WO2023246691A1 - 抗乙型肝炎病毒的抗体及其制备和应用 - Google Patents

抗乙型肝炎病毒的抗体及其制备和应用 Download PDF

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WO2023246691A1
WO2023246691A1 PCT/CN2023/101035 CN2023101035W WO2023246691A1 WO 2023246691 A1 WO2023246691 A1 WO 2023246691A1 CN 2023101035 W CN2023101035 W CN 2023101035W WO 2023246691 A1 WO2023246691 A1 WO 2023246691A1
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antibody
cells
amino acid
seq
mutations
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French (fr)
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祁永和
郝东霞
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华辉安健（北京）生物科技有限公司
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Definitions

  • the invention belongs to the field of antibody drugs.
  • the present invention provides an antibody variant that specifically targets hepatitis B virus (HBV), the antibody variant has enhanced Fc effector functions, such as ADCC and/or ADCP activity, relative to the parent antibody, and Optionally further has an extended half-life in vivo.
  • the invention also provides methods and cells for producing said antibody variants.
  • Pharmaceutical compositions comprising the antibody variants, uses of the antibody variants, and combinations of the antibody variants with other anti-HBV drugs are also provided.
  • Hepatitis B virus (HBV) infection is the main cause of liver disease.
  • Hepatitis B (who.int) https://www.who.int/news-room/fact- sheets/detail/hepatitis-b).
  • universal vaccination of hepatitis B vaccine can effectively reduce new infections, and interferons and nucleoside (acid) anti-hepatitis B drugs can effectively inhibit virus replication, they cannot completely eliminate hepatitis B virus and achieve the goal of curing chronic hepatitis B.
  • A14 antibody is a fully human IgG1 monoclonal antibody targeting hepatitis B virus pre-S1. It can not only inhibit hepatitis B virus from entering hepatocytes, but also has the ability to eliminate hepatitis B virus or infected cells through Fc-mediated immune response ( WO2016188386A1), is a potential inhibitor for the treatment of chronic hepatitis B (WO2021013135A1).
  • antibody drugs are usually produced using animal cells as production host cells.
  • Fc effector functions of IgG-type antibodies such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities are affected by glycosylation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the structure of sugar chains in glycosylation is determined by glycosyltransferase genes involved in sugar chain synthesis and glycolytic enzymes involved in sugar chain hydrolysis in the production host cell.
  • researchers have proposed that the effector function of IgG-type antibodies, especially IgG1 subtype antibodies, can be changed by changing glycosylation modifications.
  • the inventors of US8067232B2 found that antibodies lacking fucosylation showed higher ADCC activity, and the smaller the 1,6-fucose sugar chain content, the stronger the ADCC activity.
  • the patent provides mammalian cells, such as CHO cells, in which ⁇ -1,6-fucosyltransferase is inactivated by homologous recombination and demonstrates that antibodies produced using such cells have enhanced ADCC activity.
  • US7662925 discloses a variety of Fc mutations that may change the binding affinity of IgG1 antibodies to human Fc ⁇ receptors.
  • the inventor conducted a lot of exploration and developed an anti-hepatitis B virus antibody variant with enhanced Fc effector function and extended half-life based on the A14 antibody, thus completing the present invention.
  • the present invention relates to an IgG1-type antibody variant that is capable of specifically binding to the Pre-S1 domain of hepatitis B virus (HBV) and has enhanced binding to activating Fc ⁇ receptors (Fc ⁇ Rs) relative to the parent antibody. ), reduced affinity to inhibitory Fc ⁇ receptors (Fc ⁇ R), and thus have enhanced Fc-mediated effector functions, such as enhanced ADCC and/or ADCP activity.
  • HBV hepatitis B virus
  • the antibody variant comprises one or more amino acid mutations in the heavy chain constant region relative to the heavy chain constant region of human IgG1 as set forth in the amino acids of SEQ ID NO: 31, relative to the parent antibody, And optionally, the sugar chain containing 1,6-fucose is reduced or deleted in the variant antibody.
  • the amino acid mutation is located in the Fc region of the antibody variant, which has a sequence corresponding to amino acids 114 to 330 of SEQ ID NO: 31.
  • the amino acid mutation is selected from one or more of the following group: G236A, S239D, I332E, F243L, R292PY300L, V305I, P396L, L235V, wherein the amino acid position is determined according to EU numbering .
  • the antibody variants comprising amino acid mutations in the Fc region have an extended half-life relative to unmodified antibodies.
  • the antibody variant further comprises one or more amino acid mutations selected from the group consisting of: M252Y, S254T, T256E, M428L, and N434S.
  • the present invention provides production cells for preparing afucosylated modified antibodies, the cells being FUT8-inactivated mammalian cells, such as 293F cells, CHOK1 cells, CHOZNGS-/- cells or CHO K1Q cells ; and a sequence encoding an antibody is introduced into the cell.
  • the cells are Fut8 gene knockout 293F cells, CHOK1 cells, CHOZN GS-/- cells or CHO K1Q cells.
  • said sequence introduced encodes an antibody variant of the first aspect.
  • the present invention provides a method for preparing Fut8 gene knockout production cells, comprising introducing (a) a coding sequence of a Fut8-targeting gRNA and a Cas9 nuclease into a mammalian cell, or (b) targeting Fut8 Complex of gRNA and Cas9 nuclease.
  • the present invention provides sgRNA for preparing Fut8 gene knockout cells.
  • the invention provides a method of preparing an antibody, comprising culturing the cells of the second aspect in a culture medium to produce a culture comprising the antibody.
  • the present invention provides an antibody prepared by the method of the fifth aspect.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody of the first aspect.
  • the present invention provides an expression vector comprising the isolated nucleic acid molecule of the seventh aspect.
  • the invention provides a host cell comprising the isolated nucleic acid molecule of the seventh aspect or the expression vector of the eighth aspect.
  • the invention provides a pharmaceutical composition comprising the antibody variant of the first aspect or the antibody of the sixth aspect.
  • the invention provides use of an antibody variant of the first aspect or an antibody of the sixth aspect for the preparation of a medicament for the treatment of hepatitis B or D virus infection in a subject in need thereof. , or diseases caused by the infection, such as chronic hepatitis B, or trigger Fc effector functions.
  • the present invention provides a method for treating hepatitis B or D virus infection or a disease caused by the infection, such as chronic hepatitis B, or inducing ADCC in a subject, comprising administering to the subject The antibody variant of the first aspect or the antibody of the sixth aspect is administered.
  • the present invention provides a combination of the antibody variant of the first aspect or the antibody of the sixth aspect and another antiviral drug.
  • Figure 1A-B is the plasmid map of pLenti-sgFut8-EGFP (A) and the plasmid map of pLenti-OCT1-cas9-IRES-BSD (B).
  • Figure 2 is a gel image of semi-quantitative detection of gene editing efficiency.
  • Figure 3 is a graph analyzing the binding ability of 293F wild-type cells, Fut8 gene-edited cells and FITC-lentil agglutinin (LCA) by FACS.
  • Figure 4 shows the results of Sanger sequencing analysis of the DNA sequence of sgRNA targeting the Fut8 region in FCC# cells.
  • Figure 5 is a graph analyzing the binding ability of CHOK1 wild-type cells, Fut8 gene-edited cells and PE-LCA by FACS.
  • Figure 6 shows the results of in vitro cleavage of Fut8 DNA fragments using sgcgFut8-2 gRNA/Cas9 complex.
  • Figure 7 shows the efficiency of using sgcgFut8-2 gRNA/Cas9 to edit the Fut8 gene in CHOZNGS-/- cells verified by T7E1 digestion.
  • Figure 8 shows the results of flow cytometry analysis of FITC-LCA combined with CHOZN GS-/-Fut8 gene-edited cells.
  • Figure 9 is the result of analyzing the ability of CHOZN GS-/-Fut8_KO-8# to bind FITC-LCA by flow cytometry.
  • Figure 10 is the result of Sanger sequencing analysis of the Fut8 DNA sequence targeted by sgcgFut8-2 in CHOZN GS-/-Fut8_KO-8# cells.
  • Figure 11 shows the results of verifying the efficiency of sgcgFut8-2 gRNA/Cas9 in editing the Fut8 gene in CHO K1Q cells through T7E1 enzyme digestion.
  • Figure 12 is the results of flow cytometric analysis of the ability of wild-type CHO K1Q and its defucosylated cell line to bind FITC-LCA.
  • Figure 13A-C shows the DNA sequence results of the Fut8 region targeted by sgRNA in defucosylated CHO K1Q cells analyzed by Sanger sequencing.
  • A CHO K1Q-Fut8 KO-1#;
  • B CHOK1Q-Fut8 KO-2#;
  • C CHO K1Q-Fut8 KO-3#.
  • Figure 14 shows the in vitro ADCC and ADCP activities of individual A14 mutant antibodies.
  • Figure 15 shows the dose-dependent in vitro ADCC activity of each candidate mutant antibody.
  • Figure 16 shows the in vitro ADCP activity evaluation of individual A14 mutant antibodies.
  • Figure 17 shows the results of SDS-PAGE analysis of in vitro recombinantly expressed human Fc ⁇ Rs protein.
  • Figure 18 shows the results of in vitro ADCC and ADCP activity evaluation of the mutant antibodies of Example 5.
  • Figure 19 is the result of SDS-PAGE analysis of expressed and purified A14 WT and afuco-A14 GA LS.
  • Figure 20 shows the results of the in vitro ADCC and ADCP activity evaluation of afuco-A14 GA LS.
  • Figure 21 shows the results of analyzing the affinity of afuco-A14 GA LS with human FcRn by Biacore.
  • Figure 22 shows the results of analyzing the binding activity of afuco-A14 GA LS to the target antigen Pre-S1 by ELISA.
  • Figure 23 shows the in vitro antiviral activity of afuco-A14 GA LS.
  • Figure 24 shows the half-life of afuco-A14 GA LS in hFcRn KI mice.
  • Antibody in the context of the present invention has the art-recognized meaning of an immunoglobulin with a Y-shaped structure produced by the immune system in response to a foreign substance, such as a pathogen.
  • the classical structure of an antibody is a homodimer, with each monomer containing a heavy chain and a light chain connected by a disulfide bond.
  • the light chain consists of one variable region (V L ) and one constant region (C L ), while the heavy chain consists of one variable region (V H ) and three constant regions ( CH 1 , CH 2 and CH 3 )composition.
  • Each variable region contains three complementarity-determining regions (CDRs), which constitute the antigen-binding site responsible for complementarity with the antigen.
  • the antigen-binding fragment composed of VL, CL , VH and CH1 is called “ Fab ".
  • the “Fc region” has multiple functions, including binding to effector cells through the Fc receptor (FcR) on effector cells such as macrophages or natural killer cells to activate immune effector pathways.
  • FcR Fc receptor
  • the Fc region also contains glycosylation sites, and the sugar chains connected to these glycosylation sites can also affect the Fc effector function of the antibody.
  • Neutralizing antibodies or “NAbs” refer to antibodies that can “neutralize” the biological effects of foreign substances such as pathogens to protect cells or the body.
  • HBV refers to hepatitis B virus.
  • HDV refers to hepatitis D virus.
  • Pre-S1 is the domain located at the N-terminus of the product encoded by the S gene on the surface of hepatitis B virus.
  • the S gene contains three reading frames, namely pre-S1, pre-S2 and S parts.
  • the S gene can encode three protein products with different lengths.
  • the largest product is called L (large) protein, which contains three domains: Pre-S1, Pre-S2 and S. It is longer than the other two products. (M protein containing Pre-S2 and S domains, and S protein containing only S domain)
  • the extra part is the N-terminal pre-S1 domain.
  • A14 is a fully human monoclonal antibody targeting the Pre-S1 domain of hepatitis B virus previously developed by the inventor of the present application. It can inhibit hepatitis B virus from entering hepatocytes and has the ability to mediate through Fc The ability of the immune response to eliminate HBV and/or HBV-infected cells.
  • A14 is an IgG1 type antibody.
  • the antibody is also a potential inhibitor for the treatment of chronic hepatitis B. More information about the A14 antibody can be found in PCT invention disclosure documents WO2016188386A1 and WO2021013135A1.
  • IgG1 is a subtype (sometimes called a subclass) of the immunoglobulin IgG isotype. Different IgG subtypes have different positions and numbers of disulfide bonds.
  • Antibody variant is an antibody that has been modified relative to the “parent antibody”.
  • the modification includes, but is not limited to, modification of the antibody sequence and modification of the glycosylation modification of the antibody.
  • Parent antibody refers to the antibody before modification, in specific embodiments, the A14 antibody.
  • Fc variant or “Fc antibody variant” refers to an antibody that has been engineered relative to the Fc region of the parent antibody.
  • EU numbering is a way of numbering antibody amino acids. The basis of this numbering method is based on the isolation and purification of an IgG1 immunoglobulin by Gerald M. Edelman and others in 1968 and named it Eu. By measuring its amino acid sequence, each amino acid position was numbered, thus forming Eu numbering system. Although the optimized systems of Kabat, Chothia, IMGT, etc. are currently used to encode the amino acid sequence of antibodies, since the Eu numbering is based on IgG1 antibodies, it is also suitable for encoding the IgG1 antibody A14 of the present invention. The amino acid sequence is numbered. Therefore, in the present invention, EU numbering is used to illustrate the positions of amino acid mutations.
  • Fc receptor is a receptor present on the surface of various cells (such as effector cells), which can specifically bind to the Fc region of an antibody.
  • Fc ⁇ receptor or “Fc ⁇ R” is a class of Fc receptors, all members of which belong to the immunoglobulin superfamily and are further divided into Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIB, Fc ⁇ RIIIA, and Fc ⁇ RIIIB. Each member has different affinities for different subtypes of IgG and has different functions.
  • Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIIa and Fc ⁇ RIIIb are “activating Fc ⁇ -receptors”; while Fc ⁇ RIIb is the only “inhibitory Fc ⁇ -receptor” in the human body. receptor).
  • effector function of an antibody in the context of the present invention mainly refers to Fc-mediated effector functions, including ADCC and ADCP, etc.
  • ADCC is the abbreviation of antibody-dependent cell-mediated cytotoxicity, which is a cell-mediated immune mechanism. Specifically, effector cells bind to the Fc segment of antibodies that specifically bind to target cells through receptors and release cytotoxic substances to kill target cells. ADCC is an important indicator for evaluating antibody efficacy.
  • ADCP antibody-dependent cellular phagocytosis
  • Defucosylation refers to the reduction or complete disappearance of fucose contained in the sugar chain of the antibody glycoprotein through modification.
  • fucose contained in the sugar chain of the antibody glycoprotein through modification.
  • “fucosylated” means that the antibody variant has fewer fucose-containing sugar chains relative to its parent antibody, or even no fucose-containing sugar chains at all.
  • Such antibody variants can be represented by the abbreviation "afuco-" as a prefix.
  • afucosylated means that the cell is capable of devoid of fucose in the sugar chains of the antibody when used to produce the antibody, or "afucosylated cell line” Such production cell lines.
  • FUT8 refers to alpha-(1,6)-fucosyltransferase (alpha-(1,6)-fucosyltransferase), and its encoding gene is the "Fut8 gene”. FUT8 is involved in the glycosylation process after antibody expression, adding fucose to the oligosaccharide chain.
  • FITC-LCA fluorescein isothiocyanate (FITC)-labeled lentil agglutinin (LCA).
  • PE-LCA refers to phycoerythrin-tagged lentil agglutinin. LCA has a strong affinity for fucose and can be used to detect fucose levels after fluorescent labeling with FITC or phycoerythrin.
  • Treatment means the improvement, alleviation or elimination of a disease or its symptoms (i.e. cure). In some cases, treatment includes preventive treatment.
  • “Synergy” means that when two or more drugs are administered together, they produce an additive or synergistic effect, preferably a synergistic effect, that is, the effect when administered together is better than the sum of the effects of each drug when administered alone.
  • the parent antibody of the present invention is an antibody that specifically binds to the Pre-S1 domain of HBV. Compared with parent antibodies and antibody variants, the main differences lie in the amino acid sequence of the Fc region and the degree of fucosylation.
  • the parent antibody of the invention has the constant region of an unengineered human IgG.
  • the parent antibody has an unmodified heavy chain constant region of human IgG, preferably an unmodified Fc region of human IgG.
  • the human IgG is human IgGl.
  • the parent antibody of the invention is the A14 antibody.
  • the parent antibody of the present invention may also have the same six CDR sequences (such as the amino acid sequences shown in SEQ ID NOs: 32-37) as the A14 antibody, or even the same heavy chain variable region (SEQ ID NO:1) and the light chain variable region (SEQ ID NO:5), but the heavy chain constant region in particular
  • the Fc region is derived from human IgG1 without any modification.
  • the heavy chain constant sequence of human IgG1 as shown in SEQ ID NO: 31 is used as the basis, and the EU numbering system is used. Describe the type and location of amino acid mutations.
  • the human IgG1 heavy chain constant region is a sequence of 330 amino acids from alanine at position 118 to lysine at position 447, of which positions 118 to 214 are CH1 ( Corresponding to amino acid positions 1-97 of SEQ ID NO:31), positions 215 to 230 are the hinge region (corresponding to amino acid positions 98-113 of SEQ ID NO:31), and positions 231 to 340 are CH2 (corresponding to SEQ ID NO:31 ID NO: 31, amino acid positions 114-223), and positions 341 to 447 are CH3 (corresponding to amino acid positions 224-330 of SEQ ID NO: 31).
  • the position of the mutated amino acid in the EU numbering system is recorded instead of its position number in SEQ ID NO: 31.
  • the number in the EU number will be 117 greater than the number of positions of the amino acid in SEQ ID NO:31.
  • the amino acid type corresponding to the amino acid position in SEQ ID NO:31 is used as the amino acid before mutation and is placed before the position number (on the left), and the amino acid present in the mutated variant antibody is placed After the position number (right side).
  • the same position can be mutated into different amino acids, separate multiple optional amino acids with "/".
  • G236A mutation of the present invention refers to the mutation of glycine at position 236 to alanine according to EU numbering; and relative to the amino acid sequence of SEQ ID NO:31, this mutation is equivalent to Glycine at position 119 was mutated to alanine.
  • S239D/Q/E means that the serine at position 239 is mutated to aspartic acid, glutamine or glutamic acid according to EU numbering; and relative to the amino acid sequence of SEQ ID NO:31, this The mutation is equivalent to mutating serine at position 122 to aspartic acid, glutamine or glutamic acid.
  • Fc ⁇ R-mediated effects including ADCC and ADCP activities Response function is related to the ability of antibody-mediated clearance of target cells.
  • the antibody variants of the present invention improve the ADCC and/or ADCP activity of the original antibody through different means, thereby further improving the efficacy of the antibody.
  • Natural killer cells are the main immune effector cells involved in ADCC.
  • monocytes and eosinophils can also mediate ADCC.
  • the strength of antibody-mediated ADCC is related to many factors, such as the affinity of the antibody to the antigen, the affinity of the antibody Fc to the receptor Fc ⁇ RIIIa, and the characteristics of immune effector cells. Improving the affinity between Fc and Fc ⁇ RIIIa by modifying the glycosylation and amino acid sequence of the Fc segment of the antibody is an effective way to enhance ADCC activity.
  • FUT8 ⁇ -1,6 fucosyltransferase encoded by the Fut8 gene.
  • FUT8 forms the core fucose of the Fc segment oligosaccharide by transferring the fucose residue from guanosine diphosphate-fucose (GDP-Fuc) to the last position of the innermost acetylglucosamine (GlcNAc) of the Fc segment oligosaccharide chain. sugar.
  • ⁇ -1,6-fucosyltransferase is a key glycosyltransferase in the fucosylation synthesis pathway.
  • various methods have been used to reduce the level of fucosylation modification of IgG antibodies, such as using Fut8 gene knockout or low-expression antibody production cell lines, or using transgenes to increase the level of fucosylation in antibody production cell lines. Express GnTIII etc.
  • Knockout of the Fut8 gene in antibody-producing cells can be carried out in different ways, such as small interfering RNA (siRNA), short hairpin RNA, homologous recombination and other methods known in the art.
  • knockout of the Fut8 gene in antibody-producing cells is performed by nuclease-mediated homologous recombination.
  • Nuclease-mediated homologous recombination includes but is not limited to CRISPR/Cas9, TALEN, and ZFN.
  • the knockout of the Fut8 gene in antibody-producing cells is performed by the CRISPR/Cas9 system. The specific CRISPR/Cas9 editing method is described in detail later.
  • antibody variants of the invention comprise amino acid mutations in the Fc region relative to the parent antibody to improve ADCC activity.
  • Amino acid mutations used to improve ADCC activity can enhance the effect of ADCC by increasing the affinity of the antibody to activating receptors (such as human Fc ⁇ RIIIa), and/or by reducing the affinity of the antibody to inhibitory receptors (such as human Fc ⁇ RIIb).
  • the antibody variants of the invention may comprise one or more amino acid mutations for improving ADCC activity selected from the group consisting of: L235V, S239E/D/Q, F243L, R292P, Y300L, V305I, A330L, I332E, P396L.
  • Antibody variants with amino acid mutations can be introduced through molecular biology methods. Specifically, the antibody variant containing the amino acid mutation is obtained by synthesizing a nucleotide sequence encoding the amino acid mutation, inserting it into a suitable construct, and introducing the construct into a host cell for expression. Amino acid mutations used to improve other different effects below, such as amino acid mutations used to improve ADCP and half-life, can also be introduced by the same method.
  • Antibody variants can have both a knockout of the Fut8 gene and amino acid mutations that improve ADCC activity, or they can have only one of them, resulting in improvements in mediating ADCC activity.
  • ADCP activity in humans can also be tuned by engineering the Fc. ADCP activity can be enhanced by increasing the affinity to the activating receptor human Fc ⁇ RIIIa or decreasing the affinity to the inhibitory receptor human Fc ⁇ RIIb.
  • G236A One amino acid mutation that enhances ADCP activity is G236A, which reduces affinity to the inhibitory receptor Fc ⁇ RIIb.
  • the antibody variants of the invention comprise modifications that improve both ADCC and ADCP functions, including afucosylation modifications and amino acid mutations in the Fc region.
  • an antibody variant of the invention has, relative to its parent antibody:
  • the antibody variants of the invention have modifications as shown in items 3-33 in Table 6.
  • the antibody variant of the invention has mutations selected from the group (a)-(f) relative to its parent antibody:
  • the antibody variant of the invention has reduced 1,6-fucose-containing sugar chains or a lack of 1,6-fucose-containing sugar chains relative to its parent antibody. , and has the G236A amino acid mutation.
  • the half-life of an antibody is known to be altered in a number of ways.
  • the half-life is altered by altering the affinity of the antibody for the FcRn receptor.
  • the affinity of the resulting antibody variant for the FcRn receptor is enhanced by introducing amino acid mutations in the Fc region of the antibody, thereby extending half-life.
  • the antibody variants of the invention comprise one or more amino acid mutations selected from the group consisting of: M252Y, S254T, T256E, M428L, N434S relative to the parent antibody thereof.
  • the antibody variant of the invention comprises an amino acid mutation selected from the following group (g) or (h) relative to its parent antibody:
  • the antibodies of the invention possess both improved Fc-mediated effector function and extended half-life.
  • an antibody variant of the invention relative to its parent antibody, comprises:
  • One or more amino acid mutations optionally selected from the group consisting of: M252Y, S254T, T256E, M428L, N434S.
  • an antibody variant of the invention has, relative to its parent antibody, a mutation selected from a group of (a)-(f) and one mutation selected from (g) or (h) , or differing relative to the parent antibody only by a mutation selected from the group of (a)-(f) and a mutation selected from (g) or (h):
  • the antibody variant of the invention has reduced 1,6-fucose-containing sugar chains or lacks 1,6-fucose-containing sugar chains relative to the parent antibody, and G236A, M428L and N434S amino acid mutations.
  • the antibody variants of the invention have affinity for the Pre-S1 region of hepatitis B virus and/or viral activity comparable to that of the parent antibody despite one or more modifications relative to the parent antibody.
  • the antibody variant of the present invention only changes the Fc region compared to the parent antibody, but does not change the variable region and complementarity determining region (CDR), and therefore has a comparable activity against hepatitis B virus, specifically Pre-S1, as the parent antibody. protein binding affinity.
  • the antibody variants of the invention have comparable IC50 values relative to the parent antibody.
  • “Comparable” activity or value means approximately the same or similar.
  • "activity comparable to that of the parent antibody” means that the relative activity (IC 50 value) of the variant antibody of the invention and its parent antibody does not differ by more than 20%.
  • Mammalian cell lines commonly used in the art for antibody production can be used in the present invention for the production of the antibodies of the present invention.
  • Cells particularly suitable for the present invention are those useful as host cells for the development and production of recombinant protein drugs in the biopharmaceutical industry.
  • producer cell lines suitable for use in the present invention include, but are not limited to: 293F, CHO-K1, CHOZN GS -/-, and CHOK1Q. These engineered cells can be used in the development and production of the pharmaceutical industry. The cell doubling time is maintained at 12 to 40 hours, and the cell viability is ⁇ 90%.
  • 293F cells are derived from primary human embryonic kidney cells, which have high transfection efficiency and can be used to culture cells with high protein expression in suspension culture in serum-free medium. 293F cells proliferate quickly and can be cultured at high density.
  • CHO-K1, CHOZN GS -/- and CHOK1Q are all adult Chinese hamster ovary (CHO) cells.
  • CHO-K1 are unmodified wild-type CHO cells.
  • the CHOZN GS -/- cell line uses ZFN (zinc finger nuclease) technology to knock out the glutamine synthetase gene, making the survival of the cell line dependent on the supplement of exogenous glutamine. Therefore, there is no need to add additional supplements during the entire screening process.
  • Highly expressed target clones can be obtained by applying MTX or MSX pressure.
  • CHOK1Q is a wild-type CHO-K1 cell that has not been genetically modified, but has been modified by suspension, high osmotic pressure, high ammonium ions, and high lactic acid conditioning.
  • the CRISPR/Cas method used in the present invention has the advantages of simple operation, high targeting accuracy, high editing efficiency, no exogenous DNA insertion into the host genome, and high safety.
  • CRISPR/Cas9 gene editing technology uses guide RNA (sgRNA) to guide Cas9 endonuclease to cleave the target double-stranded DNA in the genome, causing double-stranded breaks in the target sequence, which are then repaired under the action of the DNA repair mechanism in the cell. Substitutions, deletions, insertions, etc. occur on the genome, thereby achieving knockout of the target gene.
  • the CRISPR/Cas9 system is now widely used for gene editing in various organisms. We use CRISPR/Cas9 technology to target the Fut8 gene in 293F, CHOZN GS -/- or CHOK1, CHOK1Q cells to obtain antibodies that can produce defucosylation and improve the ADCC activity of the antibodies.
  • nucleotide sequence of SEQ ID NO:10 can be used as sgRNA to knock out the Fut8 gene.
  • the nucleotide sequence selected from SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21 can be used as sgRNA to knock out the Fut8 gene.
  • an sgRNA having the nucleotide sequence of SEQ ID NO: 20 is used.
  • the antibody or antibody variant of the present invention is an antibody for use in humans.
  • the antibody or antibody variant of the invention can be used to treat or prevent hepatitis B, especially for the treatment of hepatitis B. Treat or prevent chronic hepatitis B.
  • the antibody or antibody variant of the present invention can be used as an adjuvant treatment for the treatment of hepatitis B, that is, as an auxiliary means for first-line treatment drugs to further improve the therapeutic effect.
  • Anti-hepatitis B virus drugs that can be combined with the antibody of the present invention include immunomodulatory drugs, such as interferon, thymosin- ⁇ 1, and cytokines; nucleoside (acid) analogs, such as Entecavir and Telbivudine (Telbivudine), Adefovir or Adefovir dipivoxil, Tenofovir, Lamivudine.
  • immunomodulatory drugs such as interferon, thymosin- ⁇ 1, and cytokines
  • nucleoside (acid) analogs such as Entecavir and Telbivudine (Telbivudine), Adefovir or Adefovir dipivoxil, Tenofovir, Lamivudine.
  • an antibody or antibody variant of the invention can be used in combination with entecavir.
  • the antibodies or antibody variants of the invention may also be used to treat or prevent hepatitis D.
  • the antibodies or antibody variants of the invention can be administered to a subject by conventional delivery means.
  • the antibodies of the invention are administered to the subject parenterally, preferably by intravenous infusion or subcutaneous injection.
  • the invention also relates to the following items:
  • An antibody variant that specifically binds to the Pre-S1 domain of hepatitis B virus (HBV) and has enhanced Fc-mediated effector function compared to the parent antibody.
  • HBV hepatitis B virus
  • the antibody variant according to item 1 or 2 which has enhanced affinity for activating Fc ⁇ receptors and/or reduced affinity for inhibitory Fc ⁇ receptors.
  • sugar chain containing 1,6-fucose is reduced or deleted, and/or one or more amino acid modifications selected from the following group: L235V, S239E/D/Q, F243L, R292P, Y300L, V305I, A330L, I332E, P396L; and
  • the antibody variant according to item 7 which has a reduced sugar chain containing 1,6-fucose or a deletion of a sugar chain containing 1,6-fucose, and has a G236A amino acid mutation (GA mutation).
  • the antibody variant of item 10 which comprises one or more amino acid mutations selected from the group consisting of: M252Y, S254T, T256E, M428L, N434S relative to the parent antibody.
  • One or more amino acid mutations optionally selected from the group consisting of: M252Y, S254T, T256E, M428L, N434S.
  • the antibody variant according to item 14 which has reduced sugar chains containing 1,6-fucose or lacks sugar chains containing 1,6-fucose relative to the parent antibody, and G236A, M428L and N434S amino acid mutations.
  • the antibody variant described in any one of items 1-15, the parent antibody and the antibody variant have the CDR sequence shown in SEQ ID NOs: 32-37.
  • the parent antibody is the A14 antibody, which has a heavy chain variable region sequence as shown in SEQ ID NO: 1, and a heavy chain constant region as shown in SEQ ID NO: 38 Sequences, such as the light chain variable region sequence shown in SEQ ID NO:5, and the light chain constant region sequence shown in SEQ ID NO:6.
  • HBV hepatitis B virus
  • An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody variant of any one of items 1-18 or the antibody of any one of items 19-20.
  • An expression vector comprising the isolated nucleic acid molecule described in item 21 or 22.
  • a host cell comprising the isolated nucleic acid molecule described in item 21 or 22 or the expression vector described in item 23.
  • a mammalian cell characterized in that FUT8 in the cell is inactivated, and the isolated nucleic acid molecule described in item 21 or 22 or the expression vector described in item 23 is introduced into the cell.
  • a method for preparing antibodies comprising the following steps:
  • a method for preparing Fut8 gene knockout mammalian cells comprising introducing into the mammalian cells:
  • introduction via (b) is preferred.
  • the method of item 30, wherein the mammalian cells are selected from the group consisting of 293F cells, CHOK1 cells, CHOZN GS-/- cells, and CHO K1Q cells.
  • nucleotide sequence of the gRNA comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequence of the sgRNA is selected from the group consisting of SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:39 The nucleotide sequence shown.
  • sgRNA for knocking out the Fut8 gene which has a nucleotide selected from the group consisting of SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:39 sequence.
  • the sgRNA described in item 35 which is used to knock out the Fut8 gene in 293F cells and has the nucleotide sequence shown in SEQ ID NO: 10.
  • the sgRNA described in item 35 which is used to knock out the Fut8 gene in CHO-K1 cells and derivative cells thereof and has SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO :The nucleotide sequence shown in 39.
  • a method for preparing antibodies comprising the following steps:
  • step (1) (2) culturing the mammalian cells obtained in step (1) to produce a culture containing the antibody;
  • step (3) Collect the antibody from the culture of step (2).
  • a pharmaceutical composition comprising the antibody variant described in any one of Items 1-18, the antibody described in any one of Items 19-20 or 39, and a pharmaceutically acceptable carrier.
  • a method of treating hepatitis B or D virus infection or a disease caused by said infection in a subject, or inducing an ADCC effect comprising administering to said subject a therapeutically effective amount of any of items 1-18
  • the antibody variant described in one item, the antibody described in any one of items 19-20 or 39, or the pharmaceutical composition of item 40 comprising administering to said subject a therapeutically effective amount of any of items 1-18.
  • any one of items 44-47 comprising administering to the subject another drug for treating or preventing infection by hepatitis B virus (HBV) Diseases caused, especially chronic diseases caused by hepatitis B virus (HBV) infection.
  • HBV hepatitis B virus
  • a pharmaceutical combination for treating hepatitis B or D virus infection or diseases caused by said infection including: (a) the antibody variant described in any one of items 1-18, items 19-20 or The antibody of any one of 39; and (b) another medicament for the treatment or prevention of diseases caused by hepatitis B virus (HBV) infection, especially caused by hepatitis B virus (HBV) infection Chronic disease.
  • HBV hepatitis B virus
  • Example 1 Establishment of afucoprotein-producing cell line
  • This example describes the establishment of a fucosylated cell line. Specifically, the Crispr/Cas9 nuclease editing system was used to knock out the Fut8 gene in the 293F cell line, so that the antibodies produced by the edited cells lacked fucosylation modification.
  • the sgRNA targeting the target gene required by the Crispr/Cas9 system was designed.
  • two sgRNA sequences targeting the Fut8 gene (NM_178155.2) in 293F cells purchasedd from Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • the sequences are shown below as SEQ ID NO:9 and SEQ ID NO:10.
  • the sgRNA targeting sequence is underlined, and the PAM sequence (protospacer adjacent module sequence; protospacer adjacent motif) is represented in bold.
  • Two primer pairs were subsequently synthesized (sgFut8-1-F1 and sgFut8-1-R1; sgFut8-2-F1 and sgFut8-2-R1, see Table 1).
  • the double-stranded product was cloned into the BfuAI-digested pLenti-sgRNA-EGFP vector (purchased from Addgene) to obtain sgRNA expression plasmids pLenti-sgFut8-1-EGFP and pLenti-sgFut8-2- respectively.
  • T7E1 In the T7E1 experiment, two pairs of primers as shown in Table 1 below were used for the two sgRNAs (T7-sgFut8-1-F1 and T7-sgFut8-1-R1; T7-sgFut8-2-F1 and T7-sgFut8 -2-R1). Since T7E1 endonuclease can recognize mismatched bases on DNA and cut the identified site, T7E1 can be used to conduct semi-quantitative detection of gene editing efficiency after gene editing to determine the efficiency of gene editing and determine The results are shown in Figure 2. As shown in Figure 2, it was confirmed that the two designed sgRNAs can effectively edit the Fut8 gene in cells.
  • monoclonal cells were obtained from the T7E1-verified cells by limiting dilution method, and cell binding experiments based on flow cytometry (FACS) were performed on the cloned cells. Verify whether the monoclonal cells are gene-edited monoclonal cells through functional phenotypic analysis.
  • Lens Culinaris Agglutinin (LCA) is a lectin that can specifically recognize and bind fucose-modified polysaccharides.
  • Fluorescein isothiocyanate (FTIC)-labeled LCA (FITC-LCA) It can be used to detect fucose modification and modification levels of cell surface proteins.
  • FACS results are shown in Figure 3.
  • the unedited 293F wild-type cells bind very strongly to FITC-LCA, and the FITC signal value is the strongest; while the successfully edited cells, such as the cell clone code-named FCC#, bind to LCA significantly. reduce.
  • the cell clone code-named F28# has no difference in its ability to bind LCA compared with wild-type cells, indicating that F28# is a cell that has not been successfully edited.
  • Sanger sequencing was further performed on FCC# cells to determine the specific gene editing form. The results showed that they were cells with homozygous Fut8 knockout (Figure 4).
  • FCC#-293F cell a 293F cell with stable knockout of the Fut8 gene (called "FCC#-293F cell"), which can be used to express fucose-free antibodies.
  • This cell can be used as a host cell for the production of defucosylated antibodies.
  • the sgRNA fragment was connected into the pLenti-sgRNA-EGFP vector, and then co-transfected into CHO-K1 cells through PEI together with the Cas9-expressing plasmid pLenti-OCT1-Cas9-IRES-BSD (purchased From the National Experimental Cell Resource Sharing Platform). Two days after transfection, flow cytometry was used to screen EGFP-positive cells (i.e., sgRNA and Cas9 co-expressing cells). After continuing to culture for 5 days, a cell binding test was performed with Rhodamine (Rho)-labeled LCA. The results are shown in Figure 5.
  • Rhodamine Rho
  • All four designed sgRNAs can effectively edit the Fut8 gene in CHO-K1 cells, and the ability of cells co-expressing sgcgFut8 and Cas9 to bind Rho-LCA is greatly reduced, indicating that the cell surface protein Sugar levels are significantly reduced. This result illustrates the successful editing of the Fut8 gene and effectively silences the expression of FUT8 protein.
  • sgcgFut8-2 In order to establish a fucosylated CHOZNGS-/- stably transduced cell line, one of the four verified sgRNAs (sgcgFut8-2) described in 2.1 was selected for gene editing. To deliver sgRNA/Cas9 into cells, a DNA-free, viral vector-free, and transfection reagent-free approach was used. The primer sequence information used in the study is shown in Table 3.
  • a gRNA/Cas9 ribonucleoprotein (RNP) complex is formed by co-incubating the sgRNA synthesized by in vitro transcription with the Cas9 protein with a signal peptide.
  • This RNP complex was then electroporated into CHOZNGS-/- cells (purchased from Merck Batch No: 2113-53667), allowing it to target the double-stranded DNA cleavage on the cell genome to edit the Fut8 gene.
  • the delivery method of RNP complex is delivered by electroporation.
  • LCA acts as a lectin that specifically binds fucose. After binding to fucose-modified proteins on the cell membrane surface, LCA is endocytosed into cells and causes cell death. Based on this principle, LCA pressure screening was combined to improve the success rate of Fut8 knockout cells.
  • the underline is the sequence of the T7 promoter, the italics are the gRNA targeting sequence, and the normal font is the gRNA conserved backbone sequence.
  • PCR reaction conditions were: pre-denaturation at 98°C for 2 minutes; then denaturation at 98°C for 10 seconds, annealing at 60°C for 10 seconds, extension at 72°C for 10 seconds, a total of 35 cycles; and finally extension at 72°C for 4 minutes.
  • the PCR product after the PCR reaction was subjected to 2% agarose gel electrophoresis, and the product with the expected size of 123 bp was recovered by cutting the gel. Then T7-sgcgFut8-2 sequencing was used to confirm the correct synthesis of the sgDNA template.
  • sgDNA As a template, prepare the following reaction system (Table 5) according to the instructions of the T7 in vitro transcription kit (NEB), and incubate at 72°C for 16 hours. The gRNA product obtained after transcription is purified to obtain gRNA.
  • the defucosylated CHO K1Q cell line was established using the same method used to establish the fucosylated CHOZN GS-/- cell line. A total of 3 independent experiments were conducted.
  • the Fut8 gene in CHOK1Q cells (purchased from Zhongshan Kangsheng) was successfully edited by electroporation of sgcgFut8-2 gRNA/Cas9 RNP.
  • the target DNA fragment is cleaved into two smaller fragments by the T7E1 enzyme.
  • Figure 11 also shows that after LCA pressure screening, the efficiency of gene editing is further improved because the content of the target DNA fragment is further reduced after being cleaved by T7E1 enzyme.
  • the cells electrotransduced with sgcgFut8-2 gRNA/Cas9 after cloning LCA pressure screening were verified by FITC-LCA cell binding assay.
  • the ability of CHO K1Q-Fut8 KO-1#, 2# and 3# obtained from three independent experiments to bind to FITC-LCA was significantly reduced, indicating that the ability of the cells to produce fucose-modified polysaccharides or proteins was reduced.
  • Sanger sequencing was performed on the Fut8 region targeted by sgcgFut8-2.
  • the sequencing results further confirmed that CHO K1Q-Fut8 KO-1#, 2# and 3# are Fut8 homozygous knockout CHO K1Q cell lines.
  • This embodiment involves modifying anti-hepatitis B virus neutralizing antibodies, especially the Fc region of the antibodies, to improve their ADCC and ADCP activities.
  • the inventor used genetic engineering technology to introduce amino acid mutations into the heavy chain Fc region of the expressed A14 antibody, and obtained a series of A14 Fc variants; the inventor constructed these A14 Fc variants into the eukaryotic expression plasmid pCAGGS vector (Addgene) , and expressed in the FCC#-293F cells (Fut8 gene deleted) constructed in Example 1, and a variety of defucosylated antibodies were obtained. A total of 33 different A14 Fc variant.
  • the Jurkat/NFAT-luc2p/Fc ⁇ RIIIa F158 and Jurkat/NFAT-luc2p/Fc ⁇ RIIa R131 transgenic cell lines expressing Fc ⁇ receptors were constructed by the inventor based on Jurkat parental cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Cell Center).
  • the target cells were CHO (CHO-59C) stably expressing the A14 binding epitope, and the ADCC and ADCP activities of the A14 Fc variants were evaluated.
  • An Fc variant (DANA) antibody that has lost its binding activity to Fc ⁇ receptors was also set up in the experiment as a control.
  • the concentrations of A14 Fc mutant antibodies were set to 30 ng/ml and 100 ng/ml respectively, and the ratio of effector cells to target cells was 6:1. After incubation at 37°C for 6 hours, the cell expression was detected. Relative luciferase activity (RLU) ( Figure 14).
  • the luciferase activity value RLU induced by A14 DANA was set to 1, and the relative ADCC and ADCP activities induced by other A14 Fc mutations were compared.
  • afuco-A14 GA excels in mediating both ADCC and ADCP activities.
  • afuco-A14 GA is not only better than afuco-A14 that only undergoes defucosylation modification, or Fc amino acid mutant variant that only undergoes GA mutation, it is also better than that in mediating ADCC and ADCP.
  • the starting protein amount of the antibody was 10 ⁇ g/ml, and 11 gradient dilutions of different concentrations were prepared at a 3-fold dilution ratio.
  • the Jurkat/NFAT-luc2p/Fc ⁇ RIIIa F158 transgenic cell line (which stably expresses NFAT-luc2p and Fc ⁇ RIIIa F158 in Jurkat cells, where Jurkat cells were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) was used as effector cells and target cells. It is CHO (CHO-59C) that stably expresses the A14 binding epitope. The ratio of effector cells to target cells is 6:1.
  • ADCP experiments afuco-A14, afuco-A14 GA, and A14-GASD mutant antibodies were further analyzed, and A14 WT was used as a control.
  • the effector cells used were macrophages derived from mouse bone marrow cells induced to differentiate in vitro; the target cells were 293F cells (293F-59C) expressing A14-binding epitopes. Effector Cells with Red Fluorescent Alexa 647 anti-mouse F4/80 monoclonal antibody was labeled, and the target cells were labeled with the green fluorescent dye CFSE. Set up negative control (PBS). The target cells were incubated with A14 Fc mutant antibody (10 ⁇ g/mL) and then added to the effector cell culture plate. Both effector cells and target cells were 2 ⁇ 10 5 cells/well. The ratio of effector cells to target cells (E:T) The ratio is 1:1.
  • the afuco-A14-GA antibody was selected as the leading candidate molecule for immune effect enhancement.
  • human Fc ⁇ R receptors with a C-terminal 6xHis tag were recombinantly expressed in 293F cells and purified through Ni columns.
  • Biacore was used to analyze the affinity of several A14 Fc variants obtained through preliminary screening to various human Fc ⁇ Rs, including activating receptors hFc ⁇ R1a, hFc ⁇ R2a H131, hFc ⁇ R2a R131, hFc ⁇ R3a V158, hFc ⁇ R3a F158, and inhibitory receptor hFc ⁇ R2b.
  • V158 and H131 are high-affinity receptor SNPs
  • F158 and R131 are low-affinity receptor SNPs.
  • ADCC activity of antibody variants is similar to that of hFc ⁇ R3a
  • the affinity of V158 and hFc ⁇ R3a F158 is related; the ADCP activity of the antibody variant is related to its affinity to hFc ⁇ R2aH131 and hFc ⁇ R2a R131.
  • the results obtained are summarized in Table 7.
  • afuco-A14 GA was selected as a leading candidate molecule for immune effect enhancement. On this basis, the half-life of the antibody was optimized.
  • afuco-A14 GA LS and afuco-A14 GA YTE variants were further obtained through genetic engineering, aiming to extend the half-life of the antibodies in the body.
  • This example further tested the various properties and functions of the afuco-A14 GA LS antibody and compared it with the unmodified A14 antibody.
  • A14 WT and afuco-A14 GA LS antibodies were expressed in 293F and FCC# cells respectively. After protein A purification, protein quantification was performed. Then 2 ⁇ g were taken for SDS-PAGE electrophoresis analysis. The results are shown in Figure 19.
  • Biacore was used to analyze the affinity of afuco-A14 GA LS with hFc ⁇ RIa, hFc ⁇ RIIa H131, hFc ⁇ RIIa R131, hFc ⁇ RIIb, hFc ⁇ RIIIa V158 and hFc ⁇ RIIIa F158.
  • Protein G is amino-coupled to the CM5 chip, and then the A14 WT antibody and afuco-A14 GA LS antibody are captured respectively.
  • Different concentrations of recombinant hFc ⁇ R are used as mobile phases through the flow path at pH 7.0 to interact with the antibodies on the chip. Binding dissociation occurred, and the affinities were calculated using a 1:1 Langmuir binding model fit to the data, and the results are shown in Table 8.
  • the same Jurkat/NFAT-luc2p/Fc ⁇ RIIIa F158 and Jurkat/NFAT-luc2p/Fc ⁇ RIIa R131 transgenic cell lines as in 3.2 were used as effector cells, and the target cells were CHO (CHO-59C) stably expressing the A14-binding epitope.
  • the ratio of effector cells to target cells is 6:1.
  • the initial concentration of the antibody was 10 mg/ml, and it was diluted 3 times.
  • a total of 11 gradient dilution samples with different concentrations were obtained to compare the ADCC and ADCP activities of afuco-A14 GA LS and A14 WT antibodies.
  • Negative control wells (wells containing only PBS without antibodies) and background well controls were set up in the experiment.
  • the half-life of an antibody in the body is mainly determined by the affinity of the antibody Fc to FcRn on the surface of human endothelial cells.
  • the binding of FcRn to antibody IgG is pH-dependent and only binds at weakly acidic pH around 6.0, but does not bind at neutral pH. Therefore, the binding activity of afuco-A14 GA LS to human FcRn under acidic conditions was analyzed by Biacore.
  • the recombinantly expressed hFcRn was amino-coupled to the CM5 chip, and then different concentrations of A14 WT antibody and afuco-A14 GA LS antibody were used as mobile phases to bind and dissolve the hFcRn on the chip through the flow path under pH 6.0 conditions.
  • the 1:1 Langmuir binding model was used to fit the data and the affinity was calculated. The results are shown in Figure 21.
  • HepG2-hNTCP cells prepared by the inventor as host cells for HBV infection Tie.
  • Serial dilutions of afuco-A14 GA LS and A14 WT were mixed with equal volumes of recombinant HBV genotype D virus (according to Yan, H., et al., Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.
  • Elife, 2012.1: p.e00049 (described in preparation) was mixed and used to infect HepG2-hNTCP cells. Cell culture supernatants were collected on the fifth day after infection.
  • the HBeAg antigen ELISA detection kit (Beijing Wantai Biopharmaceutical Co., Ltd.) was used to detect the secretion amount of HBeAg in the cell culture supernatant, and the HBeAg secretion amount in the virus-infected control group (only PBS without added antibodies) was used as the base. Calculate the percent inhibition at different antibody concentrations. Using GraphPad Prism software, with the logarithm of concentration as the abscissa and the inhibition rate as the ordinate, use a four-parameter fitting regression model to draw a curve and calculate the IC50 value. The results showed that afuco-A14 GALS and A14 WT had comparable IC50s of approximately 11.0 ng/ml ( Figure 23).
  • mice The half-life of afuco-A14 GA LS was further studied in mice.
  • Human FcRn knock-in (hFcRn KI) mice expressing human FcRn were used in this experiment.
  • the experiment set up A14 WT and afuco-A14 GA LS groups, with 6 mice in each group, half male and half female.
  • the antibody dose is 10 mg/kg, and the route of administration is intraperitoneal injection.
  • Blood was collected from the mice at the following time points: before administration, 10 minutes, 1 hour, 3 hours, 10 hours, 1 day, 3 days, 7 days, 10 days, 14 days, 17 days, 21 days after administration. 28 days, 35 days.
  • ELISA method was used to detect the blood drug concentration in mouse serum.
  • afuco-A14 GA was evaluated in the liver humanized chimeric hFRG (Fah -/- Rag2 -/- /IL2rg -/- ) mouse model. Effects of LS administered alone and in combination with entecavir.
  • Entecavir is a hepatitis B virus reverse transcriptase inhibitor that treats hepatitis B by inhibiting viral replication.
  • the disadvantage of entecavir is that viral replication rebounds once the drug is discontinued.
  • a control group normal saline
  • an afuco-A14 GA LS monotherapy group 10 mg/kg
  • entecavir (ETV) monotherapy group ETV, 0.1 mg/kg
  • afuco-A14 GA LS 10 mg/kg
  • entecavir combination treatment group 0.1 mg/kg
  • Mice were administered the drug starting on the 28th day (D28) after HBV infection (HBV D genotype, 2 ⁇ 10 9 GE/mouse) (afuco-A14 GA LS was administered by subcutaneous injection, and ETV was administered orally). Thereafter, afuco-A14 GA LS was administered subcutaneously once a week (D35, D42, D49, D56, D63, D70), while ETV was administered once a day until D70. D91 was observed after drug withdrawal.
  • the plasma of experimental mice was collected through venous blood collection, and then qPCR was used to quantitatively detect the HBV DNA level in the mouse plasma.
  • Experimental results showed that compared with the control group given physiological saline, the HBV DNA in the serum of mice in the drug treatment group was significantly reduced.
  • the viral DNA levels in the afuco-A14 GA LS monotherapy group and ETV monotherapy group decreased by approximately 7.3-fold and 436-fold, respectively.
  • Combining afuco-A14 GA LS with ETV further reduced the level of serum virus titer, reaching 855 times.
  • the viral titers in each treatment group rebounded after drug withdrawal, but were still lower than the baseline D28 viral titers.
  • the mean virus titers of each group at the end of the trial (D91, 21 days after drug withdrawal) compared with the starting point of treatment (D28) were reduced by: afuco-A14 GA LS single drug was 2.43 times; ETV single drug was 7.15 times; afuco -A14 GA LS combined with ETV is 12.6 times more effective.

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Abstract

本发明提供一种抗乙型肝炎病毒的抗体，其具有增强的Fc介导的效应功能，如ADCC和ADCP活性，并任选地进一步具有延长的半衰期。本发明还公开用于生产所述抗体的方法和细胞，以及所述抗体的用途。

Description

抗乙型肝炎病毒的抗体及其制备和应用 技术领域
本发明属于抗体药物领域。本发明提供一种特异性靶向乙型肝炎病毒(hepatitis B virus,HBV)的抗体变体，所述抗体变体相对于亲本抗体具有增强Fc的效应功能，如ADCC和/或ADCP活性，并且任选地进一步具有延长的体内半衰期。本发明还提供用于生产所述抗体变体的方法和细胞。还提供包含所述抗体变体的药物组合物，所述抗体变体的用途，以及所述抗体变体与其他抗HBV药物的联用。
背景技术
乙型肝炎病毒(HBV)感染是引起肝脏疾病的主要原因。全球有2.96亿慢性乙肝感染者，每年约有82.0万人死于乙肝病毒感染引起的肝硬化或肝癌(Hepatitis B(who.int)https://www.who.int/news-room/fact-sheets/detail/hepatitis-b)。虽然乙肝疫苗的普及接种能够有效降低新发的感染，干扰素、核苷(酸)类抗乙肝药物可以有效抑制病毒的复制，但不能完全清除乙肝病毒，达到治愈慢性乙肝的目标。因此绝大多数慢性乙肝患者需要长期服用，甚至持续病毒学抑制的患者仍有可能发展为肝癌(Grossi,G.,et al.(2017).HepatitisB virus long-term impact of antiviral therapy nucleot(s)ide analogues(NUCs).Liver Int 37 Suppl 1:45-51)。
A14抗体是一种靶向乙肝病毒pre-S1的全人源IgG1型单克隆抗体，其不仅能够抑制乙肝病毒进入肝细胞，而且具有Fc介导的免疫反应从而清除乙肝病毒或感染细胞的能力(WO2016188386A1)，是治疗慢性乙肝的潜在抑制剂(WO2021013135A1)。
在制药工业中，抗体药物通常以动物细胞作为生产宿主细胞进行生产。已知IgG型抗体的Fc效应功能(effector functions)如抗体依赖性细胞介导的细胞毒性(ADCC)和补体依赖性细胞毒性(CDC)活性受到糖基化的影响。另外，还认为糖基化中糖链的结构由生产宿主细胞中参与糖链合成的糖基转移酶基因以及参与糖链水解的糖酵解酶决定。研究者提出可以通过改变糖基化修饰来改变IgG型抗体，特别是IgG1亚型抗体的效应功能。
US8067232B2的发明人发现缺乏岩藻糖基化的抗体表现出更高的 ADCC活性，并且1,6-岩藻糖糖链含量越少其ADCC活性越强。该专利提供了通过同源重组使α-1,6-岩藻糖基转移酶失活的哺乳动物细胞，如CHO细胞，并证明了使用这样的细胞生产的抗体具有增强的ADCC活性。
US7662925公开了多种可能改变IgG1抗体与人Fcγ受体结合亲和力的Fc突变。
然而，鉴于抗体蛋白分子结构和功能的复杂性，以及糖链结构的多样性和复杂性，改变哪些氨基酸位点或哪种糖基化修饰能够将某种抗体的效应功能以及其他性质调整到理想的程度，仍然是事先难以预见的。因此，对于具有改进性质的乙肝病毒抗体及其生产方法还存在未被满足的需求。
发明内容
发明人进行了大量的探索，在A14抗体的基础上，开发出了Fc效应功能增强和半衰期延长的抗乙肝病毒抗体变体，从而完成了本发明。
因此，第一方面，本发明涉及一种IgG1型抗体变体，其能够特异性结合乙肝病毒(HBV)的Pre-S1结构域，并且相对于亲本抗体具有增强的与激活型Fcγ受体(FcγRs)的亲和力，降低的与抑制型Fcγ受体(FcγR)的亲和力，从而具有增强的Fc介导的效应功能，如增强的ADCC和/或ADCP活性。
在具体的方案中，相对于亲本抗体，所述抗体变体在重链恒定区相对于如SEQ ID NO:31的氨基酸所示的人IgG1的重链恒定区包含一种或多种氨基酸突变，并且任选地所述变体抗体中包含1,6-岩藻糖的糖链减少或缺失。在具体的方案中，所述氨基酸突变位于所述抗体变体的Fc区，其具有对应于SEQ ID NO:31的第114至330位氨基酸的序列。在优选的方案中，所述氨基酸突变选自下组中的一种或多种：G236A、S239D、I332E、F243L、R292PY300L、V305I、P396L、L235V，其中所述氨基酸位置是根据EU编号方式确定的。
进一步地，所述在Fc区包含氨基酸突变的抗体变体相对于未经改造的抗体具有延长的半衰期。在具体的实施方案中，所述抗体变体进一步包含选自下组中的一种或多种氨基酸突变：M252Y、S254T、T256E、M428L和N434S。
第二方面，本发明提供用于制备去岩藻糖基化修饰抗体的生产细胞，所述细胞为FUT8失活的哺乳动物细胞，如293F细胞、CHOK1细胞、CHOZNGS-/-细胞或CHO K1Q细胞；并且所述细胞中引入了编码抗体的序列。优选 地，所述细胞为Fut8基因敲除的293F细胞、CHOK1细胞、CHOZN GS-/-细胞或CHO K1Q细胞。优选地，引入的所述序列编码第一方面的抗体变体。
第三方面，本发明提供用于制备Fut8基因敲除的生产细胞的方法，包括向哺乳动物细胞中引入(a)靶向Fut8的gRNA和Cas9核酸酶的编码序列，或(b)靶向Fut8的gRNA和Cas9核酸酶的复合物。
第四方面，本发明提供sgRNA，其用于制备Fut8基因敲除的细胞。
第五方面，本发明提供制备抗体的方法，包括在培养基中培养第二方面的细胞以产生包含抗体的培养物。
第六方面，本发明提供通过第五方面的方法制备的抗体。
第七方面，本发明提供一种分离的核酸分子，其包含编码第一方面的抗体的核苷酸序列。
第八方面，本发明提供包含第七方面的分离的核酸分子的表达载体。
第九方面，本发明提供包含第七方面的分离的核酸分子或第八方面的表达载体的宿主细胞。
第十方面，本发明提供药物组合物，其包含第一方面的抗体变体或第六方面的抗体。
第十一方面，本发明提供第一方面的抗体变体或第六方面的抗体用于制备药物的用途，所述药物用于在有需要的受试者中治疗乙型或丁型肝炎病毒感染，或所述感染引发的疾病，如慢性乙型肝炎，或引发Fc效应功能。
第十二方面，本发明提供在受试者中治疗乙型或丁型肝炎病毒感染或所述感染引发的疾病，如慢性乙型肝炎，或引发ADCC作用的方法，包括向所述受试者施用第一方面的抗体变体或第六方面的抗体。
第十三方面，本发明提供第一方面的抗体变体或第六方面的抗体与另一种抗病毒药物的联用。
附图说明
图1A-B是pLenti-sgFut8-EGFP的质粒图谱(A)和pLenti-OCT1-cas9-IRES-BSD的质粒图谱(B)。
图2是基因编辑效率半定量检测的凝胶图。
图3是通过FACS分析293F野生型细胞、Fut8基因编辑细胞与FITC-小扁豆凝集素(LCA)的结合能力的曲线图。
图4显示了Sanger测序分析FCC#细胞sgRNA靶向Fut8区域的DNA序列的结果。
图5是通过FACS分析CHOK1野生型细胞、Fut8基因编辑细胞与PE-LCA的结合能力的曲线图。
图6显示在体外用sgcgFut8-2 gRNA/Cas9复合物切割Fut8 DNA片段的结果。
图7显示通过T7E1酶切验证使用sgcgFut8-2 gRNA/Cas9编辑CHOZNGS-/-细胞中Fut8基因的效率。
图8是通过流式细胞术分析FITC-LCA结合CHOZN GS-/-Fut8基因编辑细胞的结果。
图9是通过流式细胞术分析CHOZN GS-/-Fut8_KO-8#结合FITC-LCA的能力的结果。
图10是通过Sanger测序分析CHOZN GS-/-Fut8_KO-8#细胞中sgcgFut8-2靶向的Fut8 DNA序列的结果。
图11显示通过T7E1酶切验证sgcgFut8-2 gRNA/Cas9编辑CHO K1Q细胞中Fut8基因的效率的结果。
图12是通过流式细胞术分析野生型CHO K1Q及其去岩藻糖细胞系结合FITC-LCA的能力的结果。
图13A-C显示通过Sanger测序分析去岩藻糖CHO K1Q细胞中sgRNA靶向的Fut8区域的DNA序列结果。(A)CHO K1Q-Fut8 KO-1#；(B)CHOK1Q-Fut8 KO-2#；(C)CHO K1Q-Fut8 KO-3#。
图14显示各个A14突变抗体的体外ADCC和ADCP活性。
图15显示各个候选突变抗体的剂量依赖性的体外ADCC活性。
图16显示各个A14突变抗体的体外ADCP活性评价。
图17显示SDS-PAGE分析体外重组表达人源FcγRs蛋白的结果。
图18显示对实例5的突变抗体进行的体外ADCC和ADCP活性评价的结果。
图19是通过SDS-PAGE分析表达纯化的A14 WT和afuco-A14 GA LS的结果。
图20显示对afuco-A14 GA LS的体外ADCC和ADCP活性评价的结果。
图21显示通过Biacore分析afuco-A14 GA LS与人FcRn的亲合力的结果。
图22显示通过ELISA分析afuco-A14 GA LS与靶抗原Pre-S1的结合活性的结果。
图23显示afuco-A14 GA LS的体外抗病毒活性。
图24显示afuco-A14 GA LS在hFcRn KI小鼠中的半衰期。
发明详述
定义
“抗体”在本发明的上下文中具有本领域公知的含义，即免疫系统响应外来物质如病原体而产生的、具有Y型结构的免疫球蛋白。经典结构的抗体为一种同型二聚体，每个单体包含通过二硫键连接的一条重链(heavy chain)和一条轻链(light chain)。轻链由一个可变区(VL)和一个恒定区(CL)组成，而重链由一个可变区(VH)和三个恒定区(CH1、CH2和CH3)组成。每个可变区含有三个互补决定区(CDR)，组成负责与抗原互补的抗原结合位点。VL、CL、VH和CH1组成的抗原结合片段被称为“Fab”。剩余的部分，即Y型结构下方的“树干”部分，被称为“Fc”区，由抗体重链的恒定区CH2和CH3结构域组成。
“Fc区”具有多种功能，包括通过效应细胞如巨噬细胞或自然杀伤细胞上的Fc受体(FcR)与效应细胞结合，激活免疫效应通路。另外，Fc区还包含糖基化位点，这些糖基化位点上连接的糖链也能够影响抗体的Fc效应功能。
“中和抗体(neutralizing antibody)”或“NAb”是指能够“中和”外来物质如病原体的生物学作用从而保护细胞或机体的抗体。
“HBV”指乙型肝炎病毒。“HDV”指丁型肝炎病毒。
“Pre-S1”是乙肝病毒表面S基因编码产物中位于N末端的结构域。S基因含有三个读码框，分别为pre-S1、pre-S2和S三个部分。相应地，S基因可编码三种长度不同的蛋白产物，其中最大的产物被称为L(large)蛋白，其同时包含Pre-S1、Pre-S2和S三个结构域，比另外两种产物(包含Pre-S2和S结构域的M蛋白，以及仅含S结构域的S蛋白)多出的部分即为N末端的pre-S1结构域。
“A14”是本申请的发明人之前开发的一种靶向乙肝病毒Pre-S1结构域的全人源单克隆抗体，其能够抑制乙肝病毒进入肝细胞，并具有通过Fc介导 的免疫反应清除乙肝病毒和/或受到乙肝病毒感染的细胞的能力。A14是一种IgG1型抗体。另外，该抗体还是一种治疗慢性乙型肝炎的潜在抑制剂。关于A14抗体的更多信息可以参见PCT发明公开文本WO2016188386A1和WO2021013135A1。
“IgG1”是免疫球蛋白IgG同种型中的一种亚型(有时也称作亚类)。不同的IgG亚型具有与不同的二硫键位置和数目。
“抗体变体”是相对于“亲本抗体”而言经过改造的抗体，所述改造包括但不限于对抗体序列的改造、对抗体糖基化修饰的改造。“亲本抗体”指改造前的抗体，在具体的实施方案中指A14抗体。
“Fc变体”或“Fc抗体变体”指相对于亲本抗体Fc区域经过改造的抗体。
“EU编号”是一种对抗体氨基酸进行编号的方式。该编号方式的基础是基于1968年Gerald M.Edelman等人分离纯化得到了一种IgG1免疫球蛋白并将其命名为Eu，通过测定其氨基酸序列，对各个氨基酸位置进行了编号，由此形成了Eu编号体系。虽然当前对抗体的氨基酸序列进行编码时，更常使用的是经过优化的Kabat、Chothia、IMGT等系统，但由于Eu编号是基于IgG1类抗体做出的，也适合于对本发明中IgG1类抗体A14的氨基酸序列进行编号。因此，本发明中以EU编号来说明氨基酸突变的位置。
“Fc受体”是存在于多种细胞(如效应细胞)表面的受体，其能够特异性结合抗体的Fc区。
“Fcγ受体”或“FcγR”是Fc受体中的一类，其全部成员均属于免疫球蛋白超家族，并进一步分为FcγRI、FcγRIIA、FcγRIIB、FcγRIIIA、FcγRIIIB。各个成员之间对于IgG的不同亚型的亲和力不同，并具有不同功能。在人的多种Fcγ受体中，FcγRI、FcγRIIa、FcγRIIIa和FcγRIIIb为“激活型Fcγ受体”(activating Fcγ-receptor)；而FcγRIIb为人体中唯一的“抑制型Fcγ受体”(inhibitory Fcγ-receptor)。
抗体的“效应功能”在本发明的上下文中主要指Fc介导的效应功能，包括ADCC和ADCP等。
“ADCC”是抗体依赖性细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicity)的缩写，其为细胞介导的一种免疫机制。具体来说，效应细胞借由受体与特异性结合到靶细胞上的抗体的Fc段结合，并释放细胞毒性物质来杀伤靶细胞。ADCC是评价抗体功效的一个重要指标。
“ADCP”是抗体依赖性细胞吞噬作用(antibody-dependent cellularphagocytosis)的缩写。
“去岩藻糖基化”、“去岩藻糖基化修饰”或“去岩藻糖”指通过改造使抗体糖蛋白的糖链中含有的岩藻糖减少或完全消失。例如，在描述抗体变体时，“去岩藻糖基化”表示该抗体变体相对于其亲本抗体而言，具有更少的包含岩藻糖的糖链，或者甚至完全没有包含岩藻糖的糖链，这样的抗体变体可以通过作为前缀的缩写“afuco-”表示。在描述细胞时，“去岩藻糖基化”表示该细胞在用于生产抗体时，能够使该抗体的糖链中缺乏岩藻糖，或者用“去岩藻糖蛋白的细胞系”来描述这样的生产细胞系。
“FUT8”指α-(1,6)-岩藻糖基转移酶(alpha-(1,6)-fucosyltransferase)，其编码基因为“Fut8基因”。FUT8参与抗体表达之后的糖基化过程，将岩藻糖添加到寡糖链中。
“FITC-LCA”指异硫氰酸荧光素(FITC)标记的小扁豆凝集素(LCA)。“PE-LCA”指藻红蛋白标记的小扁豆凝集素。LCA与岩藻糖具有较强的亲和力，用FITC或藻红蛋白进行荧光标记之后，可用于检测岩藻糖的水平。
“治疗”指疾病或其症状得到改善、缓解或消除(即治愈)。在一些情况中，治疗包括预防性治疗。
“协同作用”指两种或多种药物共同施用时，产生了加合或增效作用，优选增效作用，即共同施用时的效果优于各种药物单独施用时的效果之和。
亲本抗体
本发明的亲本抗体是一种特异性结合HBV的Pre-S1结构域的抗体。亲本抗体与抗体变体相比，主要的区别在于Fc区的氨基酸序列，以及岩藻糖基化的程度。
在优选的实施方案中，本发明的亲本抗体具有未经改造的人IgG的恒定区。例如，所述亲本抗体具有未经改造的人IgG的重链恒定区，优选未经改造的人IgG的Fc区。例如，所述人IgG是人IgG1。
在具体的实施方案中，本发明的亲本抗体是A14抗体。
应该理解的是，本发明的亲本抗体也可以与A14抗体具有相同的六个CDR序列(如SEQ ID NOs:32-37所示的氨基酸序列)，甚至具有相同的重链可变区(SEQ ID NO:1)和轻链可变区(SEQ ID NO:5)，但重链恒定区特别是 Fc区来源于人IgG1且未经任何改造。
突变的命名规则
在本发明的上下文中描述Fc变体中所含的突变时，如果没有特殊说明，则均以如SEQ ID NO:31所示的人IgG1的重链恒定序列作为基础，并采用EU编号体系，对氨基酸突变的类型和位置进行描述。具体而言，根据EU编号体系，人IgG1重链恒定区是从第118位的丙氨酸到第447位的赖氨酸组成的共330个氨基酸的序列，其中第118至214位为CH1(对应于SEQ ID NO:31的氨基酸位置1-97)，第215至230位为铰链区(对应于SEQ ID NO:31的氨基酸位置98-113)，第231至340位为CH2(对应于SEQ ID NO:31的氨基酸位置114-223)，并且第341至447位为CH3(对应于SEQ ID NO:31的氨基酸位置224-330)。
在本发明中描述氨基酸突变位置时，以被突变的氨基酸在EU编号体系中的位置，而非其在SEQ ID NO:31中的位置数来记录。如上所示，对于同一氨基酸而言，EU编号的数字会比该氨基酸在SEQ ID NO:31中的位置数大117。在描述氨基酸突变的类型时，将SEQ ID NO:31中对应氨基酸位置的氨基酸类型作为突变前的氨基酸，放在位置号之前(左侧)，而将突变后的变体抗体中存在的氨基酸放在位置号之后(右侧)。当同一位置可以突变为不同的氨基酸时，以“/”隔开多种可选的氨基酸。
基于上述规则，以本发明的G236A突变为例，其指代根据EU编号在第236位的甘氨酸突变为丙氨酸；而相对于SEQ ID NO:31的氨基酸序列而言，该突变相当于将第119位的甘氨酸突变为丙氨酸。又如，S239D/Q/E是指在根据EU编号在第239位的丝氨酸突变为天冬氨酸、谷氨酰胺或谷氨酸；而相对于SEQ ID NO:31的氨基酸序列而言，该突变相当于将第122位的丝氨酸突变为天冬氨酸、谷氨酰胺或谷氨酸。
为了方便表述，在本发明的上下文中有时以缩写表示本发明的几种抗体变体相对于亲本抗体(A14)的突变或突变组合，甚至也用这些缩写表示这些抗体变体本身。缩写具体指代的突变或突变组合的类型如表6所示。
改变抗体的ADCC和ADCP活性
对于治疗性抗体而言，包括ADCC和ADCP活性在内的FcγR介导的效 应功能与抗体介导的清除靶细胞的能力相关。本发明的抗体变体通过不同的手段提高原始抗体的ADCC和/或ADCP活性，从而进一步提高抗体的功效。
自然杀伤细胞(NK)是参与ADCC的主要免疫效应细胞。除此之外，单核细胞和嗜酸性粒细胞也可以介导ADCC。在人体中，抗体介导的ADCC强弱和许多因素有关，如抗体与抗原的亲和力、抗体Fc与受体FcγRIIIa亲和力、免疫效应细胞的特性等。通过改造抗体Fc段的糖基化及氨基酸序列来提高Fc与FcγRIIIa的亲和力，是增强ADCC活性的有效途径。已有研究表明在IgG抗体的Fc片段的CH2结构域中，对Asn297处的糖链结构进行去除岩藻糖的修饰后，可以提高IgG抗体的Fc片段与NK细胞表面FcγRIIIa之间的亲和力，从而显著增强抗体的ADCC效应，改善抗体的临床疗效。
在哺乳动物细胞中，抗体Fc片段寡糖链中核心岩藻糖的添加是由Fut8基因编码的α-1,6-岩藻糖基转移酶(α-1,6 fucosyltransferase,FUT8)直接催化完成的。FUT8通过将岩藻糖残基从二磷酸鸟苷-岩藻糖(GDP-Fuc)转移至Fc片段寡糖链最内侧乙酰氨基葡萄糖(GlcNAc)的末位，形成Fc段寡糖的核心岩藻糖。α-1,6-岩藻糖基转移酶是岩藻糖化合成途径中的关键糖基转移酶。为了生产ADCC效应增强的抗体，人们已采用多种方法来降低IgG抗体岩藻糖化修饰水平，如采用Fut8基因敲除或低表达的抗体生产细胞系，或在抗体生产细胞系中通过转基因来高表达GnTIII等。
抗体生产细胞中的Fut8基因的敲除可以通过不同的方式进行，例如小干扰RNA(siRNA)、短发夹RNA、同源重组等本领域已知的方法。在本发明优选的实施方案中，抗体生产细胞中Fut8基因的敲除通过核酸酶介导的同源重组进行。核酸酶介导的同源重组包括但不限于CRISPR/Cas9、TALEN、ZFN。在更优选的实施方案中，抗体生产细胞中Fut8基因的敲除通过CRISPR/Cas9体系进行。具体的CRISPR/Cas9编辑方法在后文中详细描述。
在一些实施方案中，本发明的抗体变体相对于亲本抗体在Fc区中包含氨基酸突变以改善ADCC活性。用于改进ADCC活性的氨基酸突变可以通过增加抗体与激活型受体(如人FcγRIIIa)的亲和力，和/或通过降低抗体与抑制型受体(如人FcγRIIb)的亲和力来实现增强ADCC的效果。
在具体的实施方案中，本发明的抗体变体可以包含选自下组的一种或多种用于改进ADCC活性的氨基酸突变：L235V、S239E/D/Q、F243L、R292P、Y300L、V305I、A330L、I332E、P396L。
具有氨基酸突变的抗体变体可以通过分子生物学方法引入。具体而言，通过合成编码包含所述氨基酸突变的核苷酸序列，并将其插入到合适的构建体中，并将构建体导入宿主细胞中进行表达，从而获得包含氨基酸突变的抗体变体。下文中用于改善其他不同效果的氨基酸突变，如用于改进ADCP和半衰期的氨基酸突变也可以通过同样的方法引入。
抗体变体可以同时具有Fut8基因的敲除和改进ADCC活性的氨基酸突变，也可以仅具有其中之一，从而在介导ADCC活性方面得到改进。
抗体在人体中介导的ADCP活性也可以通过改造Fc进行调整。通过增加与激活型受体人FcγRIIIa的亲和力或降低与抑制型受体人FcγRIIb的亲和力能够增强ADCP活性。
一个能够增强ADCP活性的氨基酸突变是G236A，其能够降低与抑制型受体FcγRIIb的亲和力。
在优选的实施方案中，本发明的抗体变体同时包含提高ADCC和ADCP功能的修饰，包括去岩藻糖基化修饰和Fc区的氨基酸突变。例如，本发明的抗体变体相对于其亲本抗体而言具有：
(1)减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，和/或选自下组的一种或多种氨基酸修饰：L235V、S239E/D/Q、F243L、R292P、Y300L、V305I、A330L、I332E、P396L；和
(2)任选地G236A氨基酸突变。
在具体的实施方案中，本发明的抗体变体具有如表6中3-33项所示的修饰。
在优选的实施方案中，本发明的抗体变体相对于其亲本抗体而言具有选自下组(a)-(f)的突变：
(a)具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，
(b)具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，且具有G236A氨基酸突变(GA突变)；
(c)具有G236A和S239D氨基酸突变(GASD突变)；
(d)具有F243L、R292P、L235V、Y300L和P396L突变(FLRPLVYLPL突变)；
(e)具有F243L、R292P、Y300L、V305I和P396L突变(FLRPYLVIPL突变)；
(f)具有G236A、I332E突变(GAIE突变)。
在一个特别优选的实施方案中，本发明的抗体变体相对于其亲本抗体而言具有具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，且具有G236A氨基酸突变。
改变抗体的半衰期
作为一种蛋白质分子，已知可以通过多种方式来改变抗体的半衰期。
在本发明的具体实施方案中，通过改变抗体与FcRn受体的亲和力来改变半衰期。在具体的实施方案中，通过在抗体的Fc区引入氨基酸突变来增强所得抗体变体与FcRn受体的亲和力，从而延长半衰期。
在具体的实施方案中，本发明的抗体变体相对于其亲本抗体包含选自下组的一种或多种氨基酸突变：M252Y、S254T、T256E、M428L、N434S。
在更具体的实施方案中，本发明的抗体变体相对于其亲本抗体包含选自下组(g)或(h)的氨基酸突变：
(g)M428L和N434S氨基酸突变(LS突变)；
(h)M252Y、S254T、T256E氨基酸突变(YTE突变)。
组合修饰
在优选的实施方案中，本发明的抗体同时具有改进的Fc介导的效应功能和延长的半衰期。
在具体的实施方案中，本发明的抗体变体相对于其亲本抗体包含：
(1)减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，和/或选自下组的一种或多种氨基酸突变：L235V、S239E/D/Q、F243L、R292P、Y300L、V305I、A330L、I332E、P396L；
(2)任选地G236A氨基酸突变；和
(3)任选地选自下组的一种或多种氨基酸突变：M252Y、S254T、T256E、M428L、N434S。
在具体的实施方案中，本发明的抗体变体相对于其亲本抗体而言，具有选自(a)-(f)中的一组的突变和一个选自(g)或(h)的突变，或相对于亲本抗体的区别仅在于选自(a)-(f)中的一组的突变和选自(g)或(h)的突变：
(a)具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，
(b)具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，且具有G236A氨基酸突变(GA突变)；
(c)具有G236A和S239D氨基酸突变(GASD突变)；
(d)具有F243L、R292P、L235V、Y300L和P396L氨基酸突变(FLRPLVYLPL突变)；
(e)具有F243L、R292P、Y300L、V305I和P396L氨基酸突变(FLRPYLVIPL突变)；
(f)具有G236A、I332E氨基酸突变(GAIE突变)；
(g)M428L和N434S氨基酸突变(LS突变)；
(h)M252Y、S254T、T256E氨基酸突变(YTE突变)。
在最具体的实施方案中，本发明的抗体变体相对于亲本抗体而言，具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，以及G236A、M428L和N434S氨基酸突变。
在优选的实施方案中，尽管本发明的抗体变体相对于亲本抗体进行了一种或多种修饰，但具有与亲本抗体相当的对乙肝病毒Pre-S1区域的亲和力和/或病毒活性。例如，本发明的抗体变体相对于亲本抗体仅改变了Fc区，而未改变可变区和互补决定区(CDR)，因此具有与亲本抗体相当的对乙肝病毒，具体而言对Pre-S1蛋白的结合亲和力。例如，在体外抗病毒活性评价中，本发明的抗体变体相对于亲本抗体具有相当的IC50值。“相当的”活性或值是指大致相同或相近。例如，在体外抗病毒活性测定中，“与亲本抗体相当的活性”是指本发明的变体抗体与其亲本抗体的相对活性(IC50值)的差异不超过20％。
生产细胞系
本发明可以使用本领域通常用于抗体生产的哺乳动物细胞系，用于生产本发明的抗体。特别适合于本发明的细胞是那些可用作生物制药行业重组蛋白药物的开发和生产的宿主细胞。在优选的实施方案中，需要对本发明的生产细胞系进行Fut8基因的敲除。
在具体的实施方案中，适合用于本发明的生产细胞系包括但不限于：293F、CHO-K1,CHOZN GS-/-和CHOK1Q。这些工程细胞均可用于制药行业的开发和生产，细胞倍增时间维持在12～40h，细胞活率≥90％。
293F细胞来源于原代人胚肾细胞，其具有转染效率高，能够在无血清培养基中悬浮培养高表达蛋白的细胞。293F细胞增殖快，并且可以进行高密度培养。
CHO-K1、CHOZN GS-/-和CHOK1Q均是成年中国仓鼠卵巢细胞(CHO)。CHO-K1是未经改造的野生型CHO细胞。CHOZN GS-/-细胞株通过ZFN(锌指核酸酶)技术敲除了谷氨酰胺合成酶基因，使得细胞株的存活依赖于外源谷氨酰胺的补充，从而在整个筛选过程无需通过填加额外的MTX或MSX压力便可获得高表达的目的克隆。CHOK1Q是未经基因改造的野生型CHO-K1细胞，但经过悬浮、高渗透压、高铵离子、高乳酸驯化改造。
制备方法
如前文所述，在抗体生产细胞系中敲除Fut8基因可以通过多种不同的方式完成。本发明采用CRISPR/Cas方法相对于其他方式而言，具有操作简便、靶向精准性高、编辑效率高、无外源DNA插入宿主基因组、安全性高的优势。
CRISPR/Cas9基因编辑技术利用向导RNA(guide RNA,sgRNA)来引导Cas9核酸内切酶切割基因组中的目标双链DNA，造成靶序列的双链断裂，随后在细胞内的DNA修复机制作用下在基因组上产生替换、删除、插入等，从而可实现目的基因的敲除。CRISPR/Cas9系统现已被广泛用于各种生物体内的基因编辑。我们利用CRISPR/Cas9技术靶向敲除293F、CHOZN GS-/-或CHOK1、CHOK1Q细胞内的Fut8基因，从而获得可以生产去岩藻糖化的抗体，提高抗体的ADCC活性。
在使用293F细胞作为生产细胞系时，可以使用SEQ ID NO:10的核苷酸序列作为sgRNA来敲除Fut8基因。
在使用CHO-K1及其衍生细胞系作为生产细胞系时，可以使用选自SEQID NO:19、SEQ ID NO:20、SEQ ID NO:21的核苷酸序列作为sgRNA来敲除Fut8基因。优选地，使用具有SEQ ID NO:20的核苷酸序列的sgRNA。
用途
在没有特殊说明的情况下，本发明的抗体或抗体变体是用于人的抗体。
本发明的抗体或抗体变体可以用于治疗或预防乙型肝炎，特别是用于治 疗或预防慢性乙型肝炎。
本发明的抗体或抗体变体可以作为辅助治疗(adjuvant treatment)用于治疗乙型肝炎，即作为一线治疗药物的辅助手段，进一步改善治疗效果。
本发明的抗体可以和其他乙肝治疗联用。可以与本发明的抗体联用的抗乙肝病毒药物包括免疫调节剂类药物，如干扰素、胸腺素-α1、细胞因子；核苷(酸)类似物，如恩替卡韦(Entecavir)、替比夫定(Telbivudine)、阿德福韦(Adefovir)或阿德福韦酯(Adefovir dipivoxil)、替诺福韦(Tenofovir)、拉米夫定(Lamivudine)。例如，可以与目前已经获得临床批准的抗乙肝病毒药物进行联用。在具体的实施方案中，本发明的抗体或抗体变体可以与恩替卡韦联用。
在一些情况下，本发明的抗体或抗体变体也可以用于治疗或预防丁型肝炎。
本发明的抗体或抗体变体可以通过常规递送手段施用给受试者。在优选的实施方案中，本发明的抗体通过胃肠外方式施用给受试者，优选通过静脉输注或皮下注射。
本发明还涉及如下各项：
1.一种特异性结合乙型肝炎病毒(HBV)的Pre-S1结构域的抗体变体，其与亲本抗体相比具有增强的Fc介导的效应功能。
2.项1所述的抗体变体，所述Fc介导的效应功能为ADCC和/或ADCP活性。
3.项1或2所述的抗体变体，其具有增强的与激活型Fcγ受体的亲和力和/或降低的与抑制型Fcγ受体的亲和力。
4.项1-3中任一项所述的抗体变体，所述抗体变体和亲本抗体为IgG1型抗体。
5.项1-4中任一项所述的抗体变体，所述抗体相对于其亲本抗体具有选自下组的特征：
(1)包含1,6-岩藻糖的糖链减少或缺失，和/或选自下组的一种或多种氨基酸修饰：L235V、S239E/D/Q、F243L、R292P、Y300L、V305I、A330L、I332E、P396L；和
(2)任选地G236A氨基酸突变。
6.项5所述的抗体变体，其相对于亲本抗体具有如表6中3-33项所示 的修饰。
7.项5所述的抗体变体，其相对于亲本抗体具有选自下组(a)-(f)中任一组的突变：
(a)具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，
(b)具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，且具有G236A氨基酸突变(GA突变)；
(c)具有G236A和S239D氨基酸突变(GASD突变)；
(d)具有F243L、R292P、L235V、Y300L和P396L突变(FLRPLVYLPL突变)；
(e)具有F243L、R292P、Y300L、V305I和P396L突变(FLRPYLVIPL突变)；
(f)具有G236A、I332E突变(GAIE突变)。
8.项7所述的抗体变体，其具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，且具有G236A氨基酸突变(GA突变)。
9.项5-8中任一项所述的抗体变体，其中包含1,6-岩藻糖的糖链的减少或缺失通过敲除用于生产所述抗体变体的宿主细胞中的Fut8基因实现。
10.项1-9中任一项所述的抗体变体，其相对于所述亲本抗体具有延长的半衰期。
11.项10所述的抗体变体，其相对于所述亲本抗体包含选自下组的一种或多种氨基酸突变：M252Y、S254T、T256E、M428L、N434S。
12.项11所述的抗体变体，其相对于所述亲本抗体包含选自下组(g)或(h)的氨基酸突变：
(g)M428L和N434S氨基酸突变(LS突变)；
(h)M252Y、S254T、T256E氨基酸突变(YTE突变)。
13.项1-12中任一项所述的抗体变体，其相对于亲本抗体具有：
(1)减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，和/或选自下组的一种或多种氨基酸突变：L235V、S239E/D/Q、F243L、R292P、Y300L、V305I、A330L、I332E、P396L；
(2)任选地G236A氨基酸突变；和
(3)任选地选自下组的一种或多种氨基酸突变：M252Y、S254T、T256E、M428L、N434S。
14.项13所述的抗体变体，其相对于其亲本抗体而言，具有一个选自(a)-(f)的突变和一个选自(g)或(h)的突变：
(a)具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，
(b)具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，且具有G236A氨基酸突变(GA突变)；
(c)具有G236A和S239D氨基酸突变(GASD突变)；
(d)具有F243L、R292P、L235V、Y300L和P396L氨基酸突变(FLRPLVYLPL突变)；
(e)具有F243L、R292P、Y300L、V305I和P396L氨基酸突变(FLRPYLVIPL突变)；
(f)具有G236A、I332E氨基酸突变(GAIE突变)；
(g)M428L和N434S氨基酸突变(LS突变)；
(h)M252Y、S254T、T256E氨基酸突变(YTE突变)。
15.项14所述的抗体变体，其相对于亲本抗体而言，具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，以及G236A、M428L和N434S氨基酸突变。
16.项1-15任一项所述的抗体变体，所述亲本抗体和抗体变体具有如SEQ ID NOs:32-37所示的CDR序列。
17.项16所述的抗体变体，所述亲本抗体为A14抗体，其具有如SEQ ID NO:1所示的重链可变区序列，如SEQ ID NO:38所示的重链恒定区序列，如SEQ ID NO:5所示的轻链可变区序列，和如SEQ ID NO:6所示的轻链恒定区序列。
18.项1-17任一项所述的抗体变体，与亲本抗体相比，其具有相似的抗乙肝病毒能力，例如具有相似的IC50。
19.一种特异性结合乙型肝炎病毒(HBV)的Pre-S1结构域的抗体，其包含：
(1)如SEQ ID NOs:32、33和34所示的三个重链CDR序列，和如SEQ ID NOs:35、36和37所示的三个轻链CDR序列；和/或
(2)包含如SEQ ID NO:2所示的氨基酸序列的重链恒定区。
20.项19所述的抗体，其包含：
(1)如SEQ ID NO:1所示的重链可变区序列和如SEQ ID NO:5所示的 轻链可变区序列；和/或
(2)包含如SEQ ID NO:2所示的氨基酸序列的重链恒定区。
21.一种分离的核酸分子，其包含编码项1-18任一项所述的抗体变体或项19-20任一项所述的抗体的核苷酸序列。
22.项21所述的分离核酸分子，其包含如SEQ ID NO:4所示的核苷酸序列以编码重链恒定区。
23.一种表达载体，其包含项21或22所述的分离的核酸分子。
24.一种宿主细胞，其包含项21或22所述的分离的核酸分子或项23所述的表达载体。
25.一种哺乳动物细胞，其特征在于所述细胞中的FUT8被失活，并且在所述细胞中引入了项21或22所述的分离的核酸分子或项23所述的表达载体。
26.项25所述的哺乳动物细胞，通过敲除所述细胞的Fut8基因使FUT8失活。
27.项25或26所述的哺乳动物细胞，其选自下组：293F细胞、CHOK1细胞、CHOZN GS-/-细胞和CHO K1Q细胞。
28.项27所述的哺乳动物细胞，其为293F细胞。
29.一种制备抗体的方法，包括如下步骤：
(1)在培养基中培养项25-28中任一项的哺乳动物细胞以获得包含所述抗体的培养物；和
(2)从所述培养物中回收所述抗体。
30.一种用于制备Fut8基因敲除的哺乳动物细胞的方法，包括向所述哺乳动物细胞中引入：
(a)靶向Fut8的gRNA和Cas9核酸酶的编码序列，或
(b)靶向Fut8的gRNA和Cas9核酸酶的复合物，
其中优选通过(b)引入。
31.项30所述的方法，其中所述哺乳动物细胞选自下组：293F细胞、CHOK1细胞、CHOZN GS-/-细胞和CHO K1Q细胞。
32.项31所述的方法，其中所述gRNA的核苷酸序列包含选自下组的用于靶向Fut8基因的核苷酸序列：
如SEQ ID NO:10中所示的核苷酸序列的核苷酸位置1至20,
如SEQ ID NO:19中所示的核苷酸序列的核苷酸位置1至20,
如SEQ ID NO:20中所示的核苷酸序列的核苷酸位置1至20,
如SEQ ID NO:21中所示的核苷酸序列的核苷酸位置1至20,或
如SEQ ID NO:39中所示的核苷酸序列的核苷酸位置21至40。
33.项32所述的方法，其中所述sgRNA的核苷酸序列选自如SEQ ID NO:10、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21或SEQ ID NO:39所示的核苷酸序列。
34.通过项30-33中任一项所述的方法制备的哺乳动物细胞。
35.用于敲除Fut8基因的sgRNA，其具有选自如SEQ ID NO:10、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21或SEQ ID NO:39所示的核苷酸序列。
36.项35所述的sgRNA，其用于在293F细胞中敲除Fut8基因并且具有如SEQ ID NO:10所示的核苷酸序列。
37.项35所述的sgRNA，其用于在CHO-K1细胞及其衍生细胞中敲除Fut8基因并且具有如SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21或SEQ ID NO:39所示的核苷酸序列。
38.一种制备抗体的方法，包括如下步骤：
(1)向项34的哺乳动物细胞中引入项21或22所述的分离的核酸分子或项23所述的表达载体；
(2)培养步骤(1)获得的哺乳动物细胞以产生包含所述抗体的培养物；和
(3)从步骤(2)的培养物回收所述抗体。
39.通过项29的方法或项38的方法制备的抗体。
40.一种药物组合物，其包含项1-18任一项所述的抗体变体、项19-20或39任一项所述的抗体，和药学上可接受的载体。
41.项1-18任一项所述的抗体变体、项19-20或39任一项所述的抗体在制备药物中的用途，所述药物用于在有需要的受试者中治疗乙型或丁型肝炎病毒感染或所述感染引发的疾病，或引发ADCC作用。
42.项41所述的用途，所述受试者选自下组：确认感染乙型肝炎病毒或丁型肝炎病毒的受试者，曾经暴露于乙型肝炎病毒或丁型肝炎病毒的受试者，处于乙型肝炎病毒或丁型肝炎病毒暴露的高风险中的受试者，患有慢性乙型肝炎的受试者。
43.项41或42所述的用途，所述药物用于治疗慢性乙型肝炎。
44.一种在受试者中治疗乙型或丁型肝炎病毒感染或所述感染引发的疾病，或引发ADCC作用的方法，包括向所述受试者施用治疗有效量的项1-18任一项所述的抗体变体、项19-20或39任一项所述的抗体，或项40的药物组合物。
46.项44或45所述的方法，所述受试者选自下组：确认感染乙型肝炎病毒或丁型肝炎病毒的受试者，曾经暴露于乙型肝炎病毒或丁型肝炎病毒的受试者，处于乙型肝炎病毒或丁型肝炎病毒暴露的高风险中的受试者，患有慢性乙型肝炎的受试者。
47.项44-46任一项所述的方法，所述抗体变体或抗体通过静脉输注或皮下注射施用。
48.项44-47任一项所述的方法，所述方法包括向所述受试者施用另一种药物，所述另一种药物用于治疗或预防由乙型肝炎病毒(HBV)感染引起的疾病，特别是由乙型肝炎病毒(HBV)感染引起的慢性疾病。
49.项48所述的方法，所述另一种药物是免疫调节剂或核苷类似物。
50.一种用于治疗乙型或丁型肝炎病毒感染或所述感染引发的疾病的药物组合，包括：(a)项1-18任一项所述的抗体变体、项19-20或39任一项所述的抗体；和(b)另一种药物，其用于治疗或预防由乙型肝炎病毒(HBV)感染引起的疾病，特别是由乙型肝炎病毒(HBV)感染引起的慢性疾病。
51.项50所述的药物组合，所述另一种抗病毒药物是免疫调节剂或核苷类似物。
实施例
为了更全面地理解和应用本发明，下文将参考实施例和附图详细描述本发明，所述实施例仅是意图举例说明本发明，而不是意图限制本发明的范围。本发明的范围由后附的权利要求具体限定。
实施例1.去岩藻糖蛋白的生产细胞系的建立
本实施例描述建立去岩藻糖蛋白的细胞系的建立。具体而言，使用Crispr/Cas9核酸酶编辑系统敲除293F细胞系中的Fut8基因，从而使该编辑后的细胞产生的抗体缺乏岩藻糖基化修饰。
首先设计了Crispr/Cas9体系所需的针对目的基因的sgRNA。利用sgRNA在线设计网站http://crispr.dbcls.jp/，设计了靶向293F细胞(购自北京义翘神州科技股份有限公司)内的Fut8基因(NM_178155.2)的2条sgRNA序列，具体序列如下文中SEQ ID NO:9和SEQ ID NO:10所示。在如下序列中，sgRNA靶向序列用下划线表示，PAM序列(原间隔序列临近模块序列；protospacer adjacent motif)用粗体表示。
sgFut8-1:(SEQ ID NO:9)
sgFut8-2:(SEQ ID NO:10)
随后合成了两对引物对(sgFut8-1-F1和sgFut8-1-R1；sgFut8-2-F1和sgFut8-2-R1，参见表1)。通过体外退火合成双链后，将双链产物克隆至经BfuAI酶切的pLenti-sgRNA-EGFP载体(购自Addgene)，分别得到sgRNA表达质粒pLenti-sgFut8-1-EGFP和pLenti-sgFut8-2-EGFP(图1A)，再与表达Cas9的质粒pLenti-OCT1-cas9-IRES-BSD通过(购自Addgene)PEI共转染293F细胞(购自北京义桥神州科技股份有限公司)(图1B)。待细胞转染后48h进行流式细胞术分选EGFP表达阳性的细胞，并继续培养至转染后7天，收集细胞进行T7E1(NEB)的验证。在T7E1实验中，针对两个sgRNA分别使用了如下表1中所示的两对引物(T7-sgFut8-1-F1和T7-sgFut8-1-R1；T7-sgFut8-2-F1和T7-sgFut8-2-R1)。由于T7E1核酸内切酶能够识别DNA上错配的碱基并对识别出的该位点进行切割，因此基因编辑后可使用T7E1来进行基因编辑效率的半定量检测，确定基因编辑的效率，测定结果示于图2。如图2所示，确认了设计的2条sgRNA都能有效编辑细胞内的Fut8基因。
表1.用于敲除293F细胞中的Fut8基因的引物序列
接下来，对经过T7E1验证后的细胞，通过有限稀释法获得了单克隆化细胞，并对克隆化的细胞进行基于流式细胞术(FACS)的细胞结合实验。通过功能性表型分析验证单克隆细胞是否为基因编辑后的单克隆细胞。小扁豆凝集素(Lens Culinaris Agglutinin,LCA)是一种可以特异性地识别和结合带有岩藻糖修饰的多糖的凝集素，异硫氰酸荧光素(FTIC)标记的LCA(FITC-LCA)可以用于检测细胞表面蛋白的岩藻糖修饰及修饰水平。FACS结果示于图3。如图3所示，未经编辑的293F野生型细胞与FITC-LCA的结合非常强，FITC信号值最强；而成功编辑后的细胞，如代号为FCC#的细胞克隆，与LCA的结合显著降低。又如，代号为F28#的细胞克隆与野生型细胞相比在结合LCA的能力上没有差别，说明F28#为未编辑成功的细胞。进一步对FCC#细胞进行Sanger测序确定具体的基因编辑形式。结果显示其为纯合敲除Fut8的细胞(图4)。
以上结果表明，获得的FCC#克隆为Fut8基因稳定敲除的293F细胞(称为“FCC#-293F细胞”)，可用于去岩藻糖抗体的表达。该细胞可以作为宿主细胞用于生产去岩藻糖化的抗体。
实施例2.去岩藻糖基化CHOK1、CHOZNGS-/-和CHO K1Q细胞系的建立
2.1 sgRNA设计和验证
利用sgRNA在线设计网站http://crispr.dbcls.jp/，根据NCBI数据库公布的CHO-K1细胞(购自中国医学科学院基础医学研究所，细胞中心)的Fut8基因序列(Gene ID:100751648,NCBI参考号:NM_003613860)，设计了4条针对该基因的sgRNA序列(表2)。使用如实施例1所述的方法，将sgRNA片段连接入pLenti-sgRNA-EGFP载体，然后分别与表达Cas9的质粒pLenti-OCT1-Cas9-IRES-BSD一起通过PEI共转染CHO-K1细胞(购自国家实验细胞资源共享平台)。转染后2天用流式细胞仪筛选EGFP阳性的细胞(即sgRNA和Cas9共表达细胞)，继续培养5天后，用罗丹明(Rhodamine,Rho)标记的LCA进行细胞结合试验。结果如图5所示，设计的4条sgRNA均能有效地编辑CHO-K1细胞中的Fut8基因，并且sgcgFut8和Cas9共表达的细胞结合Rho-LCA的能力大大降低，说明细胞表面蛋白的岩藻糖水平明显降低。 该结果说明了对Fut8基因的成功编辑，有效地沉默了FUT8蛋白的表达。
表2.靶向CHO-K1细胞Fut8基因的sgRNA靶向序列
2.2去岩藻糖CHOZNGS-/-稳转细胞系建立
为了建立去岩藻糖CHOZNGS-/-稳转细胞系，选择了2.1中所述经验证的4条sgRNA中的一条sgRNA(sgcgFut8-2)进行了基因编辑操作。为了将sgRNA/Cas9递送至细胞中，采用了无DNA、无病毒载体，以及无转染试剂的方法。研究中使用的引物序列信息参见表3。
表3.引物序列清单
通过将体外转录合成的sgRNA与带有信号肽的Cas9蛋白共孵育形成gRNA/Cas9核糖核蛋白(RNP)复合物。然后将这种RNP复合物电转入CHOZNGS-/-细胞(购自Merck Batch No：2113-53667)，使其在细胞基因组上靶向切割DNA双链，进行Fut8基因的编辑。不同于实施例1中通过质粒递送sgRNA和Cas9编码序列的方法，通过电转递送RNP复合物的递送方法 的优势在于操作简便，基因编辑特异性高，没有DNA插入突变的风险，且没有转染试剂引起的细胞毒性，如图8所示。LCA作为可特异性结合岩藻糖的凝集素，其在结合细胞膜表面的岩藻糖修饰蛋白后，被内吞进入细胞导致细胞死亡。基于此原理，结合LCA加压筛选，以提高Fut8基因敲除细胞获得的成功率。
2.2.1 sgDNA模板制备
设计如下模板sgDNA序列(SEQ ID NO:39)：
其中下划线为T7启动子的序列，斜体为gRNA靶向序列，正常字体为gRNA保守骨架序列。
如下表4所示配制Overlap PCR反应体系用来制备如上所示sgDNA模板。PCR反应条件为：98℃预变性2分钟；然后98℃变性10秒，60℃退火10秒，72℃延伸10秒，共35个循环；最后为72℃再次延伸4分钟。PCR反应后的PCR产物经2％琼脂糖凝胶电泳，切胶回收预期大小为123bp的产物。然后用T7-sgcgFut8-2测序鉴定，确定了sgDNA模板的正确合成。
表4.Overlap PCR制备sgDNA模板反应体系
2.2.2 T7体外转录gRNA
以sgDNA为模板，按照T7体外转录试剂盒的说明(NEB)配制如下反应体系(表5)，并在72℃孵育16小时。转录后获得的gRNA产物经纯化后得到gRNA。
表5.T7体外转录制备gRNA
2.2.3 gRNA/Cas9复合物的制备
将上述制备的sgcgFut8-2 gRNA与EnGen Cas9-NLS(20uM，NEB)蛋白以2:1的摩尔比例在室温孵育10-15分钟，形成gRNA/Cas9复合物。体外的CRISPR/Cas9切割试验表明(图6)，合成的sgcgFut8-2 gRNA与EnGen Cas9-NLS形成复合物后能有效地切割含有sgcgFut8-2靶向序列的CHOK1 Fut8 DNA片段，说明建立的基因编辑系统能有效的工作。
2.2.4通过电转筛选和建立Fut8敲除CHOZN GS-/-细胞系
接下来采用Gene Pulser Xcell电转仪将2.2.3中制备的gRNA/Cas9复合物电转至CHOZN GS-/-细胞。共进行了3个独立实验。
电转后1周，收集部分细胞并提取其基因组DNA。用T7E1-sgcgFut8-2F和T7E1-sgcgFut8-2R引物通过PCR扩增了含有sgcgFut8靶向区域的基因组DNA片段。然后进行T7E1酶切验证sgcgFut8-2 gRNA/Cas9介导的基因编辑效率。结果表明，sgcgFut8-2 gRNA成功引导Cas9切割了CHOZN GS-/-细胞中的Fut8基因(图7)。
为了富集Fut8基因编辑的细胞，采用LCA进行加压筛选。筛选12天后，用FACS分析FITC-LCA结合细胞的能力。结果显示，野生型的CHOZNGS-/-细胞能高亲和力结合FITC-LCA；而在sgcgFut8-2 gRNA/Cas9电转的细胞中，有部分细胞群丧失了结合FITC-LCA的能力。该结果说明存在Fut8基 因的成功编辑。进一步经LCA加压筛选后，结合FITC-LCA的细胞数显著降低，说明Fut8基因成功编辑的细胞得到了有效的富集(图8)。
进一步通过克隆化LCA加压筛选后的sgcgFut8-2 gRNA/Cas9电转的细胞，并通过FITC-LCA细胞结合试验(图9)和Sanger测序sgcgFut8-2靶向的Fut8区域(图10)，获得了Fut8纯和敲除的CHOZN GS-/-细胞系(Fut8_KO-8#)。
2.3去岩藻糖CHO K1Q稳转细胞系的建立
采用与去岩藻糖CHOZN GS-/-细胞系建立所用相同的方法,建立了去岩藻糖CHO K1Q细胞系。共进行了3个独立实验。
经T7E1验证，通过电转sgcgFut8-2 gRNA/Cas9 RNP成功编辑了CHOK1Q细胞(购自中山康晟)中的Fut8基因。如图11所示，目标DNA片段被T7E1酶切割为两个较小的片段。图11还显示，经LCA加压筛选后，进一步提高了基因编辑的效率，因为经T7E1酶切割后目标DNA片段的含量进一步减少。
对于经克隆化LCA加压筛选后的sgcgFut8-2 gRNA/Cas9电转的细胞，通过FITC-LCA细胞结合试验进行了验证。如图12所示，三个独立实验获得的CHO K1Q-Fut8 KO-1#、2#和3#结合FITC-LCA的能力显著降低，说明细胞生产岩藻糖修饰多糖或蛋白的能力降低。此外，对sgcgFut8-2靶向的Fut8区域进行了Sanger测序。如图13所示，测序结果进一步确定了CHO K1Q-Fut8 KO-1#、2#和3#为Fut8纯合敲除的CHO K1Q细胞系。
实施例3.增强中和抗体的ADCC和ADCP
本实施例涉及通过改造抗乙肝病毒中和抗体，特别是抗体的Fc区，从而提高其ADCC和ADCP活性。
3.1制备A14 Fc变体
发明人利用基因工程技术在表达的A14抗体的重链Fc区域引入了氨基酸突变，获得了一系列A14 Fc变体；发明人将这些A14 Fc变体构建到真核表达质粒pCAGGS载体(Addgene)中，并在实施例1构建的FCC#-293F细胞(Fut8基因缺失)内表达，获得了多种去岩藻糖化抗体。总共构建了33个不同 的A14 Fc变体。
首先，通过Nanodrop one测定蛋白在280nm处的吸收值，根据获得的每ml细胞培养液中抗体的量，对各个A14抗体变体的表达量进行了评价，并将结果示于表6。表6中的每个“+”代表10μg/ml。根据表6的结果，选择了表达水平较高的A14抗体变体进行后续实验。
表6.构建的A14变体及其表达量情况
3.2 A14 Fc变体的ADCC和ADCP活性评价
利用发明人以Jurkat亲本细胞(购自中国医学科学院基础医学研究所，细胞中心)为基础构建的表达Fcγ受体的Jurkat/NFAT-luc2p/FcγRIIIa F158和Jurkat/NFAT-luc2p/FcγRIIa R131转基因细胞系作为效应细胞，靶细胞为稳定表达A14结合表位的CHO(CHO-59C)，进行了对A14 Fc变体的ADCC和ADCP活性的评价。实验中同时设置了丧失了与Fcγ受体结合活性的Fc变体(DANA)抗体作为对照。在进行ADCC和ADCP活性评价时，分别设置A14 Fc突变抗体浓度为30 ng/ml和100 ng/ml，效应细胞与靶细胞的比例为6:1，在37℃孵育6h后，检测细胞表达的相对荧光素酶活性(RLU)(图14)。
实验中以A14 DANA诱导的荧光素酶活性值RLU设为1，比较其它A14 Fc突变诱导的相对ADCC和ADCP活性。
如图14所示，afuco-A14 GA在介导ADCC和ADCP两种活性方面都表现出色。在介导ADCC和ADCP方面，afuco-A14 GA不仅都好于只进行了去岩藻糖基化修饰的afuco-A14，或是只进行了GA突变的Fc氨基酸突变变体，其还优于在去岩藻糖基化修饰和GA突变基础上进行了其他突变的抗体变体，如afuco-A14 GAIE、afuco-A14 GAALIE。这个结果是出乎预料的。例如，从图14中可见，在介导ADCC方面，GAIE的效果是显著好于GA的，但进一步加入去岩藻糖基化修饰之后，afuco-A14 GAIE的效果则不如afuco-A14 GA。
3.3 A14 Fc变体ADCC活性的量效关系评价
根据3.2中初步评价的结果，对ADCC和ADCP相对活性较强的A14Fc变体afuco-A14、afuco-A14 GA、A14-GASD、A14-FLRPYLVIPL、A14-FLRPLVYLPL、A14-GAIE进一步进行了ADCC活性的量效关系评价。使用了未经修饰的A14抗体(A14 WT)作为对照。
抗体的起始蛋白量为10μg/ml，并按3倍的稀释比例制备11种不同浓度的梯度稀释品。在实验中选用了Jurkat/NFAT-luc2p/FcγRIIIa F158转基因细胞系(在Jurkat细胞稳定表达NFAT-luc2p和FcγRIIIa F158，其中Jurkat细胞购自中国科学院典型培养物保藏委员会细胞库)作为效应细胞，靶细胞为稳定表达A14结合表位的CHO(CHO-59C)。效应细胞和靶细胞的比例为6:1。实验中设置阴性对照孔(即不含抗体仅含PBS的孔)和背景孔对照。利用 GraphPad Prism软件，以相对光信号值(RLU)为纵坐标，抗体浓度的对数值(Log10)为横坐标，使用四参数拟合回归模型绘制曲线，并计算了抗体ADCC效应的EC50值。评价的候选抗体和结果如图15所示，其中去岩藻糖化修饰的A14抗体变体afuco-A14和afuco-A14 GA表现出最优的ADCC活性。
3.4 A14 Fc变体的ADCP活性评价
在ADCP实验中，进一步分析了afuco-A14、afuco-A14 GA、A14-GASD突变抗体，并以A14 WT作为对照。
所用效应细胞为来自小鼠骨髓细胞在体外诱导分化的巨噬细胞；靶细胞为表达A14结合表位的293F细胞(293F-59C)。效应细胞用带红色荧光Alexa647的抗小鼠F4/80单抗标记，靶细胞用绿色荧光染料CFSE标记。设置阴性对照(PBS)。将靶细胞与A14 Fc突变抗体(10μg/mL)一起孵育后加入到效应细胞培养板中，效应细胞和靶细胞均为2×105细胞/孔，效应细胞与靶细胞(E:T)的比例为1:1。在37℃下孵育2小时后，弃去培养上清，洗去未被巨噬细胞吞噬的靶细胞。使用共聚焦显微镜NikonA1采集荧光照片并计数吞噬指数，即100个巨噬细胞(红色荧光)中阳性靶细胞(绿色荧光)数目。结果表明，afuco-A14 GA介导巨噬细胞对靶细胞293F-59C的吞噬作用(ADCP)最强(图16)。
综合以上ADCC和ADCP的结果，并结合抗体的表达水平,选择了afuco-A14-GA抗体为免疫效应增强的先导候选分子。
实施例4.A14 Fc突变抗体与人FcγRs受体的亲和力测定
4.1体外重组表达人源FcγRs蛋白
为了研究A14 Fc变体与FcγR受体之间的亲和力，在293F细胞中重组表达了C端带6xHis标签的人FcγR受体，并通过Ni柱进行了纯化。
4.2 A14 Fc突变抗体与FcγRs受体的亲和力测定
运用Biacore分析了初筛获得的几株A14 Fc变体与多种人FcγR的亲和力，包括激活型受体hFcγR1a、hFcγR2a H131、hFcγR2a R131、hFcγR3a V158、hFcγR3a F158，以及抑制型受体hFcγR2b。V158、H131是高亲和力受体SNP，F158、R131是低亲和力的受体SNP。抗体变体的ADCC活性与其同hFcγR3a V158、hFcγR3a F158的亲和力相关；抗体变体的ADCP活性与其同hFcγR2aH131、hFcγR2a R131的亲和力相关。将获得的结果总结在表7中。
表7.Biacore分析A14 Fc变体与人FcγRs的亲和力(KD,M)
从表7的数据可见，afuco-A14 GA相对于野生型A14(A14 WT)具有增强的与激活性受体hFcγR2a H131、hFcγR2a R131、hFcγR3a V158和hFcγR3aF158的亲和力。
综合以上结果，选择了afuco-A14 GA为免疫效应增强的先导候选分子。在此基础上，对抗体的半衰期进行了优化改造。
实施例5.基因工程改造获得半衰期延长的A14 Fc变体
在根据之前的研究评估获得的afuco-A14 GA抗体基础上，进一步通过基因工程改造获得了afuco-A14 GA LS和afuco-A14 GA YTE变体，旨在延长抗体在体内的半衰期。
利用基于Jurkat细胞的ADCC和ADCP荧光素酶报告系统，考察了引入如上所示新的突变后，抗体的ADCC和ADCP活性是否受到影响。
结果如图18所示。相比于A14 WT以及前体抗体afuco-A14 GA，在抗体的Fc片段引入YTE突变后，抗体的ADCC和ADCP活性均显著降低，影响了前体抗体的免疫效应活性。与之不同，LS突变的引入没有明显改变ADCC和ADCP活性。说明afuco-A14 GA LS是最佳的免疫效应增强和半衰期延长的候选分子。
实施例6.afuco-A14 GA LS分子的理化性质、生物活性和体内半衰期 研究
本实施例进一步测试了afuco-A14 GA LS抗体的多种性质和功能，并与未经改造的A14抗体进行了比较。
6.1 SDS-PAGE分析表达的A14-WT和afuco-A14 GA LS
在293F和FCC#细胞中分别表达A14 WT和afuco-A14 GA LS抗体，经protein A纯化后，进行蛋白定量。然后分别取2μg进行SDS-PAGE电泳分析。结果见图19。
6.2 afuco-A14 GA LS与人FcγRs的亲和力分析
按照实施例4中所述的方法，运用Biacore对afuco-A14 GA LS与hFcγRIa、hFcγRIIa H131、hFcγRIIa R131、hFcγRIIb、hFcγRIIIa V158和hFcγRIIIa F158的亲和力进行了分析。具体而言，将Protein G经氨基偶联到CM5芯片，然后分别捕获A14 WT抗体和afuco-A14 GA LS抗体，不同浓度重组的hFcγR作为流动相在pH 7.0条件下通过流路与芯片上的抗体发生结合解离，然后使用1:1 Langmuir结合模式拟合数据计算亲和力，结果示于表8。
如表8所示，相比于A14 WT，afuco-A14 GA LS与激活性受体hFcγRIIa和hFcγRIIIa的亲和力明显增强。
表8.afuco-A14 GA LS与人FcγRs的亲和力分析(KD,M)
6.3 afuco-A14 GA LS的ADCC和ADCP活性评价
使用与3.2中相同的Jurkat/NFAT-luc2p/FcγRIIIa F158和Jurkat/NFAT-luc2p/FcγRIIa R131转基因细胞系作为效应细胞，靶细胞为稳定表达A14结合表位的CHO(CHO-59C)。效应细胞和靶细胞的比例为6:1。抗体的起始浓度为10mg/ml，按3倍进行倍比稀释，共获得11种不同浓度的梯度稀释样品，进行afuco-A14 GA LS和A14 WT抗体的ADCC和ADCP活性比较。实验中设置了阴性对照孔(不含抗体仅含PBS的孔)和背景孔对照。利用 GraphPad Prism软件，以相对萤光信号值(RLU)为纵坐标，抗体浓度的对数值(Log10)为横坐标，使用四参数拟合回归模型绘制曲线，并计算了抗体ADCC效应的EC50值，结果示于图20。
结果显示，afuco-A14 GA LS相比于A14明显增强了ADCC和ADCP活性，增强倍数分别至少为18.6倍和4.7倍。
6.4 afuco-A14 GA LS与人FcRn的结合活性评价
抗体在体内的半衰期主要由抗体Fc与人内皮细胞表面的FcRn的亲和力决定的。FcRn与抗体IgG的结合是pH依赖性的，只在弱酸性pH6.0左右结合，而在中性pH无结合。因此，通过Biacore分析了afuco-A14 GA LS在酸性条件下与人FcRn的结合活性。
具体而言，将重组表达的hFcRn经氨基偶联到CM5芯片，然后不同浓度的A14 WT抗体和afuco-A14 GA LS抗体作为流动相在pH 6.0条件下通过流路与芯片上的hFcRn发生结合解离，然后使用1:1 Langmuir结合模式拟合数据计算亲和力，结果示于图21。
如图21所示，观察到afuco-A14 GA LS相比于A14在酸性条件下的亲和力更强，KD值提高了3.4倍。
6.5 afuco-A14 GA LS抗体与靶抗原的亲和力分析
由于afuco-A14 GA LS的序列改造限于Fc区域，而与A14 WT具有相同的Fab用于识别HBV Pre-S1表位，因此理论上二者与靶抗原Pre-S1的结合活性一致。尽管如此，还是通过实验确认了afuco-A14 GA LS与靶抗原结合的能力。
运用ELISA结合活性检测方法，测定了afuco-A14 GA LS与抗原多肽NC36b(pre-S1)的相对结合活性，结果示于图22。抗原多肽由北京中科亚光生物科技有限公司合成，其序列如WO2016188386A1第019段所描述。图22的结果显示，afuco-A14 GA LS与Pre-S1的结合活性与A14 WT相当，没有明显改变。
6.6体外抗病毒活性评价
使用以发明人制备的人HepG2-hNTCP细胞为宿主细胞的HBV感染体 系。系列稀释afuco-A14 GA LS和A14 WT，将其与等体积的重组HBV D基因型病毒(按照Yan,H.,et al.,Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife,2012.1:p.e00049描述制备)混匀后，用于感染HepG2-hNTCP细胞。于感染后第五天收集细胞培养上清。利用HBeAg抗原ELISA检测试剂盒(北京万泰生物药业股份有限公司)检测细胞培养上清中HBeAg的分泌量，并以病毒感染对照组(未添加抗体仅含PBS)的HBeAg分泌量为基数，计算不同抗体浓度下的抑制百分率。利用GraphPad Prism软件，以浓度的对数为横坐标，以抑制率为纵坐标，使用四参数拟合回归模型绘制曲线，计算IC50值。结果显示，afuco-A14 GALS和A14 WT具有相当的IC50，约为11.0 ng/ml(图23)。
6.7 afuco-A14 GA LS在hFcRn KI小鼠中的体内半衰期研究
进一步在小鼠体内研究了afuco-A14 GA LS的半衰期。本实验中使用了表达人FcRn的人FcRn敲入(hFcRn KI)小鼠。实验设置了A14 WT和afuco-A14 GA LS组，每组6只小鼠，雌雄各半。抗体剂量为10mg/kg，且给药途径为腹腔注射。
在如下时间点对小鼠进行采血：给药前，给药后10分钟，1小时，3小时，10小时，1天，3天，7天，10天，14天，17天，21天，28天，35天。用ELISA方法检测小鼠血清中的血药浓度。结果如图24显示，其中A14 WT的T1/2为5.77±1.16天，AUCInf为515.13±84.82天*μg/mL，末端清除率(CL)为19.79±2.78mL/天/kg；而afuco-A14 GA LS的T1/2为15.41±2.96天，AUCInf为995.48±197.64天*μg/mL，CL为10.39±2.08mL/天/kg。从结果可见，相比于A14 WT，afuco-A14 GA LS的半衰期延长了约2.67倍。
6.8 afuco-A14 GA LS在小鼠体内的抗病毒活性评价
为研究afuco-A14 GA LS对已建立的HBV感染的治疗效果，在肝脏人源化嵌合体hFRG(Fah-/-Rag2-/-/IL2rg-/-)小鼠模型中评价了afuco-A14 GA LS单独给药以及与恩替卡韦联合给药的效果。恩替卡韦是一种乙肝病毒逆转录酶抑制剂，通过抑制病毒复制来治疗乙肝。恩替卡韦的缺点在于一旦停药后，病毒复制会出现反弹。
实验中分别设置了对照组(生理盐水)、afuco-A14 GA LS单药治疗组(10 mg/kg)、恩替卡韦(entecavir,ETV)单药治疗组(ETV，0.1mg/kg)、afuco-A14 GA LS(10mg/kg)和恩替卡韦联合治疗组(0.1mg/kg)。小鼠于HBV感染后(HBV D基因型，2×109 GE/只)第28天(D28)开始给药(afuco-A14 GA LS为皮下注射给药，ETV为口服给药)。之后afuco-A14 GA LS每周皮下给药一次(D35、D42、D49、D56、D63、D70)，而ETV每天给药一次至D70。停药后观察至D91。
通过静脉采血收集实验小鼠的血浆，然后采用qPCR定量检测小鼠血浆中的HBV DNA水平。实验结果显示，相比于给予生理盐水的对照组，给药组小鼠血清中的HBV DNA均显著降低。具体而言，在末次给药时(D70)，afuco-A14 GA LS单药治疗组和ETV单药治疗组的病毒DNA水平分别下降了约7.3倍和436倍。afuco-A14 GA LS联合ETV后进一步下降了血清病毒滴度的水平，达到了855倍。各治疗组病毒滴度在停药后均出现反弹，但仍低于基线D28时病毒滴度水平。各组病毒滴度均值在试验终点(D91，停药后21天)与治疗起点(D28)相比下降倍数分别为：afuco-A14 GA LS单药为2.43倍；ETV单药为7.15倍；afuco-A14 GA LS与ETV联合用药为12.6倍。这些结果说明，本发明的抗体变体与ETV的联用效果好于单独使用它们中任意一种所产生的效果，并且这种在抗病毒效果上的优势可以延续到停药长达21天之后。
序列信息


Claims (20)

  1. 一种特异性结合乙型肝炎病毒(HBV)的Pre-S1结构域的抗体变体，其与亲本抗体相比具有增强的Fc介导的效应功能，例如ADCC和/或ADCP活性。
  2. 权利要求1所述的抗体变体，所述抗体变体和亲本抗体为IgG1型抗体。
  3. 权利要求1或2所述的抗体变体，所述抗体相对于其亲本抗体具有选自下组的特征：
    (1)减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，和/或选自下组的一种或多种氨基酸修饰：L235V、S239E/D/Q、F243L、R292P、Y300L、V305I、A330L、I332E、P396L；和
    (2)任选地G236A氨基酸突变。
  4. 权利要求3所述的抗体变体，其相对于亲本抗体具有选自下组(a)-(f)中任一组的突变：
    (a)具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，
    (b)具有减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，且具有G236A氨基酸突变(GA突变)；
    (c)具有G236A和S239D氨基酸突变(GASD突变)；
    (d)具有F243L、R292P、L235V、Y300L和P396L突变(FLRPLVYLPL突变)；
    (e)具有F243L、R292P、Y300L、V305I和P396L突变(FLRPYLVIPL突变)；
    (f)具有G236A、I332E突变(GAIE突变)。
  5. 权利要求3或4所述的抗体变体，其中包含1,6-岩藻糖的糖链的减少或缺失通过敲除用于生产所述抗体变体的宿主细胞中的Fut8基因实现。
  6. 权利要求1-5中任一项所述的抗体变体，其相对于所述亲本抗体具有延长的半衰期。
  7. 权利要求6所述的抗体变体，其相对于所述亲本抗体包含选自下组的一种或多种氨基酸突变：M252Y、S254T、T256E、M428L、N434S。
  8. 权利要求7所述的抗体变体，其相对于所述亲本抗体包含选自下组 (g)或(h)的氨基酸突变：
    (g)M428L和N434S氨基酸突变(LS突变)；
    (h)M252Y、S254T、T256E氨基酸突变(YTE突变)。
  9. 权利要求1-8中任一项所述的抗体变体，其相对于亲本抗体具有：
    (1)减少的包含1,6-岩藻糖的糖链或缺失包含1,6-岩藻糖的糖链，和/或选自下组的一种或多种氨基酸突变：L235V、S239E/D/Q、F243L、R292P、Y300L、V305I、A330L、I332E、P396L；
    (2)任选地G236A氨基酸突变；和
    (3)任选地选自下组的一种或多种氨基酸突变：M252Y、S254T、T256E、M428L、N434S。
  10. 权利要求1-9任一项所述的抗体变体，所述亲本抗体和抗体变体具有如SEQ ID NOs:32-37所示的CDR序列。
  11. 权利要求16所述的抗体变体，所述亲本抗体为A14抗体，其具有如SEQ ID NO:1所示的重链可变区序列，如SEQ ID NO:38所示的重链恒定区序列，如SEQ ID NO:5所示的轻链可变区序列，和如SEQ ID NO:6所示的轻链恒定区序列。
  12. 一种特异性结合乙型肝炎病毒(HBV)的Pre-S1结构域的抗体，其包含：
    (1)如SEQ ID NOs:32、33和34所示的三个重链CDR序列，和如SEQ ID NOs:35、36和37所示的三个轻链CDR序列；和
    (2)包含如SEQ ID NO:2所示的氨基酸序列的重链恒定区。
  13. 一种分离的核酸分子，其包含编码权利要求1-18任一项所述的抗体变体或权利要求12所述的抗体的核苷酸序列，优选包含如SEQ ID NO:4所示的核苷酸序列以编码重链恒定区。
  14. 一种表达载体，其包含权利要求13所述的分离的核酸分子。
  15. 一种宿主细胞，其包含权利要求13所述的分离的核酸分子或权利要求14所述的表达载体。
  16. 一种哺乳动物细胞，其特征在于所述细胞中的FUT8被失活，并且在所述细胞中引入了权利要求13所述的分离的核酸分子或权利要求14所述的表达载体。
  17. 权利要求16所述的哺乳动物细胞，其选自下组：293F细胞、CHOK1 细胞、CHOZN GS-/-细胞和CHO K1Q细胞。
  18. 一种制备抗体的方法，包括如下步骤：
    (1)在培养基中培养权利要求16或17中任一项的哺乳动物细胞以获得包含所述抗体的培养物；和
    (2)从所述培养物中回收所述抗体。
  19. 一种用于制备Fut8基因敲除的哺乳动物细胞的方法，包括向所述哺乳动物细胞中引入：
    (a)靶向Fut8的gRNA和Cas9核酸酶的编码序列，或
    (b)靶向Fut8的gRNA和Cas9核酸酶的复合物，
    其中优选通过(b)引入。
  20. 权利要求19所述的方法，其中所述sgRNA的核苷酸序列选自如SEQ ID NO:10、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、或SEQ ID NO:39所示的核苷酸序列。
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