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WO2022025129A1 - Nouveau procédé de fabrication d'immunoglobuline anti-hbs - Google Patents

Nouveau procédé de fabrication d'immunoglobuline anti-hbs Download PDF

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Publication number
WO2022025129A1
WO2022025129A1 PCT/JP2021/027933 JP2021027933W WO2022025129A1 WO 2022025129 A1 WO2022025129 A1 WO 2022025129A1 JP 2021027933 W JP2021027933 W JP 2021027933W WO 2022025129 A1 WO2022025129 A1 WO 2022025129A1
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seq
antibody
amino acid
acid sequence
hbv
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Japanese (ja)
Inventor
正博 佐竹
里佳 古田
智津 豊田
隆太郎 飛田
輝人 安居
武春 南谷
宏樹 秋葉
浩平 津本
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Japanese Red Cross Society
National Institutes of Biomedical Innovation Health and Nutrition
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Japanese Red Cross Society
National Institutes of Biomedical Innovation Health and Nutrition
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses

Definitions

  • the present invention relates to providing a recombinant anti-HBs human immunoglobulin preparation.
  • HBV hepatitis B virus
  • HBIG anti-HBs human immunoglobulin preparations
  • blood donor plasma having extremely high titers against HBs antigen, which is a neutralizing epitope of HBV (anti-HBs antibody).
  • the vaccine is additionally inoculated to human individuals who have already acquired anti-HBs antibody by HBV vaccine, and the anti-HBs antibody titer is increased.
  • a project to secure raw material plasma by donating blood is being carried out, and the Japanese Red Cross Society has been entrusted with this project and has been implementing it so far [Sugauchi F, et al., Hepatol Res. 2006; 36: 107- 14].
  • An object of the present invention is to provide a safe and effective antibody preparation by fundamentally solving the problem of unachieved domestic self-sufficiency of raw blood of anti-HBs human immunoglobulin preparation.
  • the present invention is an anti-HBs antigen-specific monoclonal that can have neutralizing activity against HBV from in-vivo peripheral blood circulation B cells of a human individual whose antibody titer is increased by acquiring anti-HBs antibody by inoculation with HBV vaccine. It was decided to solve the above-mentioned problems by screening an antibody, isolating the gene of the antibody, and providing a recombinant anti-HBs human immunoglobulin preparation.
  • the present application provides the following aspects in order to solve the above-mentioned problems: [1]: (1) Heavy chain complementarity determining regions, CDR1 (GFMFSGHS, SEQ ID No: 1), CDR2 (IGSTGEFI, SEQ ID No: 2), and CDR3 (AREQGTRGRYYYYGLDV, SEQ ID No: 3), and Light chain complementarity determining regions, CDR1 (SQSVSTY, SEQ ID No: 4), CDR2 (DAF, SEQ ID No: 5), and CDR3 (QQRGHWPLT, SEQ ID No: 6); (2) Heavy chain complementarity determining regions, CDR1 (SGGSISGHY, SEQ ID No: 7), CDR2 (IHYSGIT, SEQ ID No: 8), and CDR3 (ARGDATYGY, SEQ ID No: 9), and light chain complementarity.
  • CDR1 GFMFSGHS, SEQ ID No: 1
  • CDR2 IGSTGEFI, SEQ ID No: 2)
  • CDR1 Sex determining regions, CDR1 (SQSLLHRNGYNY, SEQ ID No: 10), CDR2 (LGS, SEQ ID No: 11), and CDR3 (MQALRTPWT, SEQ ID No: 12); (3) Heavy chain complementarity determining regions, CDR1 (SGFSFSNYG, SEQ ID No: 13), CDR2 (IWRDGSHQ, SEQ ID No: 14), and CDR3 (AREDPAIVLPVLDH, SEQ ID No: 15), and light chain complementarity.
  • Sex determining regions CDR1 (QRINSY, SEQ ID No: 16), CDR2 (GAS, SEQ ID No: 17), and CDR3 (QQGYSTPLLS, SEQ ID No: 18); Contains the complementarity determining regions of any heavy or light chain selected from the group consisting of, has binding to HBsAg of HBV, has neutralizing activity against HBV, is not derived from blood source, Antigens or antibody derivatives; [2]: Recombinant antibody or antibody derivative according to [1]; [3]: The HBsAg is one or more selected from the group consisting of L antigen, M antigen, S antigen, HBsAg adr type, adw type, ayr type, and ayw type of HBsAg, [1].
  • Amino acid sequence, including substitutions), insertions, or deletions The antibody or antibody derivative according to any one of [1] to [4], which is selected from the above; [6]: The amino acid sequence of the light chain variable region VL domain of the antibody or antibody derivative is (1) Of the amino acid sequence of SEQ ID No: 20 or the amino acid sequence of SEQ ID No: 20, CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6). Amino acid sequences containing substitutions (eg, conservative substitutions), insertions, or deletions of one or several amino acids in parts other than).
  • the antibody or antibody derivative is (1) An antibody or antibody derivative containing a heavy chain (SEQ ID No: 25) and a light chain (SEQ ID No: 26); (2) An antibody or antibody derivative containing a heavy chain (SEQ ID No: 27) and a light chain (SEQ ID No: 28); (3) An antibody or antibody derivative containing a heavy chain (SEQ ID No: 29) and a light chain (SEQ ID No: 30); Item 6.
  • [8] A pharmaceutical composition for neutralizing HBV, which comprises the antibody or antibody derivative according to any one of [1] to [7] and does not contain blood-derived components; [9]: The pharmaceutical composition according to [8], which comprises a plurality of types of the antibody or antibody derivative according to any one of [1] to [7]; [10]: The pharmaceutical composition according to [8] or [9] for preventing reactivation of hepatitis B or preventing medical infection of HBV; [11]: A step of bringing a biological sample collected from a subject into contact with the antibody or antibody derivative according to any one of [1] to [7] in vitro. A step of detecting and measuring HBV in a sample bound to the antibody or antibody derivative.
  • a method for detecting and measuring the presence / abundance of HBV in a biological sample including [12]: A kit for detecting and measuring the presence / abundance of HBV in a subject, which comprises the antibody or antibody derivative according to any one of [1] to [7].
  • an antibody expected to have safe, effective and stable activity that can fundamentally solve the problems of unachieved domestic self-sufficiency and the problems of blood products related to anti-HBs human immunoglobulin preparations derived from blood raw materials. Formulations can be provided.
  • FIG. 1 is a diagram showing a virus infection neutralization experiment in HepG2-hNTCP-C4 cells infected with genotype D HBV.
  • FIG. 2 is a diagram showing a virus infection neutralization experiment in primary human hepatocytes PXB cells infected with genotype C HBV.
  • FIG. 3 is a diagram showing an electrophoretic image in which the binding characteristics of the recombinant anti-HBs antibody were evaluated.
  • FIG. 4 is a diagram confirming that the recombinant anti-HBs antibody of the present invention also binds to the HBs antigen aggregate by the ELISA method.
  • FIG. 1 is a diagram showing a virus infection neutralization experiment in HepG2-hNTCP-C4 cells infected with genotype D HBV.
  • FIG. 2 is a diagram showing a virus infection neutralization experiment in primary human hepatocytes PXB cells infected with genotype C HBV.
  • FIG. 3 is a diagram showing an electrophoretic image in which the binding
  • FIG. 5 is a diagram analyzing the interaction of the recombinant anti-HBs antibody candidate antibody of the present invention with the recombinant HBs antigen using surface plasmon resonance (SPR) technology.
  • FIG. 6 is a diagram showing the results of confirming the non-specific binding (cross-reactivity) of the recombinant anti-HBs antibody to the human-derived component.
  • FIG. 7 is a diagram showing the results of immunostaining of human tissues using recombinant anti-HBs antibody.
  • FIG. 7a shows a histological image in which HBsAg was detected.
  • FIG. 7 is a diagram showing the results of immunostaining of human tissues using recombinant anti-HBs antibody.
  • FIG. 7b shows that no staining was observed on various normal human tissues.
  • HBsAg An antigen that is one of the proteins that make up the outer shell of HBV and is tested when determining the presence or absence of HBV infection. Antibodies to this HBsAg are found in vivo when HBV has been infected in the past but the virus has been eliminated, or when HBV vaccine is given and the antibody becomes positive (+);
  • HBIG Human anti-HBs Immunoglobulin. Commercially available HBIG is a pharmaceutical product obtained by purifying IgG from human plasma, which has a high anti-HBs antibody titer. Although it has an immediate effect, its effect lasts for about 3 months, which is relatively short.
  • the present invention is not derived from a blood source, has binding to HBsAg of HBV, has neutralizing activity against HBV, contains complementarity determining regions of heavy and light chains having a specific amino acid sequence. It has been shown that the subject of the present invention can be solved by providing an antibody or an antibody derivative.
  • This antibody or antibody derivative can be any antibody or antibody derivative as long as it has binding property to HBs antigen of HBV, has neutralizing activity against HBV, and is not derived from blood raw materials. It is included in the scope of the present invention.
  • the antibody or antibody derivative used in the present invention has various problems peculiar to blood preparations such as blood-derived antibody preparations conventionally used for the treatment of hepatitis virus (particularly, infectivity problems and immunity problems).
  • Viruses such as HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) that may be contained in the blood (risk of being mixed in blood preparations) for the purpose of solving the problem. It is characterized by being free of pathogens such as, and free of blood-derived components that may cause immune abnormalities in the administered individual, such as antigenic proteins contained in blood and antibodies that bind to human proteins. ..
  • immortalized cells derived from antibody-producing cells are obtained under conditions that do not contain blood-derived components, and are prepared from the cells under culture conditions that do not contain blood-derived components.
  • Antibodies and recombinant antibodies that can be prepared by recombinantly expressing the antibody protein using the DNA defining the antibody protein obtained from the immortalized cells can be used.
  • the antibody in the present invention is derived from antibody-producing cells by obtaining and immortalizing a cell clone that produces an antibody against HBs antigen derived from HBV from the blood of an individual to which the HBV vaccine has been administered in the past. It can be obtained by obtaining immortalized cells and selecting cells having an action of neutralizing HBV in vivo from the immortalized antibody-producing cells.
  • the present invention comprises obtaining an mRNA and producing a cDNA from an immortalized antibody-producing cell having an action of neutralizing HBV selected by the above-mentioned method according to a well-known method, and then producing an antibody protein. It has neutralizing activity obtained by obtaining the specified DNA sequence and expressing it in a mammalian expression system containing no blood-derived components using a vector to produce it as recombinant immunoglobulin (IgG). Monoclonal antibodies or antibody derivatives thereof can also be provided as recombinant HBIG.
  • derivatives of these antibodies can also be used.
  • an antibody variant selected from a humanized antibody, a chimeric antibody, a polyvalent antibody, and a multispecific antibody or a functional fragment thereof can be used.
  • the functional fragment for example, F (ab') 2 can be used, but the functional fragment is not limited thereto.
  • Derivatives of these antibodies can be produced according to a method well known in the art after the antibody is obtained, and can be prepared as the recombinant antibody derivative described for the antibody of the present invention described above.
  • the antibody or antibody derivative obtained by the above-mentioned method has binding property to HBs antigen derived from HBV, but in the present invention, such an antibody having binding property to HBs antigen derived from HBV.
  • screening is performed based on having an action of further neutralizing HBV from among antibody derivatives, and an antibody or antibody derivative having the neutralizing action is provided.
  • HBV is a spherical virus with a diameter of 42 nm that forms a double structure of the envelope and core.
  • proteins constituting HBV HBs antigen, HBc antigen, HBe antigen, X protein (HBx antigen) and the like are known. Since the HBc antigen is a protein in the inner core of the virus, its antibody is useful for confirming the state of infection, but it is known that it cannot be used to protect against HBV infection.
  • an antibody against the HBe antigen anti-HBe antibody
  • HBe antigen anti-HBe antibody
  • the X protein binds to enhancer and promoter regions. It is a trans-activated protein having a function of regulating the expression of various proteins, and is known not to have a function of protecting against HBV infection.
  • HBs antigen is a protein that constitutes the outer shell (envelope) of HBV, and it is known that the presence of anti-HBs antibody can protect against HBV infection.
  • HBIG is commonly used. Therefore, in the present invention, the antibody or antibody derivative needs to have binding property to HBsAg.
  • the HBsAg as a target of the antibody or antibody derivative of the present invention has three domains, S domain, Pre-S1 domain, and Pre-S2 domain, and from the combination of these, S antigen (consisting only of S domain). , -M antigen (composed of S domain and Pre-S2 domain) and-L antigen (composed of three domains, S domain, Pre-S2 domain, and Pre-S1 domain) There are three types.
  • the antibody or antibody derivative of the present invention is for the HBsAg, whether it is for the S antigen, the M antigen, or the L antigen, as long as it has a binding property to the HBs antigen. You may.
  • this HBsAg has three types of antigenic determinants, an antigenic determinant a, an antigenic determinant d or y, and an antigenic determinant r or w, and the antigenic determinant a is all. It is common to subtypes and can be classified into four subtypes, adr type, adw type, ayr type, and ayw type, depending on the combination. In Japan, adr type accounts for 70 to 90%, adw type accounts for 10 to 30%, and ayr type and ayw type are rare. This evaluation of HBs subtypes is used to elucidate the infection route of HBV and analyze coinfection.
  • the antibody or antibody derivative of the present invention targets any of these four subtypes, adr type, adw type, ayr type, and ayw type, as long as it has binding property to HBs antigen. Of these, a plurality of them may be targeted.
  • the antibody or antibody derivative of the present invention is any of the above-mentioned HBV-derived HBs antigens (for example, any of the S antigen, M antigen, and L antigen, and any of the four subtypes of adr, adw, ayr, and ayw. Anything that binds to (possibly) and has the effect of neutralizing HBV as a result can be used to achieve the object of the present invention.
  • HBIG preparations have traditionally been used for the treatment of patients suffering from HBV.
  • the antibody or antibody derivative of the present invention is characterized in that blood is superior in titer and quality stability as compared with HBIG as a raw material.
  • the antibody or antibody derivative of the present invention is intended to be used as an HBV therapeutic agent in place of the conventional HBIG, it is preferably the same as, preferably the same as, more preferably the neutralizing activity of HBV. It can have the above neutralization activity of HBV.
  • the antibody or antibody derivative of the present invention is characterized in that a plurality of antibodies or antibody derivatives can be used as a cocktail, even if a single antibody or antibody derivative has low neutralizing activity, a plurality of antibodies or antibody derivatives can be used. Can be used as a therapeutic agent in combination with the antibody or antibody derivative of.
  • the neutralizing activity of HBV is obtained by infecting cultured human liver-derived cells or primary human liver cells with HBV and adding an antibody to the cells under culture conditions. ⁇ Suppression of HBV viral load, -Inhibition of intracellular expression of HBV-derived proteins (eg, HBs antigen, HBc antigen, HBe antigen, X protein (HBx antigen), etc.), -Semi-quantitative depending on whether or not the amount of HBV-derived DNA in the cell (for example, the amount of viral DNA (capsid-associated relaxed circular DNA [rcDNA)) covered with HBV capsid in the cell) is suppressed. Can be specified in.
  • Example of antibody of the present invention> cells producing an antibody that binds to an HBs antigen derived from HBV are collected from an individual vaccinated with a known HBV vaccine in the above-mentioned ⁇ antibody or antibody derivative> and obtained.
  • the neutralizing activity against HBV was examined for the antibody or antibody derivative as described above ⁇ HBV neutralizing action>.
  • a plurality of antibodies and cells producing the antibodies were obtained from a plurality of individuals.
  • the complementarity determining regions of heavy chains and light chains having the following specific amino acid sequences: (1) Heavy chain complementarity determining regions, CDR1 (GFMFSGHS, SEQ ID No: 1), CDR2 (IGSTGEFI, SEQ ID No: 2), and CDR3 (AREQGTRGRYYYYGLDV, SEQ ID No: 3), and light chain complementarity.
  • CDR1 GFMFSGHS, SEQ ID No: 1
  • CDR2 IGSTGEFI, SEQ ID No: 2
  • CDR3 AREQGTRGRYYYYGLDV, SEQ ID No: 3
  • CDR1 Sex determining regions, CDR1 (SQSVSTY, SEQ ID No: 4), CDR2 (DAF, SEQ ID No: 5), and CDR3 (QQRGHWPLT, SEQ ID No: 6);
  • Heavy chain complementarity determining regions CDR1 (SGGSISGHY, SEQ ID No: 7), CDR2 (IHYSGIT, SEQ ID No: 8), and CDR3 (ARGDATYGY, SEQ ID No: 9), and light chain complementarity.
  • CDR1 Sex determining regions, CDR1 (SQSLLHRNGYNY, SEQ ID No: 10), CDR2 (LGS, SEQ ID No: 11), and CDR3 (MQALRTPWT, SEQ ID No: 12); (3) Heavy chain complementarity determining regions, CDR1 (SGFSFSNYG, SEQ ID No: 13), CDR2 (IWRDGSHQ, SEQ ID No: 14), and CDR3 (AREDPAIVLPVLDH, SEQ ID No: 15), and light chain complementarity.
  • CDR1 QRINSY, SEQ ID No: 16
  • CDR2 GAS, SEQ ID No: 17
  • CDR3 QQGYSTPLLS, SEQ ID No: 18
  • the present invention in one embodiment, as such an antibody or antibody derivative: (1) Of the amino acid sequence of SEQ ID No: 19 or the amino acid sequence of SEQ ID No: 19, CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3). Amino acid sequences containing substitutions (eg, conservative substitutions), insertions, or deletions of one or several amino acids in parts other than).
  • Amino acid sequence including substitutions), insertions, or deletions, The amino acid sequence of the heavy chain variable region VH domain selected from; and (1) the amino acid sequence of SEQ ID No: 20 or the amino acid sequence of SEQ ID No: 20, CDR1 (SEQ ID No: 4), CDR2 ( Amino acid sequences containing substitutions (eg, conservative substitutions), insertions, or deletions of one or several amino acids in moieties other than SEQ ID No: 5) and CDR3 (SEQ ID No: 6).
  • CDR1 SEQ ID No: 10
  • CDR2 SEQ ID No: 11
  • CDR3 SEQ ID No: 12
  • amino acid sequence containing one or several amino acid substitutions eg, conservative substitutions
  • insertions, or deletions and (3) the amino acid sequence of SEQ ID No: 24, or SEQ ID No: 24.
  • amino acid sequence of one or several amino acid substitutions (eg, conservative) in parts other than CDR1 (SEQ ID No: 16), CDR2 (SEQ ID No: 17), and CDR3 (SEQ ID No: 18).
  • Amino acid sequence, including substitutions), insertions, or deletions Amino acid sequence of light chain variable region VL domain selected from; As a more specific example, an antibody or antibody derivative containing the above can be mentioned.
  • the binding property of the antibody is determined by 6 CDRs (that is, CDR1 to CDR3 in each of the heavy chain and the light chain), and the binding property of the antibody is determined by the CDRs other than the CDRs of the heavy chain variable region and the light chain variable region.
  • the binding property of the antibody is determined by the CDRs other than the CDRs of the heavy chain variable region and the light chain variable region.
  • one or several amino acid mutations do not lose their intended binding.
  • M2-11 clone An antibody or antibody derivative containing a heavy chain variable region (SEQ ID No: 19) and a light chain variable region (SEQ ID No: 20); (2) 5A4 clone: antibody or antibody derivative containing heavy chain variable region (SEQ ID No: 21) and light chain variable region (SEQ ID No: 22); or (3) 3B6 clone: heavy chain variable region (SEQ ID) Antibodies or antibody derivatives containing No: 23) and light chain variable region (SEQ ID No: 24); or (1) M2-11 clones: heavy chain (SEQ ID No: 25) and light chain (SEQ ID No: 26).
  • ⁇ Pharmaceutical composition In one aspect of the present invention, it is also possible to provide a pharmaceutical composition containing the antibody or antibody derivative described above and for neutralizing HBV.
  • This pharmaceutical composition is used to prevent or treat the onset of symptoms caused by HBV, to prevent reactivation of hepatitis B, or to prevent the reactivation of hepatitis B, or to the mother and child of HBV, for individuals infected or suspected of being infected with HBV. It can be used to stop infections and medical infections.
  • this pharmaceutical composition may be contained in blood (there is a risk of contamination with blood preparations). Blood that does not contain pathogens such as viruses such as HBV, HCV, and HIV, and has a risk of causing immune abnormalities in administered individuals, such as antigenic proteins contained in blood and antibodies that bind to human proteins. It is characterized by not containing derived components.
  • the pharmaceutical composition in the present invention may contain one type of the above-mentioned antibody or antibody derivative, or may contain a plurality of types.
  • the antibody or antibody derivative produced in the present invention can be used for the purpose of detecting and measuring the presence / absence of HBV in a biological sample.
  • a biological sample collected from a subject is brought into contact with the antibody or antibody derivative of the present invention in vitro, and the antibody or antibody derivative of the present invention bound to HBV is obtained by a secondary antibody.
  • the presence / abundance of HBV in a biological sample can be detected / measured by detecting / measuring HBV in a sample bound to the antibody or antibody derivative.
  • HBV in a biological sample can be detected and measured by the antibody or antibody derivative of the present invention as described above, an existing anti-HBs human immunoglobulin preparation (for example, a commercially available HBIG preparation (Japan Blood Product Organization) Alternatively, it is possible to compare the performance with Nihon Pharmaceutical Co., Ltd. (sold by Takeda Pharmaceutical Co., Ltd.)).
  • the antibody or antibody derivative of the present invention can also be used as a research reagent in applications such as ELISA and Western blotting for detecting and measuring HBV in a sample.
  • applications such as ELISA and Western blotting for detecting and measuring HBV in a sample.
  • kits for detecting and measuring the presence / abundance of HBV in a subject body containing the antibody or antibody derivative of the present invention can be used as a kit for detecting and measuring the presence / abundance of HBV in a subject body containing the antibody or antibody derivative of the present invention.
  • a kit may include a labeled secondary antibody for detecting the antibody or antibody derivative.
  • Example 1 Antigen-specific single cell sorting method; AgS-SCS method) The purpose of this example was to isolate the gene for anti-HBs antibody from a human individual vaccinated with HBV vaccine.
  • B cells circulating in the peripheral blood about 60% are naive B cells that have not been stimulated with antigen, and 40% are memory B cells that express any of membrane-bound IgG, IgA, IgM, or IgE. It is known to be a cell. It is considered that the antibody sequence in Memory B cells already contains somatic hypermutation, and the binding affinity to the antigen is enhanced. Therefore, as a method for isolating the target antibody gene, only cells expressing a high-affinity antibody that specifically binds to the HBs antigen are selectively sorted from the memory B cell population that has been class-switched to IgG. We decided to isolate the anti-HBs antibody gene by adopting the antigen-specific single cell sorting method (AgS-SCS method) that isolates the antibody gene using cell cloning technology.
  • AgS-SCS method antigen-specific single cell sorting method
  • HBV vaccine booster vaccination was given to human individuals who had been vaccinated with HBV vaccine in the past.
  • the boosted HBV vaccine includes beamgen (KM biologics, Japan), which is a recombinant HBs antigen vaccine derived from yeast with HBV genotype C and serotype adr sequence, or yeast-derived group with HBV genotype A and serotype adr sequence.
  • beamgen KM biologics, Japan
  • a replacement HBsAg vaccine, Heptavax II Merck, German
  • ELISA enzyme-linked immunosorbent assay
  • the purified B cells 5.0 ⁇ 10 7 cells were stained under the following conditions, and single-cell sorted was performed on a 96-well microplate in which 8 ⁇ L of buffer for reverse transcription reaction was dispensed.
  • yeast-derived recombinant HBs antigen (HBsAg) that retains a virus-like particle (hereinafter referred to as VLP) structure with HBs antigen.
  • VLP virus-like particle
  • antibody cocktail anti-HBs polyclonal rabbit antibody (Beacle Inc), anti-rabbit IgG-Alexa488 (Jackson ImmunoReserach Laboratories, USA), anti-human IgG-APC (BD Bioscience), Anti-CD27-PE (BD Bioscience), Anti-CD19-ACP-Cy7 (BD Bioscience), Anti-CD38-PE-Cy7 (BioLegend, USA), 7-AAD7-AAD (BD Bioscience) It was mixed at the specified dose and stained with 100 ⁇ L of FACS buffer) on ice for 30 minutes.
  • antibody cocktail anti-HBs polyclonal rabbit antibody (Beacle Inc), anti-rabbit IgG-Alexa488 (Jackson ImmunoReserach Laboratories, USA), anti-human IgG-APC (BD Bioscience), Anti-CD27-PE (BD Bioscience), Anti-CD19-ACP-Cy7 (BD Bioscience), Anti-CD38-PE-Cy7 (BioLegend, USA), 7-A
  • Lymphocytes are selected from all blood cells based on the cell size by forward scattered light (FSC), cell morphology by lateral scattered light (SSC), and cell internal structure such as nuclei and granules.
  • FSC forward scattered light
  • SSC lateral scattered light
  • cell internal structure such as nuclei and granules.
  • -A live cell population (7AAD-) contained in the cells was obtained.
  • IgG + and CD19 + cells were selected and selected.
  • IgG memory B cells were obtained by selecting CD27 + and CD38- cells. From these cells, HBs antigen bound to the anti-HBs antibody expressed on the memory B cell membrane was detected by HBs antigen high gate using the anti-HBs polyclonal antibody for detection, and as a result, about 300 cells were obtained. rice field.
  • Sorted single cells are placed in individual wells of a 96-well plate containing 8 ⁇ l of reverse transcriptase buffer per well and reverse transcriptase containing reverse transcriptase primers (ATATGGATCC GGCGCGCCGT CGACTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • IgG 1 expression cloning vector pIgG 1 H7 (pcDNA3.1 (+) Hyg (Invitrogen) 962 to 991)
  • a plasmid vector in which the IgG heavy chain secretion signal peptide sequence gene, the restriction enzyme recognition sequence for variable region cloning (NotI-XhoI), and the IgG 1 constant region gene were artificially synthesized and inserted as the skeleton lacking 29 bp. ) was cloned into the restriction enzyme recognition sequence (NotI-XhoI) site for cloning to obtain the desired antibody heavy chain expression vector.
  • the light chain secretion signal is obtained by deleting the PCR product from the light chain expression cloning vector pLight (pcDNA3.1 (+) Hyg (Invitrogen) from 962 to 991 at 29 bp).
  • pLight pcDNA3.1 (+) Hyg (Invitrogen) from 962 to 991 at 29 bp.
  • the one in which the entire base sequence was determined and the open reading frame was confirmed to be normal was selected as a heavy chain + light chain pair of human kidney-derived cell line 293T cells.
  • Transient gene transfer (Lipofectamin LTX, Thermo Fisher scientific) into (ATCC CRL-3216), and the amount of target antibody in the culture supernatant after 5 days is determined by a commercially available ELISA kit (Enzygnost anti-HBs II, SIEMENS, Germany) or in. Measured by house ELISA. As a result, a strongly positive antibody (M2-11) was obtained.
  • the light chain expression vector was identified as pL-M2-11.
  • Example 2 Antibody gene isolation (EBV-hybridoma method) The purpose of this example was to isolate the anti-HBs antibody gene from a human individual vaccinated with the HBV vaccine by a method different from that of Example 1.
  • PBMC Peripheral blood mononuclear cells
  • EBV Epstein-Barr virus
  • EBV-infected immortalized B cells After mixing 1.0 ⁇ 10 6 EBV-infected immortalized B cells and JMS-3 myeloma cells, collect them and add 1 mL of 50% PEG (PEG 1540, FUJIFILM Wako Pure Chemical Corporation, Japan) over 1 minute. The cells were added dropwise, 1 mL of RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) was added over 1 minute, and 10 mL of RPMI 1640 medium was added over 1 minute, mixed, and then centrifuged for recovery.
  • PEG PEG 1540, FUJIFILM Wako Pure Chemical Corporation, Japan
  • Cells were harvested, suspended in RPMI 1640 medium supplemented with 20% FCS at a cell concentration of 2.5 ⁇ 10 5 cells / mL, dispensed 0.1 mL each into 96-well plates, and the next day, 0.1 mL HAT selective medium (0.5). The culture was continued with the addition of ⁇ M vavine), and it was confirmed that the antibody was produced in the culture supernatant after 7 to 10 days.
  • Antibody-producing cells were collected from wells where antibody production was confirmed, and the antibody-producing cells were cloned by the limiting dilution method.
  • a passive hemagglutination reaction method (Passive) was performed.
  • High-throughput screening was performed using an automatic measuring device (PK7300, Beckman Coulter, USA) by Hemmaglutination: PHA) (Mycel II antibody-HBs, Special Immunology Laboratory). Since the PHA method is characterized by the fact that the strength of antibody avidity (binding ability to specific antigens) is reflected in the antibody titer, an antibody with virus neutralizing activity can be obtained by adopting screening by this method. It is more likely to be obtained. As a result, 12 strains of HBsAg aggregation-positive hybridomas were established from PBMCs of 3 individuals.
  • RNA was purified from PHA-positive hybridomas (NucleoSpin RNA (TaKaRa)) to obtain antibody heavy chain variable regions, light chain variable regions and constant regions, and carried out using the same primers as those in Example 1 above. It was cloned into a vector by the same method as in Example 1 and expressed as a recombinant antibody by transient gene transfer (Lipofectamin LTX, Thermo Fisher scientific) into human kidney-derived cell line 293T cells (ATCC CRL-3216).
  • ELISA was performed in the same manner as in Example 1.
  • three types of antibodies (5A4, 3D1 and 3B6 ) became strongly positive in ELISA, and these were designated as recombinant HBIG candidates.
  • Chain expression vector is pL-5A4, 3D1 antibody heavy chain expression vector is pIgG 1 H7-3D1, 3D1 antibody light chain expression vector is pL-3D1, 3B6 antibody heavy chain expression vector is pIgG 1 H7-3B6, 3B6 antibody
  • the light chain expression vector was identified as pL-3B6.
  • Example 3 Recombinant antibody expression and purification The purpose of this example was to express and purify four types of recombinant HBIG candidate antibodies obtained in Examples 1 and 2.
  • Heavy chain expression vector and light chain expression vector (pIgG 1 H7-M2-11 and pL-M2-11) of M2-11 antibody obtained in Example 1, and three kinds of antibodies obtained in Example 2 (pIgG 1 H7-M2-11 and pL-M2-11).
  • Heavy chain expression vector and light chain expression vector for 5A4, 3D1 and 3B6) (pIgG 1 H7-5A4 and pL-5A4 for 5A4 antibody, pIgG 1 H7-3D1 and pL-3D1 for 3D1 antibody, pL-3D1 for 3B6 antibody pIgG 1 H7-3B6 and pL-3B6) were transiently transfected into antibody-expressing cells.
  • the culture supernatant was centrifuged to remove cell debris, and filtered through hydrophilic membranes (Durapore, Merck, USA) with pore sizes of 0.45 ⁇ m and 0.22 ⁇ m to adjust the pH to 7.4.
  • affinity purification of the antibody from the culture supernatant using HiTrap Protein G HP Columns (GE Healthcare, USA) equilibrated with PBS gel filtration using Sephadex G-25 (PD-10 column, GE Healthcare) is performed. Buffer exchange to PBS and concentration were performed by the extrafiltration method (Centricon Plus-70 Centrifugal Filter, Merck).
  • purified antibodies were obtained for the four antibodies M2-11, 5A4, 3D1 and 3B6 that were strongly positive for ELISA in Example 1 and Example 2.
  • Example 4 Evaluation of Neutralizing Activity of Recombinant Antibody This Example was performed to confirm that the purified antibody obtained in Example 3 has neutralizing activity against HBV.
  • GEq genomic equivalents
  • DMEM genomic equivalents
  • FBS fetal bovine serum
  • PEG 8000 Nacalai Tesque
  • the qPCR reaction uses nucleic acid recovered from infected cells as a template, Fast SYBER Green Master Mix (Applied Biosystems, USA), primer set (forward primer 5'-gagtgtggattcgcactcc-3'(SEQ ID No: 79), and reverse primer 5'. -This was done using gaggcgagggagttcttct-3'(SEQ ID No: 80).
  • the three types of purified antibodies, M2-11, 5A4, and 3B6, showed good neutralization activity by reducing the copy number of HBV in a concentration-dependent manner, but showed good neutralization activity in 3D1.
  • the evaluation of the 3D1 antibody was terminated here because no decrease in DNA copy number was observed and no neutralizing activity was observed (Fig. 1).
  • HBIG Hebsbrin intramuscular injection 1000 units, Japan
  • Example 5 Evaluation of binding characteristics of recombinant antibody
  • 3 types of human anti-HBs recombinant monoclonal antibodies (M2-11, 5A4, 3B6 3) whose neutralizing activity was confirmed in the screening of Example 4 This was done to confirm the binding properties of the purified antibody) to HBV.
  • HBsAg-XT Recombinant HBs antigen
  • VLP virus-like particles
  • the Blue Native-PAGE method was used. That is, the above HBs antigen was adjusted to 0.1 mg / mL with Native sample buffer (Thermo Fisher Scientific), a reducing agent (50 mM DTT) was added to half of the samples, and NativePAGE 4-16% Bis-Tris. Electrophoresis was performed on Protein Gels (Thermo Fisher Scientific). The protein after electrophoresis is transferred to a PVDF membrane (iBlot 2 Transfer Stacks, PVDF, mini, Thermo Fisher Scientific), blocked at room temperature for 1 hour, and then 2 ⁇ g / mL of the antibody to be evaluated (3 types of human anti-HBs recombination). A primary reaction was performed overnight at 4 ° C. with any of the monoclonal antibodies (three purified antibodies of M2-11, 5A4, 3B6) or the comparative control HBIG or horse polyclonal antibody).
  • the HBs antigen is detected as a monomer (about 25 kDa) and a dimer (about 50 kDa) by the anti-HBs horse polyclonal antibody used as a control, but it is not reduced.
  • a signal was observed near the sample well, indicating that VLP, which is a polymer multimer, was detected under non-reducing conditions (Fig. 3, top).
  • the three monoclonal antibodies of the present invention did not bind to the monomeric and dimeric HBs antigens at all under the reducing conditions SDS-PAGE, and hardly bound to VLP even in the non-reducing state.
  • 3B6 bound weakly to the non-reduced VLP (Fig. 3, top).
  • the epitope recognized by the three monoclonal antibodies is a conformational epitope consisting of discontinuous amino acids, is not defined as a continuous peptide sequence, and at least M2-11 /. It was shown that 5A4 and 3B6 recognize different epitopes.
  • HBsAg has 8 cysteine residues in the extracellular space, and intramolecular and intermolecular disulfide bonds by these residues greatly contribute to the VLP multimer structure. Therefore, the VLP binding of M2-11 and 5A4 is reducing agent sensitive, which strongly supports that these antibodies recognize the multimeric structure itself consisting of HBsAg.
  • Example 6 Evaluation of binding properties of candidate antibodies to antigen aggregates (ELISA) Since the HBsAg may exist as an aggregate in the living body and the higher-order structure of the antigen changes in such a case, in this example, the antibody of the present invention also binds to the HBsAg aggregate. It was decided to confirm whether or not to do so by the ELISA method.
  • Recombinant HBsAg (HBsAg-XT) was heated at 100 ° C for 10 minutes and then rapidly cooled to 4 ° C to induce HBsAg aggregates.
  • the antigen aggregate 5 ⁇ g / mL thus prepared was immobilized on a 96-well plate (Corning, 9018) and allowed to stand overnight at 4 ° C. It was then washed with PBS-10xT (PBS + 0.05% Tween20) solution and then blocked with skim milk solution (adjusted with PBS-10xT to a final concentration of 5%).
  • PBS-10xT PBS + 0.05% Tween20
  • control recombinant HBsAg and heat-recombinant HBsAg detected by M2-11 antibody are referred to as “M2-11” and “M2-11 heat”, and control recombinant HBs by 5A4 antibody.
  • Antigens and heat-recombinant HBs antigens were detected as "5A4" and "5A4 heat”. Both the M2-11 antibody and the 5A4 antibody showed the same degree of binding with or without heating.
  • the binding activity concentration of each antibody at this time was as shown in Table 3 below. From this, it was shown that the antibody of the present invention binds to the aggregate of recombinant HBs antigens as well as to the recombinant HBs antigens that are not associated.
  • Example 7 Intermolecular interaction of the candidate antibody with the recombinant HBs antigen
  • the interaction of the candidate antibody with the recombinant HBs antigen was analyzed using surface plasmon resonance (SPR) technology.
  • the heated recombinant HBs antigen (aggregate) prepared in Example 6 and the recombinant HBs antigen prepared in Example 5 were used, and the antibody was neutralized by the screening of Example 4.
  • Human anti-HBs recombinant monoclonal antibody (M2-11, 5A4) with confirmed activity was used.
  • Example 8 Evaluation of cross-reactivity of candidate antibody
  • three human anti-HBs recombinant monoclonal antibodies M2-11, 5A4, 3B6 whose neutralizing activity was confirmed in the screening of Example 4 were used. This was done to confirm that there was no non-specific binding to human-derived components (no cross-reactivity).
  • the most feared side reaction to the use of antibody drugs is -If the administered antibody has a sequence derived from an animal species other than human, it must be antigenic to humans. -The antibody used is non-specific binding to normal human tissue. Since the three monoclonal antibodies isolated this time are fully human antibodies derived from human B cells obtained from the peripheral blood of vaccinated healthy human individuals, the possibility of the former side reaction is considered to be extremely low. Also, regarding the latter, since the onset of inflammation, autoimmune diseases, etc. has not been reported at all in human individuals who provided B cells derived from these three types of monoclonal antibodies, these antibodies are self-reactive. Is unlikely to indicate.
  • ELISA ELISA was performed using the Antigen-Down ELISA Development Kit (Immunochemistry technologies, Australia).
  • the components targeted by ELISA are human insulin (Fujifilm Wako Pure Drug), which is used to evaluate non-specific binding of antibody drugs to human-derived components, and patients with autoimmune diseases.
  • Genome DNA (used as human genomic DNA extracted from human cultured cell line HeLa cells (ATCC CCL-2)), which is a typical autoantibody antigen in the above, was immobilized on a 96-well plate for ELISA.
  • the three human HBs monoclonal antibodies show non-specific binding to the human-derived component immobilized on the ELISA plate even at an antibody concentration of 10 times or more the concentration at which the binding to the specific antigen HBs is saturated. There was no (Fig. 6).
  • a recombinant anti-HBs antibody mixed solution (mixed solution of M2-11, 5A4, 3B6) diluted to 10 ⁇ g / mL, and then reacted with a secondary antibody (709-035-149, Jackson ImmunoReserach Laboratories). rice field.
  • a peroxidase substrate (ImmPACTDAB, VECTORlaboratorie, USA) was added to develop color, and counterstaining was performed with Mayer's Hematoxylin.
  • the three monoclonal antibody mixed solutions clearly detected HBsAg in the liver tissue of HBV-positive individuals at an antibody concentration of 10 ⁇ g / mL (Fig. 7a), but at the same concentration, various normal human tissues were found. No staining was observed (Fig. 7b). From these results, it was clarified that the three monoclonal antibodies that are candidates for recombinant HBIG have extremely low cross-reactivity with human-derived components.
  • Example 9 Characteristics of candidate antibody CDR sequences
  • the characteristics of the amino acid sequences of the heavy chain and light chain of each HBs monoclonal antibody obtained in the present invention were analyzed.
  • variable regions VH region and VL region
  • the amino acid sequences of the heavy chain variable regions and light chain variable regions of each of these HBs monoclonal antibodies M2-11, 5A4, and 3B6 were analyzed in more detail, and the amino acids in the complementarity determining regions (CDRs) of each chain were analyzed.
  • the sequences are underlined in the following amino acid sequences (CDR1, CDR2, CDR3 in each amino acid sequence, respectively).
  • IMTG the international ImMunoGeneTics information system
  • IMTG / V-QUEST was used to extract the CDR sequences.
  • SEQ ID NOs were assigned to each CDR amino acid sequence as shown in Table 4 below.
  • an antibody expected to have safe, effective and stable activity that can fundamentally solve the problems of unachieved domestic self-sufficiency and the problems of blood products related to anti-HBs human immunoglobulin preparations derived from blood raw materials. Formulations can be provided.

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Abstract

L'invention a pour objet de résoudre à la base le problème de non-autosuffisance sur le plan national en sang de matière de départ pour préparation pharmaceutique d'immunoglobuline humaine anti-HBs, et de fournir une préparation pharmaceutique d'anticorps sûre et efficace.　À cet effet, un anticorps monoclonal spécifique d'antigène anti-HBs pouvant présenter une activité de neutralisation vis-à-vis du VHB, est soumis à un criblage à partir de cellules B circulant dans le sang périphérique à l'intérieur du corps d'un individu humain ayant acquis des anticorps anti-HBs par injection du vaccin contre le VHB, et dont le niveau d'anticorps est augmenté, le gène de cet anticorps est isolé, et une préparation pharmaceutique d'immunoglobuline humaine anti-HBs recombinée, est fournie.
PCT/JP2021/027933 2020-07-29 2021-07-28 Nouveau procédé de fabrication d'immunoglobuline anti-hbs Ceased WO2022025129A1 (fr)

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HEHLE VERENA, BERETTA MAXIME, BOURGINE MARYLINE, AIT-GOUGHOULTE MALIKA, PLANCHAIS CYRIL, MORISSE SOLEN, VESIN BENJAMIN, LORIN VAL&: "Potent human broadly neutralizing antibodies to hepatitis B virus from natural controllers", JOURNAL OF EXPERIMENTAL MEDICINE, ROCKEFELLER UNIVERSITY PRESS, US, vol. 217, no. 10, 5 October 2020 (2020-10-05), US , pages 20200840, XP055836281, ISSN: 0022-1007, DOI: 10.1084/jem.20200840 *
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WANG QIAO; MICHAILIDIS ELEFTHERIOS; YU YINGPU; WANG ZIJUN; HURLEY ARLENE M.; OREN DEENA A.; MAYER CHRISTIAN T.; GAZUMYAN ANNA; LIU: "A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape Mutations", CELL HOST & MICROBE, ELSEVIER, NL, vol. 28, no. 2, 5 June 2020 (2020-06-05), NL , pages 335, XP086247146, ISSN: 1931-3128, DOI: 10.1016/j.chom.2020.05.010 *

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