Slac Experimental Animal Co., Ltd.) via the tail vein by a single injection at 10E11 (10 11 )GC (genome copies) / mouse and the modeling period was 30 days.

[0205] 5.2 Method for detecting HBsAg in mouse serum (1) Preparation of reaction plate: Mouse monoclonal antibody HBs-45E9 (Beijing Wantai Biopharmaceutical Co., Ltd.) was diluted to 2 μg / mL with 20 mM PB buffer (Na2HPO4 / NaH2PO4 buffer, pH 7.4), and 100 μL of coating solution was added to each well of the chemiluminescence plate, and coating was performed at 2 ° C to 8 ° C for 16 h to 24 h, and then at 37 ° C for 2 h. The plate was washed once with PBST washing solution and centrifuged dry. After washing, 200 μL of blocking solution was added to each well, and blocking was performed at 37 ° C for 2 h. Subsequently, the blocking solution was discarded, and the plate was placed in a drying chamber to dry, and then stored at 2 ° C to 8 ° C for later use.

[0206] (2) Sample dilution: The collected mouse serum was diluted 1:500 with PBS solution containing 20% ​​NBS (newborn bovine serum) for subsequent quantitative detection.

[0207] (3) Denaturation of the sample: 15 μL of the diluted serum sample was taken and thoroughly mixed with 7.5 μL of denaturation buffer (15% SDS, dissolved in 20 mM PB7.4), and reacted for 1 hour at 37° C. Then, 90 μL of neutralization buffer (4% CHAPS, dissolved in 20 mM PB7.4) was added and thoroughly mixed.

[0208] (4) Sample reaction: 100 μL of the above-mentioned denatured serum sample was added to the reaction plate and reacted for 1 hour at 37° C. The reaction plate was then washed five times with PBST and centrifuged to dry.

[0209] (5) Enzyme labeling reaction: 100 μL / well of HBs-A6A7-HRP (Beijing Wantai Biopharmaceutical Co., Ltd.) reaction solution was added to the chemiluminescence plate and reacted at 37° C. for 1 hour. The plate was then washed five times with PBST and centrifuged to dry.

[0210] (6) Luminescence reaction and measurement: The luminescence solution (100 μL / well) was added to the chemiluminescence plate, and the light intensity was detected.

[0211] (7) Calculation of HBsAg concentration in mouse serum samples: A parallel experiment was performed using a standard substance, and a standard curve was created based on the measurement results of the standard substance. The measured values ​​of the light intensity of the mouse serum samples were then substituted into the standard curve to calculate the HBsAg concentration in the serum samples to be tested.

[0212] 5.3 Detection of HBsAg in serum of AAV-HBV mice Thirty days after the establishment of the AAV-HBV mouse model, mouse blood was collected from the retro-orbital venous plexus, and then changes in HBsAg levels in mouse serum were detected. Mice were randomly stratified into groups with 5 mice per group according to HBsAg titers. 125shIgG1 was injected into HBV-adw, HBV-adr, and HBV-ayw serotype mice at a dose of 10 mg / kg via tail vein injection, and a blank control group was set up by injection of PBS. Continuous tests were performed for 12 days, and mouse blood was collected from the retro-orbital venous plexus on days 0, 1, 4, 7, and 11 after treatment to monitor the HBsAg levels in mouse serum. The results (Figures 4A and 4B) showed that 125shIgG1 could maintain serum HBsAg in the 100 IU / ml concentration range for more than one week in HBV-adw and HBV-ayw mice compared with the blank control group, demonstrating a long-term therapeutic effect. The above results demonstrated that 125shIgG1 has a broad and long-term therapeutic effect.

[0213] Example 6: Viral clearance capacity of nanobody 125s in HBV transgenic mice In this example, the viral clearance ability of 125shIgG1 in HBV transgenic mice (donated by Professor Chen Peizhe of National Taiwan University) was investigated. HBV transgenic mice are well-known model mice in the art that mimic persistent HBV infection and are widely used in the field of HBV-related basic research or drug development. This model mouse had stable HBV replication, transcription and viral protein expression levels, and its virological levels were comparable to those of patients' serum levels during the immune clearance and HBeAg-negative phases. The HBV transgenic mouse model had an intact immune system and immune tolerance to HBV, which was similar to HBV-infected individuals to some extent. Specifically, 125shIgG1 was injected into HBV transgenic mice via tail vein injection at a dose of 10 mg / kg, with 5 HBV transgenic mice in each group. A continuous study was conducted for 2 weeks, and blood was collected from the retro-orbital venous plexus of mice on days 0, 2, 3, 5, 7, 9, 11 and 14 after treatment. HBsAg levels in mouse serum were monitored according to the method of Example 5.2. The results (Figure 5) showed that after a single injection, HBsAg serum concentrations in mice were maintained in the range of 100 IU / ml for one week, and HBsAg in the serum of HBV transgenic mice returned to baseline levels after 14 days, indicating that a single injection of 125 s was sufficient to achieve the effect of long-term viral clearance.

[0214] Example 7: Identification of the epitope recognized by nanobody 125s 7.1 Binding ability of nanobody 125s to denatured HBsAg Denaturation of HBsAg: 8 mmol / L SDS was added to the HBsAg sample and boiled in a metal bath at 100°C for 10 min.

[0215] Preparation of reaction plate: Denatured HBsAg protein and normal HBsAg protein were diluted to a final concentration of 2 μg / mL with 50 mM CB buffer (NaHCO3 / Na2CO3 buffer with a final concentration of 50 mM and a pH value of 9.6), added to a 96-well ELISA plate at 100 μL / well, and coated at 2°C-8°C for 16-24 hours, then at 37°C for 2 hours. After washing the plate once with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), blocking solution (20 mM Na2HPO4 / NaH2PO4 buffer with a pH value of 7.4 containing 20% ​​calf serum and 1% casein) was added at 200 μL / well, and blocked at 37°C for 2 hours. The blocking solution was discarded, the plate was dried, placed in an aluminum foil bag, and stored at 2°C-8°C for later use.

[0216] Elisa detection of the reactivity of antibody 125s with denatured HBsAg protein: 125shIgG1 was diluted with 20 mM PBS buffer and diluted 3-fold with a starting concentration of 1 μg / mL and a total gradient of 12. Humanized antibody 162 (detailed in China Patent Application Publication No. 201610879693.5) was used as a positive control. 100 μL of diluted sample was added to each well of the HBsAg plate and reacted for 60 minutes in a 37 ° C incubator. After washing the ELISA plate five times with PBST washing solution, 100 μL of HRP-labeled goat anti-human (GAH) IgG reaction solution was added to each well and placed in a 37 ° C incubator for 30 minutes. After the enzyme labeling reaction process was completed, the ELISA plate was washed five times with PBST washing solution, and 50 μL of TMB color reagent was added to each well and reacted for 15 minutes in a 37 ° C incubator. After the color reaction process was completed, 50 μL of a stop solution was added to each well of the reacted ELISA plate, and the OD450 / 630 value of each well was detected using a microplate reader.

[0217] The results are shown in Figure 6. The results showed that 125s could bind to normal HBsAg but not to denatured HBsAg.

[0218] 7.2 Construction of pTT5hIgG1-HBsAg clone Nucleotide sequences of different lengths of HBsAg extracellular segment on the surface of HBV subtype B virus were constructed in pTT5hIgG1 vector to construct a series of fusion proteins. In the fusion proteins, the extracellular segment sequence was linked to the N-terminus of hIgG1-Fc via a linker (G4S)5. Six HBsAg extracellular segment gene sequences of different lengths listed in Table 2 were constructed in pTT5hIgG1 vector, thereby obtaining six Fc fusion proteins (pTT5hIgG1-S1, pTT5hIgG1-S2, pTT5hIgG1-S3, pTT5hIgG1-S4, pTT5hIgG1-S5, pTT5hIgG1-S6).

[0219] [Table 2]

[0220] 7.3 Expression and purification of pTT5hIgG1-S fusion protein 293F cells were used to express pTT5hIgG1-S1, pTT5hIgG1-S2, pTT5hIgG1-S3, pTT5hIgG1-S4, pTT5hIgG1-S5 and pTT5hIgG1-S6 fusion proteins. 293F cells with a viability of more than 95% were prepared, 200 ml of cells were taken and 4 × 10 cells were cultured in a 1 L cell culture flask. 6 The cells were seeded at a density of 10 ...

[0221] 7.4 Evaluation of the reactivity of antibody 125s with the six fusion proteins In this experiment, E6F6 and 129G1 were used as internal reference antibodies. For E6F6 (recognition epitope located in sa) and 129G1 (recognition epitope located in Se), please refer to Zhang TY et al. Prolonged suppression of HBV in mice by a novel antibody that targets a unique epitope on hepatitis B surface antigen. Gut. 2016 Apr;65(4):658-71. doi: 10.1136 / gutjnl-2014-308964.

[0222] Preparation of reaction plate: Six fusion proteins of HBsAg extracellular segments were diluted to a final concentration of 1 μg / mL in 50 mM CB buffer (NaHCO3 / Na2CO3 buffer with a final concentration of 50 mM and a pH value of 9.6) and added to a 96-well ELISA plate at 100 μL / well for coating at 2°C-8°C for 16-24 hours and then at 37°C for 2 hours. After washing the plate once with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), 200 μL of blocking solution (20 mM Na2HPO4 / NaH2PO4 buffer with a pH value of 7.4 containing 20% ​​calf serum and 1% casein) was added to each well and placed at 37°C for blocking for 2 hours. The blocking solution was discarded and the plate was dried, then placed in an aluminum foil bag and stored at 2°C-8°C for later use.

[0223] Elisa detection of reactivity of antibody 125s with various fusion proteins: 125smIgG2 was diluted to 1 μg / ml with 20 mM PBS buffer. The internal reference antibodies E6F6 and 129G1 were treated in the same manner as 125smIgG2. Elisa plates coated with pTT5hIgG1-S1, pTT5hIgG1-S2, pTT5hIgG1-S3, pTT5hIgG1-S4, pTT5hIgG1-S5, and pTT5hIgG1-S6 fusion proteins were taken, and 100 μL of the diluted samples were added to each well and reacted in a 37°C incubator for 60 minutes. The Elisa plate was washed five times with PBST washing solution, and 100 μL of HRP-labeled goat anti-mouse IgG reaction solution was added to each well and reacted in a 37°C incubator for 30 minutes. After the enzyme labeling reaction process was completed, the Elisa plate was washed five times with PBST washing solution, 50μL of TMB color reagent was added to each well, and the plate was placed in a 37℃ incubator for 15 minutes. After the color reaction process was completed, 50μL of stop solution was added to each well of the reacted Elisa plate, and the OD450 / 630 value of each well was detected by a microplate reader. The reactivity of 125s with pTT5hIgG1-S1, pTT5hIgG1-S2, pTT5hIgG1-S3, pTT5hIgG1-S4, pTT5hIgG1-S5, and pTT5hIgG1-S6 fusion proteins was determined based on the reading value after the reaction.

[0224] Analysis of the epitope recognized by antibody 125s: The results of Elisa detection (FIG. 7B) showed that 125smIgG2 had good reactivity with fusion proteins displaying extracellular segments S1, S4, S5 and S6, but weak reactivity with fusion proteins displaying extracellular segments S2 and S3. Sequence analysis of these extracellular segments showed that the common feature of the extracellular segments S1, S4, S5 and S6 was that they contained incomplete HBsAg aa157-aa174, and the common feature of the extracellular segments S2 and S3 was that they contained incomplete HBsAg aa118-aa156 but did not contain aa157-aa174. In addition, the results of ELISA showed that the reactivity of the fusion proteins pTT5hIgG1-S1 and pTT5hIgG1-S4 with the antibody was higher than that of the other fusion proteins, and the reactivity of the fusion proteins pTT5hIgG1-S5 and pTT5hIgG1-S6 with the antibody was higher than that of pTT5hIgG1-S2 and pTT5hIgG1-S3. Therefore, it was possible to determine that the epitope recognized by 125s is aa157-aa174, and is AFAKYLWEWASVRFSWLS. In addition, since the sequence of S4 is shorter than that of S1, it was considered that S4 is a preferred core epitope.

[0225] 7.5 Evaluation of reactivity of antibody 125s with the Cter region (aa157-aa174) of different genotypes Conservation analysis of the Cter region (aa157–aa174) of 10 HBV genotypes (A–J) is shown in Figure 7C. The red box is aa162–aa167, which is 100% conserved in different genotypes, suggesting that it may contain the major core epitope of nanobody 125s.

[0226] Example 8: Analysis of the sensitivity of antibody 125s to amino acid mutations in the extracellular segment S1 8.1 Construction of pTT5hIgG1-S1 amino acid single point mutant clones Because 125s has good binding activity with pTT5hIgG1-S1, single amino acid point mutations were made to pTT5hIgG1-S1 to generate a total of 14 mutant fusion proteins. The amino acid sequences of these 14 mutants are listed in SEQ ID NO: 22 to SEQ ID NO: 35.

[0227] 8.2 Expression and purification of pTT5hIgG1-S1 amino acid single point mutant fusion protein 293F cells were used for the expression of the above single point mutant fusion proteins. The cells were placed in a 5% CO2 incubator at 37°C for 7 days of expression. The cell supernatant was collected and purified.

[0228] 8.3 Reactivity of 125s with pTT5hIgG1-S1 single amino acid point mutant fusion protein 8.3.1 Preparation of reaction plate The reaction plate was prepared according to the method of Example 7.3.1, and the coating antigen was pTT5hIgG1-S1 amino acid single point mutant fusion protein.

[0229] 8.3.2 Elisa detection of 125s and pTT5hIgG1-S1 amino acid single point mutant fusion proteins 125smIgG2 was diluted to 1μg / mL with 20mM PBS buffer. The Elisa plate coated with pTT5hIgG1-S1 amino acid single point mutation fusion protein was taken, 100μL of diluted sample was added to each well, and reacted in a 37℃ incubator for 60 minutes. The Elisa plate was washed 5 times with PBST washing solution, 100μL of HRP-labeled goat anti-mouse IgG reaction solution was added to each well, and reacted in a 37℃ incubator for 30 minutes. After the enzyme labeling reaction process was completed, the Elisa plate was washed 5 times with PBST washing solution, 50μL of TMB color reagent was added to each well, and reacted in a 37℃ incubator for 15 minutes. After the color reaction process was completed, 50μL of stop solution was added to each well of the reaction microplate, and the OD450 / 630 value of each well was detected by a microplate reader. The reactivity of 125s with pTT5hIgG1-S1 single amino acid point mutant fusion protein was determined based on the readout value after the reaction, and the readout value was normalized to obtain the ratio of the "response value between 125s and the epitope" to the "response value between E6F6 and the epitope". In addition, since the epitope of E6F6 is not on the Cter, the mutation of 158-174 does not affect the reactivity of E6F6, and it was known that the reactivity of E6F6 with the presented epitope is related only to the coating concentration of the epitope.

[0230] The results are shown in Figure 7D. When single point mutations occurred at positions 163-165, the binding activity of 125s to the HBsAg extracellular segment fusion protein was significantly reduced, indicating that this was the core epitope. When single point mutations occurred at positions 158, 160, and 171, the binding activity of 125s to the HBsAg extracellular segment fusion protein was reduced to some extent. Mutations at positions 161, 162, 167-170, and 172-174 did not significantly affect the binding activity, indicating that these were not the core binding sites.

[0231] Example 9: Generation of humanized antibodies of 125s 9.1 Humanization of nanobody 125s The humanization of camelid nanobody 125s was based on the CDR grafting method. By searching gene databases with IMGT, we first found the human germline gene variable region sequences with the highest homology to the alpaca antibody 125s VHH region. After homology analysis, we identified IGHV3-23 * A humanized antibody heavy chain was designed using the germline gene sequence of h125s-04 as a template. At the same time, the germline gene did not contain the FR4 required for the designed portion, so the FR4 portion needed to be compared separately, and a humanized antibody heavy chain FR4 was designed using the IGHJ5*02 germline gene sequence as a template. The CDR region of the alpaca antibody 125s VHH was grafted into the FR (framework) region of the human template. At the same time, the FR region of the alpaca antibody and the human germline gene were subjected to sequence alignment, and the difference amino acids were selectively subjected to back mutation, and finally one humanized heavy chain was designed to obtain one humanized antibody, which was named h125s-26, and its VHH sequence is set forth in SEQ ID NO:13.

[0232] 9.2 Generation of humanized antibodies The variable region sequence of the humanized antibody was linked to the N-terminus of human IgG1 Fc fragment (SEQ ID NO: 14) to obtain the nanobody-Fc fusion protein h125s-26-hIgG1. Specifically, the variable region sequence of the humanized antibody was constructed in the pTT5-hIgG1 vector (containing the human Fc region described in SEQ ID NO: 14). In this experiment, gene synthesis was performed, followed by sequencing analysis (Shanghai Bioengineering Co., Ltd.). The plasmid for gene synthesis was extracted in large quantities using an endotoxin-free plasmid maximum extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.). 293F cells were used for the expression of the 125s humanized antibody. The supernatant was taken for subsequent purification, and the purification method was as described in Example 1.3.

[0233] Example 10: ELISA binding activity of 125s humanized antibody with HBsAg The 125s humanized antibody h125s-26-hIgG1 obtained in Example 9 was taken and subjected to gradient dilution with SD-1 buffer at a starting concentration of 20 μg / mL and a total gradient of 8. The ELISA detection method was the same as that described in Example 2. The results showed that the EC50 values ​​of humanized antibodies h125s-26-hIgG1 and 125s-hIgG1 for binding to HBsAg protein were 27.77 ng / mL and 25.49 ng / mL, respectively. The results were shown in Figure 8. The humanized antibody h125s-26-hIgG1 maintained the same binding activity to HBsAg as 125s-hFc.

[0234] Example 11: Viral clearance ability of 125s humanized antibody in HBV-AAV mice h125s-26-hIgG1 and 125s-hIgG1 were injected separately into HBV-AAV mice (HBV-adw serotype) at a single dose of 5 mg / kg by tail vein injection, with 5 HBV transgenic mice in each group. A continuous study was performed for 17 days, and mouse blood was collected from the retro-orbital venous plexus on days 0, 2 hours, 1, 3, 5, 7, 10, 12, 14 and 17 after treatment. HBsAg levels in mouse serum were monitored according to the method in Example 5.2. The results were shown in Figure 9. After a single injection, both h125s-26-hIgG1 and the control antibody 125s-hIgG1 had better HBsAg clearance effect. These two antibodies had similar therapeutic effects in the first 10 days. After 10 days, the HBsAg titers in mice treated with 125s-hFc recovered rapidly and returned to baseline levels on day 14. In contrast, the HBsAg titers in mice treated with humanized antibody h125s-26 recovered slowly, and the HBsAg titers did not return to baseline levels on day 17. This suggests that the humanized molecule h125s-26 has better antigen clearance ability and long-lasting antigen suppression effect in the AAV / HBV mouse model, and the humanized design is successful.

[0235] Example 12: Evaluation of immunogenicity of epitope peptide-Fc fusion proteins In this example, the immunogenicity of a fusion protein of a series of HBsAg extracellular segment polypeptides including S4 (aa157-aa174) and human IgG1-Fc region in BALB / C mice was examined. Briefly, recombinant proteins of the above series of polypeptides and human IgG1-Fc region (SEQ ID NO: 14) were prepared, and BALB / C mice were immunized with the recombinant proteins to prepare hybridoma cells. Anti-HBs antibody titers were then detected in the secretion supernatants of hybridoma cells (1B2, 1H2, 1F12, 2B6, 7C4, 9G1, 10D3). The results are shown in FIG. 10. In the immunotherapy group using the recombinant proteins, anti-HBsAg antibodies could be detected in mouse serum samples. It was suggested that the Cter segment of the HBsAg extracellular segment has immunogenicity.

[0236] Example 13: Construction and expression of SpyTag-mouse ferritin particles and SpyCatcher-Cter proteins Based on the sequence of mouse ferritin (NCBI protein name: NP_034369.1), a B11-SpyTag-ferritin expression vector was constructed, and its nucleic acid sequence was codon-optimized for HEK293 host cells, and three G4S linkers were added between the SpyTag fragment and the ferritin fragment. Similarly, the SpyCatcher fragment and the Cter fragment (SEQ ID NO: 19) were inserted into the B11 prokaryotic expression vector, and the two fragments were linked via three G4S linkers. The gene was synthesized by General Biosystems (Anhui) Co., Ltd., and a His tag was inserted for purification, finally obtaining the genes B11-SpyTag-mouse ferritin-his and B11-SpyCatcher-Cter-his. The amino acid sequence of SpyTag-mouse ferritin is set forth in SEQ ID NO: 49, and the amino acid sequence of SpyCatcher-Cter is set forth in SEQ ID NO: 50.

[0237] Expression and purification of B11-SpyTag-Mouse Ferritin: The gene was transformed into BL21 competent cells, and expression was induced with IPTG at 37°C. After sonication of the cells, the cell supernatant was collected and heat-treated in a 70°C water bath for 10 min. After ammonium sulfate precipitation, it was purified using a gel chromatography column, and the sample was stored at 4°C. Transmission electron microscopy results (Figure 11) showed that the purified SpyTag-mouse ferritin was a nanoparticle structure with uniform size, stable structure and morphology, and a diameter of approximately 20 nm.

[0238] Expression and purification of B11-SpyCatcher-Cter: The gene was transformed into BL21 competent cells, and expression was induced with IPTG at 37° C. After ultrasonic disruption of the bacterial cells, the supernatant was taken and passed through a Ni-agarose self-packing column, and the SpyCatcher-Cter protein was eluted with imidazole.

[0239] Example 14: Assembly of SpyTag-Mouse Ferritin and SpyCatcher-Cter SpyTag-ferritin and SpyCatcher-Cter were mixed in a 1:1 molar ratio and mixed by inversion overnight at 4°C. After mixing SpyTag-ferritin and SC-Cter, the mixture was filtered through a concentration tube and purified through a Superose6 Increase gel filtration column to obtain a highly purified ferritin-Cter nanovaccine. Transmission electron microscopy results (Figure 12) showed that the surface of ferritin-Cter nanoparticles exhibited a protruding structure, indicating that SpyTag-mouse ferritin and SpyCatcher-Cter were successfully assembled, demonstrating their potential as a vaccine.

[0240] Although certain specific embodiments of the present invention have been described in detail above, in light of all the teachings disclosed, it will be understood by those skilled in the art that various modifications and substitutions can be made in the details without departing from the spirit and scope of the present invention, and all of these modifications are within the scope of protection of the present invention. The scope of the present invention is limited only by the appended claims and any equivalents thereof.

Claims

1. 1. A Nanobody or antigen-binding fragment thereof capable of specifically binding to HBsAg, comprising a CDR1 having the sequence as set forth in SEQ ID NO: 1 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. one, two or three amino acid substitutions, deletions or additions), a CDR2 having the sequence as set forth in SEQ ID NO: 2 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. one, two or three amino acid substitutions, deletions or additions), and a CDR3 having the sequence as set forth in SEQ ID NO: 3 or a sequence which has one or several amino acid substitutions, deletions or additions compared thereto (e.g. one, two or three amino acid substitutions, deletions or additions).

2. 2. The Nanobody or antigen-binding fragment thereof of claim 1, comprising the sequence set forth in SEQ ID NO: 8, or a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity thereto, or a sequence that has one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto.

3. the Nanobody or antigen-binding fragment thereof is humanized, For example, the Nanobody, or antigen-binding fragment thereof, further comprises a heavy chain framework region of a human immunoglobulin (e.g. a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene), wherein the heavy chain framework region optionally comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) backmutations of human residues to camelid residues; For example, the nanobody or antigen-binding fragment thereof of claim 1, wherein the nanobody or antigen-binding fragment thereof comprises FR1 to FR4 as set forth in SEQ ID NOs: 9 to 12.

4. 4. The Nanobody or antigen-binding fragment thereof of claim 3, comprising the sequence set forth in SEQ ID NO: 13, or a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity thereto, or a sequence that has one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto.

5. A polypeptide construct capable of specifically binding to HBsAg, comprising a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 4 and an immunoglobulin Fc domain. For example, the immunoglobulin Fc domain is linked to the N-terminus and / or C-terminus (e.g. the C-terminus) of the Nanobody or antigen-binding fragment thereof, optionally via a peptide linker; For example, the immunoglobulin Fc domain is an IgG Fc domain (e.g., an IgG1, IgG2, IgG3, or IgG4 Fc domain); For example, the immunoglobulin Fc domain is a human or mouse immunoglobulin Fc domain, such as a human or mouse IgG Fc domain (e.g., a human or mouse IgG1, IgG2, IgG3, or IgG4 Fc domain); For example, a polypeptide construct, wherein said immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity thereto, or a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto.

6. 6. An isolated nucleic acid molecule encoding a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 4, or a polypeptide construct according to claim 5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. 8. A host cell comprising the isolated nucleic acid molecule of claim 6 or the vector of claim 7.

9. 10. A method for producing a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a polypeptide construct according to claim 5, comprising culturing a host cell according to claim 8 under conditions allowing protein expression, and recovering the Nanobody or antigen-binding fragment thereof, or the polypeptide construct from a culture of the cultured host cells.

10. 9. A pharmaceutical composition comprising a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, a polypeptide construct according to claim 5, an isolated nucleic acid molecule according to claim 6, a vector according to claim 7, or a host cell according to claim 8, together with a pharma- ceutical acceptable carrier and / or excipient.

11. 13. Use of a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 4, a polypeptide construct according to claim 5, an isolated nucleic acid molecule according to claim 6, a vector according to claim 7, a host cell according to claim 8, or a pharmaceutical composition according to claim 10 in the manufacture of a medicament for preventing and / or treating HBV infection in a subject (e.g. a human) or a disease associated with HBV infection (e.g. hepatitis B), for neutralizing toxicity of HBV in vitro or in a subject (e.g. a human), for reducing serum levels of HBV DNA and / or HBsAg in a subject (e.g. a human) and / or for activating a humoral immune response against HBV in a subject (e.g. a subject with chronic HBV infection or a patient with chronic hepatitis B).

12. 10. A method for use in preventing and / or treating HBV infection in a subject (e.g. a human) or a disease associated with HBV infection (e.g. hepatitis B), neutralizing the toxicity of HBV in vitro or in a subject (e.g. a human), reducing serum levels of HBV DNA and / or HBsAg in a subject (e.g. a human) and / or activating a humoral immune response against HBV in a subject (e.g. a subject with chronic HBV infection or a patient with chronic hepatitis B), comprising administering to a subject in need thereof an effective amount of a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, a polypeptide construct according to claim 5, an isolated nucleic acid molecule according to claim 6, a vector according to claim 7, or a host cell according to claim 8, or a pharmaceutical composition according to claim 10.

13. A conjugate comprising a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 4, or a polypeptide construct according to claim 5, and a detectable label, For example, a conjugate, wherein the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

14. Use of a Nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 4, or a polypeptide construct according to claim 5, or a conjugate according to claim 13 in the manufacture of a detection reagent for detecting the presence or level of HBsAg protein in a sample or for diagnosing whether a subject is infected with HBV.

15. 1. An isolated epitopic peptide or variant thereof, said epitopic peptide or variant thereof comprising an epitope located within amino acid residues 157 to 174 of HBsAg protein, said epitope comprising at least amino acid residues 163 to 165 of HBsAg protein, said variant differing from the epitopic peptide from which it is derived only by mutation (e.g., substitution, addition or deletion) of one or several (e.g., 1, 2, 3, 4 or 5) amino acid residues, and retaining the biological function of the epitopic peptide from which it is derived; For example, the epitope comprises at least amino acid residues 163 to 166 of the HBsAg protein; For example, the epitope comprises at least amino acid residues 163 to 165, as well as amino acid residues 158, 160 and 171 of the HBsAg protein; For example, the isolated epitope peptide or variant thereof, wherein the epitope comprises at least amino acid residues 163 to 166, as well as amino acid residues 158, 160 and 171 of HBsAg protein.

16. the epitope is a conformational epitope consisting of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 non-consecutive amino acid residues located within amino acid residues 157 to 174 of the HBsAg protein, and including at least amino acid residues 163 to 165 of the HBsAg protein; For example, the epitope comprises at least amino acid residues 163 to 166 of the HBsAg protein; For example, the epitope comprises at least amino acid residues 163 to 165, as well as amino acid residues 158, 160 and 171 of the HBsAg protein; For example, the epitope peptide or its variant according to claim 15, wherein the epitope comprises at least amino acid residues 163 to 166, as well as amino acid residues 158, 160 and 171 of HBsAg protein.

17. the epitope peptide consists of at least 5 (e.g. at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20) and / or at most 80 (e.g. at most 75, at most 70, at most 65, at most 60, at most 55, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20) consecutive amino acid residues of the HBsAg protein and comprises the epitope, For example, the epitope peptide is 5 to 80 (e.g., 5 to 75, 5 to 70, 5 to 65, 5 to 60, 5 to 55, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13) peptides of the HBsAg protein.

17. The epitope peptide or its variant according to claim 15 or 16, which comprises consecutive amino acid residues of at least one of the following: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 74, 75 or 80, and contains the epitope.

18. the epitope peptide comprises at least amino acid residues 157 to 174 of the HBsAg protein; For example, the epitope peptide or its variant according to any one of claims 15 to 17, wherein amino acid residues 157 to 174 of the HBsAg protein are set forth in any one of SEQ ID NO: 19 and SEQ ID NO: 38 to SEQ ID NO:

44.

19. the variant does not contain a mutation at a position corresponding to amino acid position 163 to amino acid position 165 of the HBsAg protein; For example, the variant does not contain a mutation at a position corresponding to amino acid position 163 to amino acid position 166 of the HBsAg protein, For example, the variant does not contain a mutation at a position corresponding to amino acid position 162 to amino acid position 167 of the HBsAg protein, For example, the variant further comprises no mutation at a position corresponding to amino acid position 157 of the HBsAg protein, For example, the variant further comprises no mutation at a position corresponding to amino acid position 171 of the HBsAg protein, For example, the variant further does not contain a mutation at one or more (e.g., one, two or three) of the amino acid positions corresponding to the following positions: 158, 160 of the HBsAg protein.

20. the variant comprises a mutation at one or more (e.g. one, two, three, four or five) of the amino acid positions corresponding to the following positions of the HBsAg protein: 161, 162, 167, 168, 169, 170, 172, 173, 174, For example, the mutation is an amino acid substitution, such as a conservative substitution, For example, the epitope peptide or its variant according to any one of claims 15 to 19, wherein the amino acid substitution comprises substitution of the amino acid at the position with alanine.

21. The epitope peptide or a variant thereof comprises at least AX １ X ２ X ３ X ４ X ５ WEWAX ６ X ７ X ８ X ９ S.X. １０ X １１ X １２ (SEQ ID NO:51), wherein: X １ (corresponding to amino acid position 158 of the HBsAg protein) is F or L, X ２ (corresponding to amino acid position 159 of the HBsAg protein) is A or G, X ３ (corresponding to amino acid position 160 of the HBsAg protein) is K or R, X ４ (corresponding to amino acid position 161 of the HBsAg protein) is any naturally occurring amino acid (e.g., Y, F, or A); X ５ (corresponding to amino acid position 162 of the HBsAg protein) is any naturally occurring amino acid (e.g., L or A); X ６ (corresponding to amino acid position 167 of the HBsAg protein) is any naturally occurring amino acid (e.g., S or A); X ７ (corresponding to amino acid position 168 of the HBsAg protein) is any naturally occurring amino acid (e.g., V or A); X ８ (corresponding to amino acid position 169 of the HBsAg protein) is any naturally occurring amino acid (e.g., R, H, or A); X ９ (corresponding to amino acid position 170 of the HBsAg protein) is any naturally occurring amino acid (e.g., F or A); X １０ (corresponding to amino acid position 172 of the HBsAg protein) is any naturally occurring amino acid (e.g., W or A); X １１ (corresponding to amino acid position 173 of the HBsAg protein) is any naturally occurring amino acid (e.g., L or A); X １２ (corresponding to amino acid position 174 of the HBsAg protein) is any naturally occurring amino acid (e.g., S or A); For example, X ５ is L, and X ６ is S, for example, X ９ is F and X １０ is W and X １１ is L, and X １２ is S, For example, the epitope peptide or its variant comprises at least an amino acid sequence selected from the sequences set forth in any one of SEQ ID NO: 19 and SEQ ID NO: 38 to SEQ ID NO:

44. The epitope peptide or its variant according to any one of claims 15 to 20.

22. The epitope peptide or variant thereof according to any one of claims 15 to 21, wherein the HBsAg protein comprises a sequence as set forth in SEQ ID NO: 36, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

23. The epitope peptide or its variant according to any one of claims 15 to 22, comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 16, 19, 22 to 35, and 38 to 44.

24. A recombinant protein comprising the isolated epitopic peptide or variant thereof according to any one of claims 15 to 23 and a carrier protein, wherein the recombinant protein is not a naturally occurring protein or a fragment thereof, For example, a recombinant protein in which the epitope peptide or variant thereof is linked to a carrier protein, optionally via a linker (e.g., a rigid linker or a flexible linker, such as a peptide linker comprising one or more glycines and / or one or more serines).

25. the carrier protein is selected from an immunoglobulin Fc domain, such as an IgG Fc domain (e.g., an IgG1, IgG2, IgG3 or IgG4 Fc domain); For example, the epitope peptide or variant thereof is linked to the N-terminus or C-terminus (e.g., the N-terminus) of the immunoglobulin Fc domain, optionally via a linker (e.g., a rigid or flexible linker, such as a peptide linker comprising one or more glycines and / or one or more serines); For example, the recombinant protein of claim 24, wherein the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO:

14.

26. the carrier protein is selected from proteins capable of self-assembling to form nanoparticles; For example, the protein capable of self-assembling to form nanoparticles is a ferritin, such as mouse ferritin; For example, the recombinant protein of claim 24, wherein the epitope peptide or a variant thereof is linked to the N-terminus or C-terminus (e.g., the N-terminus) of the protein capable of self-assembling to form nanoparticles via a peptide bond or an isopeptide bond.

27. the epitope peptide or variant thereof is linked via an isopeptide bond to the N-terminus or C-terminus (e.g., the N-terminus) of the protein capable of self-assembling to form nanoparticles; For example, the isopeptide bond is formed by a protein-protein bond pair, For example, the protein-protein binding pair comprises a first member (e.g., a first peptide tag) and a second member (e.g., a second peptide tag), wherein the first member and the second member are linked via the isopeptide bond, the first member is linked to the N-terminus or C-terminus of the epitope peptide or variant thereof, optionally via a first linker (e.g., a rigid linker or a flexible linker, such as a peptide linker comprising one or more glycines and / or one or more serines) to form a first protein, and the second member is linked to the N-terminus or C-terminus (e.g., the N-terminus) of the protein capable of self-assembling to form nanoparticles, optionally via a second linker (e.g., a rigid linker or a flexible linker, such as a peptide linker comprising one or more glycines and / or one or more serines) to form a second protein; For example, the first protein and / or the second protein may further comprise a protein tag (e.g., at its N-terminus or C-terminus); For example, the protein-protein binding pair is selected from the group consisting of: (i) a SpyTag:SpyCatcher pair; (ii) a SpyTag:KTag pair; (iii) an Isopeptag:pilin-C pair; (iv) a SnoopTag or SnoopTagJr:SnoopCatcher pair; (v) a SpyTag002:SpyCatcher002 pair; (vi) an RrgATag, RrgATag2 or DogTag:RrgACatcher pair; (vii) an IsopepTag N:Pilin N pair; (viii) a PsCsTag:PsCsCatcher pair; 27. The recombinant protein of claim 26, wherein, for example, the protein-protein binding pair is a SpyTag:SpyCatcher pair, for example, wherein the SpyTag comprises the sequence set forth in SEQ ID NO: 46, and for example, the SpyCatcher comprises the sequence set forth in SEQ ID NO: 47 or SEQ ID NO:

48.

28. formed by linking the first protein and the second protein via an isopeptide bond; the first protein comprises the epitope peptide or variant thereof and a first member of a SpyTag:SpyCatcher pair, the first member being linked to the epitope peptide or variant thereof (e.g., linked to the N-terminus or C-terminus of the epitope peptide or variant thereof) optionally via a first linker; the second protein comprises the protein capable of self-assembling to form nanoparticles and a second member of a SpyTag:SpyCatcher pair, the second member being linked to the protein capable of self-assembling to form nanoparticles (e.g., linked to the N-terminus of the protein capable of self-assembling to form nanoparticles), optionally via a second linker; For example, the first protein comprises the epitope peptide or a variant thereof and SpyCatcher or an active fragment thereof (e.g., an N-terminal truncation), and the second protein comprises the protein capable of self-assembling to form nanoparticles and SpyTag; For example, the recombinant protein of claim 27, wherein the first protein comprises the sequence set forth in SEQ ID NO:49 and the second protein comprises the sequence set forth in SEQ ID NO:

50.

29. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the epitopic peptide or variant thereof according to any one of claims 15 to 23, or the recombinant protein according to any one of claims 24 to 28.

30. 30. A vector comprising the isolated nucleic acid molecule of claim 29.

31. 31. A host cell comprising the isolated nucleic acid molecule of claim 29 or the vector of claim 30.

32. A method for producing the epitope peptide or variant thereof according to any one of claims 15 to 23, or the recombinant protein according to any one of claims 24 to 28, comprising culturing a host cell according to claim 31 under conditions allowing protein expression, and recovering the epitope peptide or variant thereof, or the recombinant protein from a culture of the cultured cells.

33. A multimer comprising a plurality of monomers, each monomer being independently selected from a recombinant protein according to any one of claims 24 to 28; For example, the multimer is a dimer, trimer, or tetramer; For example, the monomers are identical to each other, For example, a multimer in which the monomers are identical to each other and are selected from the recombinant protein of claim 25.

34. A particle presenting the isolated epitope peptide or a variant thereof according to any one of claims 15 to 23 on its surface, For example, the particle is a nanoparticle, a virus-like particle (VLP) or a core-like particle (CLP), For example, a particle comprising or consisting of a recombinant protein according to any one of claims 24 to 28 or a multimer according to claim 33.

35. A pharmaceutical composition comprising the epitope peptide or variant thereof according to any one of claims 15 to 23, the recombinant protein according to any one of claims 24 to 28, the isolated nucleic acid molecule according to claim 29, the vector according to claim 30, the host cell according to claim 31, the multimer according to claim 33, or the particle according to claim 34, For example, the pharmaceutical composition is a vaccine, such as a protein vaccine or a nucleic acid vaccine, For example, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier and / or excipient (eg, an adjuvant).

36. 27. Use of an epitopic peptide or variant thereof according to any one of claims 15 to 23, a recombinant protein according to any one of claims 24 to 28, an isolated nucleic acid molecule according to claim 29, a vector according to claim 30, a host cell according to claim 31, a multimer according to claim 33, a particle according to claim 34 or a pharmaceutical composition according to claim 35 in the manufacture of a preparation for use in reducing serum levels of HBV DNA and / or HBsAg in a subject (e.g. a human), inducing an immune response (e.g. a humoral immune response) against HBV in a subject (e.g. a human), and / or preventing and / or treating HBV infection or a disease associated with HBV infection (e.g. hepatitis B) in a subject (e.g. a human), comprising: For example, the preparation is a vaccine, such as a protein vaccine or a nucleic acid vaccine; For example, the use wherein the subject is a subject not infected with HBV, or a subject infected with HBV, such as a subject with chronic HBV infection or a chronic hepatitis B patient.

37. 31. A method for reducing serum levels of HBV DNA and / or HBsAg in a subject (e.g. human), inducing an immune response (e.g. humoral immune response) against HBV in a subject (e.g. human), and / or preventing and / or treating HBV infection or a disease associated with HBV infection (e.g. hepatitis B) in a subject (e.g. human), comprising administering to a subject in need thereof an effective amount of an epitope peptide or variant thereof according to any one of claims 15 to 23, a recombinant protein according to any one of claims 24 to 28, an isolated nucleic acid molecule according to claim 29, a vector according to claim 30, a host cell according to claim 31, a multimer according to claim 33, a particle according to claim 34, or a pharmaceutical composition according to claim 35.

38. An antibody or an antigen-binding fragment thereof capable of specifically binding to the epitope peptide or variant thereof according to any one of claims 15 to 23, or an epitope contained therein, the recombinant protein according to any one of claims 24 to 28, the multimer according to claim 33, or the particle according to claim 34, For example, the antibody or antigen-binding fragment thereof may be a Fab, Fab', F(ab') ２ , Fd, Fv, dAb, complementarity determining region fragment, single chain antibody (e.g. scFv), nanobody, humanized antibody, chimeric antibody, or bispecific or multispecific antibody; For example, an antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof competes with a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4 for binding to HBsAg protein.

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