WO2008018555A1 - PARTICULE HBsAg À FAIBLE ANTIGÉNICITÉ ET SON PROCÉDÉ DE PRODUCTION - Google Patents

PARTICULE HBsAg À FAIBLE ANTIGÉNICITÉ ET SON PROCÉDÉ DE PRODUCTION Download PDF

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WO2008018555A1
WO2008018555A1 PCT/JP2007/065646 JP2007065646W WO2008018555A1 WO 2008018555 A1 WO2008018555 A1 WO 2008018555A1 JP 2007065646 W JP2007065646 W JP 2007065646W WO 2008018555 A1 WO2008018555 A1 WO 2008018555A1
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hbsag
protein
msl
amino acid
cell
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PCT/JP2007/065646
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English (en)
Japanese (ja)
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Ichiro Yamada
Masaharu Seno
Hiroko Tada
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National University Corporation Okayama University
Beacle Inc.
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Priority to JP2008528881A priority Critical patent/JP5147697B2/ja
Publication of WO2008018555A1 publication Critical patent/WO2008018555A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5184—Virus capsids or envelopes enclosing drugs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a low antigenic HBsAg particle and a method for producing the same.
  • DDS drug delivery system
  • the viral coat protein used as a protein forming the hollow nanoparticles exhibits antigenicity and antibody inducing ability similar to those of viruses when administered into the human body. For this reason, 1) the virus-infected patients who already possess antiviral antibodies by administering the virus vaccine, the administered hollow nanoparticles are neutralized by the antiviral antibodies. In the case of continuous administration, the antibody against itself is induced and neutralized by the antibody, so that the administered hollow nanoparticles may not be able to exert the intended drug delivery ability. Furthermore, the concern of anaphylaxis cannot be excluded as a side effect. Therefore, overcoming this antigenicity and immunogenicity has been a challenge for pharmaceutical applications of hollow nanoparticles.
  • Patent Literature l WO03 / 82344
  • Patent Document 2 JP 2003-286198 A
  • Patent Document 3 JP 2004-2313
  • Patent Document 4 WO03 / 82330
  • An object of the present invention is to provide hollow nanoparticles having a lower antigenicity / immunogenicity and a constituent tamper thereof. It is to provide a protein, a particle comprising the protein as a constituent element, and a preparation method thereof. Means for solving the problem
  • hepatitis B virus coat protein hepatitis B virusless surface antigen protein: HBsAg
  • HBsAg hepatitis B virusless surface antigen protein
  • the present invention includes the following inventions.
  • HBsAg human hepatitis B virus surface antigen protein containing an S polypeptide part, wherein at least amino acids at positions 105 to 148 of the S polypeptide part are deleted, HBsAg Protein variant.
  • HBsAg protein variant according to Item 1, wherein the HBsAg protein variant further comprises a cell recognition moiety.
  • HBsAg protein modification strength In addition to the S polypeptide portion, it contains a non-cell recognition site or a part of the PreS portion, and further, a hepatocyte recognition portion derived from the PreS portion, an antibody, a growth factor, a cyto force-in, Item 4.
  • the HBsAg according to any one of Items 1 to 3, comprising at least one cell recognition moiety selected from the group consisting of a cell surface antigen, a tissue-specific antigen, a receptor, a molecule derived from a virus and a microorganism, and a sugar chain. Protein variant.
  • HBsAg protein variant according to any one of Items 1 to 4, which has a linker peptide at a deletion site including positions 105 to 148.
  • An HBsAg hollow particle comprising the HBsAg protein variant according to any one of Items 1 to 8 as a constituent element.
  • An expression vector containing a gene encoding the HBsAg protein variant according to any one of Items 1 to 8 is introduced into a eukaryotic cell, the cell is transformed, and the resulting transformed cell is cultured.
  • HBsAg hollow particle there is at least one selected from the group consisting of DNA, RNA, protein, lipid, carbohydrate, labeling substance, drug, and physiologically active substance that can function in cells.
  • Material carrier with the following substances.
  • hollow nanoparticles with low antigenicity can be obtained in high yield, and the hollow nanoparticles are excellent in the ability to introduce substances such as genes and proteins into cells.
  • FIG.3 Reactivity of MSL-deficient mutants with goat anti-HBsAg antibody M6_d54-3xFLAG (HBsAg positive control, PC), MSL (108_148) / PD-d54, MSL (108_148) / QE-d54 mutant expression
  • Cos7 cells transfected with the vector were analyzed by western blotting using anti-FLAG-tag antibody and western blotting using goat anti-HBsAg antibody.
  • the MSL-deficient mutant protein was detected to the same extent by the anti-FLAG antibody, but was hardly detected by the anti-HBsAg antibody.
  • mutant proteins that can be used for the formation of the above hollow nanoparticles include human hepatitis B virus coat protein (surface antigen protein, HBsAg).
  • Hepatitis B virus surface antigen L protein has the ability to form particles and recognize target cells.
  • HBsAgL protein consists of N-terminal PreS region and C-terminal S protein region.
  • the PreS region bears the ability to recognize target cells (human hepatocytes), but is replaced with another target recognition sequence when creating hollow nanoparticles with converted target tissues. Therefore, the PreS region is not a region common to all hollow nanoparticles.
  • the S protein part since the S protein part is involved in particle structure formation, it is common to all hollow nanoparticles.
  • Pre-S2 consisting of 55 amino acids added to S protein is M protein (M particle constituent protein), and Pre-Si consisting of 108 amino acids (subtype y) or 119 amino acids (subtype d) is added to M protein.
  • M protein M particle constituent protein
  • Pre-Si consisting of 108 amino acids (subtype y) or 119 amino acids (subtype d) is added to M protein.
  • L protein the protein that constitutes the L particle.
  • the numbering of amino acid positions in the Pre-Sl region is based on a subtype y of 108 amino acids.
  • HBsAg There are three types of HBsAg: adr type, adw type, and ayw type.
  • Pre-Si adr type is 108 a. A.
  • the adw type is 115 a.a. and the ayw type is 119 a.a.
  • Pre_S2 is 55 a.a for all adr, adw and ayw types. Furthermore, S is 226 a.a. for all adr, adw, and ayw types.
  • the adr type L protein (SEQ ID NO: 45) is a 389 amino acid protein consisting of Pre-S1 (108 a.a.), Pre-S2 (55 a.a.) and S (226 a.a.) forces.
  • pGLDLIIP39_RcT L protein (Pre_Sl ⁇ 108 aa, Pre-S2 ⁇ 49 aa, S ⁇ 226 aa) is positions 152 to 15 7 in the adr type L protein of SEQ ID NO: 45
  • SIFSRT prote sensitive self-sequence
  • L protein and M protein have the ability to form particles, similar to S protein. Therefore, the two regions of PreSl and PreS2 may be arbitrarily substituted, attached, deleted, or inserted. For example, by using a modified protein in which a hepatocyte recognition site contained in amino acids 3 to 77 of the Pre-Sl region is deleted, hollow particles that have lost the ability to recognize hepatocytes can be obtained. In addition, since the PreS2 region includes a site that recognizes hepatocytes via albumin! /, This albumin recognition site can also be deleted.
  • hollow bio-nanoparticles composed of hepatitis B virus protein or a protein capable of recognizing hepatocytes, such as L protein and M protein
  • cell recognition sites can be introduced to recognize any cell other than hepatocytes, and the polymer / nucleic acid complex can be introduced into various target cells.
  • cell recognition sites for recognizing specific cells include cells and tissues such as cell function regulatory molecules, cell surface antigens, tissue-specific antigens, receptors, etc. composed of polypeptides such as growth factors and cytoforce-ins.
  • Polypeptide molecules for identification, polypeptide molecules derived from viruses and microorganisms, antibodies, sugar chains and the like are preferably used. Specifically, antibodies against EGF receptor and IL 2 receptor that appear specifically in cancer cells, EGF, and receptors presented by HBV are also included.
  • a protein capable of binding an antibody Fc domain for example, a ZZ tag
  • a strept tag showing biotin-like activity to display a biorecognition molecule labeled with biotin via streptavidin can also be used.
  • the cell recognition site is a polypeptide
  • a DNA encoding a hepatitis B virus protein or a variant thereof and a DNA encoding a cell recognition site if necessary, a DNA encoding a spacer peptide
  • a hollow bio-nanoparticle recognizing an arbitrary target cell can be obtained by linking it in frame, incorporating it into a vector or the like, and expressing it in a eukaryotic cell.
  • the DNA encoding the hepatitis B virus protein or a variant thereof and the DNA encoding the ZZ tag are optionally passed through the DNA encoding the spacer peptide. Connected in-frame and incorporated into a vector etc.
  • the desired hollow bionanoparticles can be obtained by mixing the hollow bionanoparticles that are expressed in the vesicles and an antibody that can recognize the target cells.
  • the cell recognition site is a sugar chain
  • it is obtained by linking a sugar chain capable of recognizing cells such as Sialyl Lewis X to a hollow bionanoparticle having no cell recognition ability using a glycosyltransferase. That's the power S.
  • the Pre-S (Pre_Sl, Pre_S2) of the HBsAg L protein plays an important role when HBV binds to hepatocytes.
  • the S protein part has the ability to form a particle structure.
  • S protein has transmembrane 1! 6 (& 113016011 ⁇ & 116 sequence, TM1 and TM2 from the N-terminal side) at 8-26 residue and 80-98 residue, C-terminal 156-226 residue In addition, it has a membrane interaction region that exhibits very high hydrophobicity.
  • MSL major surface loop
  • An antibody recognition epitope is also mapped to the C-terminal hydrophobic region (156-226).
  • HBsAg L protein, HBsAg M protein, or HBsAg S protein When HBsAg L protein, HBsAg M protein, or HBsAg S protein is expressed in eukaryotic cells, the protein is synthesized and accumulated as a membrane protein on the endoplasmic reticulum membrane, and then aggregates between molecules, causing the endoplasmic reticulum membrane to While taking up, it is released as particles to the lumen side in the form of budding, and finally it is separated into the culture supernatant.
  • linker-amino acid / linker peptide in addition to 105-148, part or all of 101-104 and 149-154 can be deleted, and a linker-amino acid / linker peptide can be introduced as necessary.
  • Preferred linker lengths include 2 to 4 amino acids.
  • a substitution sequence derived from a restriction enzyme site introduced for convenience of DNA construction one amino acid (Gly (G)) on the N-terminal side and the C-terminal side on both sides of the MSL to be deleted) 2 amino acids Ser-Trp (SW); substitution of a total of 3 residues.
  • the MSL (108_148) PD variant MSL108-148 deficient PD linker insertion
  • MSL107_150 MSL107- 150 deficient GPDSW linker purchase.
  • substitution sequences derived from restriction enzyme sites that can be introduced on both sides of the linker can vary depending on the selection of restriction enzymes (sites), the nucleotide sequences on both sides of the deleted amino acid sequence, and the like.
  • TM2 transmembrane helix 2
  • C-terminal hydrophobic region is linked. Between these two structures, we first stop the TM2 helix structure, pull its C-terminal part out of the membrane, and then turn to introduce a sequence that goes back into the membrane and connects to the C-terminal hydrophobic region. Is desirable.
  • the length of the linker is preferably exemplified by 2 to 4 residues because the so-called folded structure has 4 residues but may not be completely folded.
  • Amino acids introduced as linkers include amino acid residues that break the helix (Gly, Pro, Asn, Tyr), amino acid residues that are many in the turn structure ( ⁇ , Glv. Pro. ASP. Ser. Tro), membrane interface Examples include combinations of amino acid residues (Sl, As ⁇ , Lys, His, Arg, Gki, Pro, Agn) that are difficult to stay (prone to go out of the membrane), and are not particularly limited.
  • Pro-Asp (PD), Gln_Glu (QE), Pro-Asp-A sn-Gly (PDNG), Pro_Ser-Ser-Ser (PSSS), Pro_Lys, Asp-Pro, and the like.
  • the Cys residue contained in the S protein portion can be substituted with other amino acids such as Ser and Ala.
  • Cys at positions 76, 90, 107, 137, and 149 can be substituted with other amino acids such as Ser and Ala.
  • Examples of preferred substitutions include Cys76 / Ala, Cys 90 / Ala, Cys 107 / Ser, Cys 137 / Ser, Cys 139 / Ser, Cys 149 / Ser, and Cys 221 / Ala.
  • Non-Patent Document 1 describes the ability to delete S-protein 107-146 for its production efficiency, usefulness as a substance introduction agent, etc.! It is not listed. Rather, it has been described that the efficiency of gene transfer is reduced by this deletion.
  • 105-148 can be deleted with respect to the major antigen loop, and 101-104, 149-154 amino acids, and also the lack of the 54 amino acid residue region on the C-terminal side of the C-terminal hydrophobic region. Loss is possible. For example, deletion of the amino acid sequence (101-154) or (105-154) is particularly preferred. For the purposes of the present invention, the more deleted regions, the more desirable.
  • Proteins that constitute particles such as HBsAg mutant protein are linked to S protein directly or through a linker, or introduced into the PreS region by targeting a molecule that recognizes a specific cell. Substances can also be introduced specifically into tissues. Examples of such molecules that recognize specific cells include cell function regulatory molecules such as growth factors and cytokines, cell surface antigens, tissue-specific antigens, molecules for identifying cells and tissues such as receptors, Molecules derived from viruses and microorganisms, antibodies, sugar chains (eg, Sialyl Lewis X) and the like are preferably used. Specifically, it includes antibodies to EGF receptor and IL2 receptor that appear specifically in cancer cells, EGF, and receptors presented by HBV.
  • cell function regulatory molecules such as growth factors and cytokines, cell surface antigens, tissue-specific antigens, molecules for identifying cells and tissues such as receptors
  • Molecules derived from viruses and microorganisms antibodies, sugar chains (eg, Sialyl Lewis X) and
  • a protein capable of binding an antibody Fc domain for example, a ZZ tag
  • a strept tag showing biotin-like activity to display a biorecognition molecule labeled with biotin via streptavidin can also be used.
  • These are appropriately selected according to the target cell or tissue.
  • Cell recognition molecules can be introduced into HBsAg mutant proteins according to known methods!
  • Examples of the low antigenic particles of the present invention include those obtained by expressing HBsAg mutein in eukaryotic cells.
  • the method for producing particles is described in Patent Documents 1 to 4, etc., and the preparation method for H BsAg is Vaccine. 2001 Apr 30; 19 (23-24): 3154-63 ⁇ Physicochemical an a immunological cnaractenzation or nepatitis B virus envelope particles exclusively c onsisting of the entire L (pre ⁇ Sl + pre ⁇ S2 + S) protein.
  • the protein is preferably expressed and accumulated as a membrane protein on the endoplasmic reticulum membrane and released as a nanoparticle.
  • eukaryotic cells include animal cells such as mammals (for example, CHO cells), insect cells (such as expression systems using baculowinoles), and yeast. Such particles are extremely safe for the human body because they do not contain any HBV genome.
  • the cell selectivity of the nanoparticle of the present invention for hepatocytes or other cells can be enhanced by introducing a cell recognition molecule into at least a part of the protein constituting the particle as necessary.
  • HBsAg represents hepatitis B virus surface antigen (H mark atitis B virus surface Antigen) which is a coat protein of HBV.
  • HBsAg contains an S protein composed of 226 amino acids.
  • M protein has 55 amino acids (Pre_S2 peptide) added to the N-terminal side of S protein, and L protein has 108 or 119 amino acids (PreSl p-marked tide) added to the N-terminal side of M protein ( The addition of 108 amino acids is shown in Figure 1).
  • a secretory signal sequence was added to the N-terminus, and a protease-sensitive sequence IJ (six amino acids at positions 152-157 of SEQ ID NO: 45) in the PreS2 region was removed. I used something.
  • an L protein was prepared by fusing a C-terminal detection epitope tag sequence. That is, the plasmid pB0747 for animal expression of a mutant (L-d54-FLAG) in which the C-terminal 54 amino acids of the wild-type L protein were deleted and the FL AG tag sequence was fused was cleaved with Xho I and the FLAG-tag sequence twice
  • the plasmid pB0982 for expression of the protein (L-d54-3xFLAG) in which the FLAG-tag sequence was fused three times was constructed by inserting and ligating the synthetic DNA (SEQ ID NO: 1 & 2).
  • L-d54_3xFLAG A plasmid pB0851 for expressing a mutant (M6_d54-3xFLAG) in which 6 of 13 Cys residues in the S region of the protein were replaced with Ser residues or Ala residues was constructed. Specifically, substitution mutations of Cys76 / Ala, Cys 90 / Ala, Cys 107 / Ser, Cys 137 / Ser, Cys 139 / Ser, Cys 147 / Ser, and Cys 149 / Ser were introduced. The subsequent amino acid sequence numbers are the S protein sequence numbers.
  • the above-mentioned MSL (108_148) / PD-d54 expression plasmid pB0941 is a saddle type, and site characteristics using 3 sets of synthetic DNAs shown in Table 1, SEQ ID NOs: 15 & 16, 17 & 18, or 19 & 20.
  • Apal site By introducing the second restriction enzyme Apal site into the position corresponding to Leu98, TyrlOO, and Glyl02, respectively, by introducing different mutations, the resulting plasmid DNA was cleaved with Apal and recombined, so that the S protein MSL Three MSL deletion L proteins (MSL (99) with deletion of residues 99-148, 101-148, or 103-148, respectively, and a PD linker sequence inserted.
  • MSL (99) Three MSL deletion L proteins
  • mutant HBsAg included in the present invention can be obtained by replacing the synthetic DNA used for site-directed mutagenesis by the same method as described above.
  • Table 3 shows the name of the MSL-deleted L protein expression vector constructed as described above, the name of the encoded MS L-deleted L protein, and the amino acid sequence around the deletion site.
  • the monkey kidney-derived cell line COS 7 was cultured in Danolebecko's modified Eagle's medium (DMEM) containing 5% ushi fetal serum (FBS) in the presence of 37 ° C and 5% C02.
  • COS7 cells were suspended in 10% FBS-containing DMEM so as to have lxlO 5 cells / ml, seeded in 2 ml in 3.5 cm dishes, and cultured for 14 to 16 hours.
  • DMEM 95 1 and the transfection reagent FuGene6 (Roche Diagnostics) 41 was mixed.
  • the entire amount was added to the COS7 cells. After culturing for 14 to 16 hours, the medium was replaced with 1.5 ml of serum-free medium CHO-S-SFMII (Invitrogen), and further cultured for 2 days, and then the culture supernatant and cells were collected.
  • serum-free medium CHO-S-SFMII Invitrogen
  • a 0-phenylenediamine solution (0.4 mg / ml o_phenylenediamine, 23 mM citrate, 51.4 mM Na2HP04, 0.0012% H 2 O 2) was used as the HRP substrate, and quantified by measuring the absorbance at 495 nm. Wild-type L particle-expressing COS7 cell culture supernatant was used as a negative control, and L-d54-3xFLAG particle-expressing COS7 cell culture supernatant was used as a positive control.
  • Tables 4 and 5 show the results of measuring each of the collected MSL-deleted L protein-expressing COS7 cell culture supernatants by 2-fold to 4-fold dilution and measuring by the above FLAG-ELISA.
  • Table 4 When comparing the effects of insertion linker sequences in MSL (108_148) deletions (Table 4), 2 amino acid residue insertion type (QE, PD) rather than 4 amino acid residue insertion type (PSSS, P DNG) ) Had more particle secretion.
  • QE, PD 2 amino acid residue insertion type
  • PSSS 4 amino acid residue insertion type
  • P DNG amino acid residue insertion type
  • the IMX HBsAg Atsy system (Dynabot) is an automated HBsAg measurement system based on an enzyme immunoassay using mouse anti-HBsAg monoclonal antibody, biotinylated anti-HBsAg polyclonal antibody and alkaline phosphatase (AP) -labeled anti-biotin antibody. Yes, it is used for blood diagnosis of HBV infection. Using this system, the reactivity of various MSL-deleted L particles secreted into the culture supernatant and anti-HBsAg antibody was measured.
  • AP alkaline phosphatase
  • MS recovered L-depleted L protein expression COS7 cell culture supernatant was diluted with Dulbecco's phosphate buffer (PBS) containing 1% FBS and measured.
  • PBS Dulbecco's phosphate buffer
  • V and misplaced MSL-deleted L particles were also determined to be HBsAg-negative. (Tables 4 and 5). Therefore, the anti-HBs Ag antibody response value per L particle amount of the MSL mutant that showed good particle secretion was 1 to 4% or less of L-d54-3xFLAG, and anti-HBsAg antibody reactivity due to MSL deletion was significantly reduced.
  • M6_d54-3xFLAG particle-expressing COS7 cells or MSL-deficient L protein expressing COS7 cells were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. After blocking PV DF membrane with 5% skim milk, analysis using biotinylated anti-FLAG M2 antibody or biotinylated anti-HBsAg polyclonal antibody (Dynabot MX HBsAg Atsy system liquid phase antibody) and HRP-labeled streptavidin (Figure 3).
  • M6_d54-3xFL AG showed a clear band around 40 kDa when detected with anti-FLAG antibody and with anti-HB sAg antibody.
  • MSL-deleted L protein mutants (MSL (108-148) / QE-d54 and MSL (108_148) / PD-d54) detected a clear band with anti-FLAG antibody, but with anti-HBsAg antibody was not detected, indicating decreased reactivity to goat anti-HBsAg polyclonal antibody.
  • MSL (105_152) / PD-d54 MSL (105_154) / PD-d54 is more reactive to goat anti-HBs polyclonal antibody than MSL (105-148) / PD-d54 ( Figure 4, Table 1).
  • Hebsbrin (Wuerfid) is an anti-HB sAg human immunoglobulin product prepared from sera of anti-HBsAg antibody positive patients. Whether this human anti-HBsAg polyclonal antibody recognizes MSL-deficient L particles by Western blotting and enzyme immunoassay. taking measurement.
  • L-d54-3xFLAG protein-expressing COS7 cells or MSL-deficient L protein SL (105_154) / PD-d54] -expressing COS7 cells were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. After blocking the PVDF membrane with 5% skim milk, it is reacted with a herbsulin solution, and further detected with an HRP-labeled-anti-HgG (H + L) antibody. The PVDF membrane is then reprobed with biotinylated anti-FLAG antibody and HRP-labeled streptavidin.
  • biotinylated anti-FLAG antibody reacts with L-d54-3xFLAG and MSL-deficient L protein to the same extent and shows a clear band around 40 kDa.
  • hebsbrin human anti-H BsAg polyclonal antibody
  • L-d54-3xFLAG particle expression COS7 cells or MSL-deficient L protein [MSL (105_154) / PD-d54] expression COS7 cell culture supernatant was added with anti-FLAG antibody-agarose gel (Sigma), 4 After reacting at ° C, recover the agarose gel. The obtained gel is washed with TBST (0.1% Tween 20, 150 mM NaCL, lOmMTris-HCl buffer ⁇ 7.5), and then TBST containing 5% skim milk is added and reacted at 4 ° C for 2 hours. After washing with TBST, add hebrin solution and react at 4 ° C for 2 hours.
  • AP labeled anti-HgG (H + L) antibody is further added and allowed to react at room temperature for 1 hour.
  • AP chromogenic substrate solution CDP-star, New England BioLabs
  • the reaction between MSL-deficient L particles and hebusulin is significantly lower than that with L-d54-FLAG particles.
  • WT-d54-3xFLAG particle-expressing COS7 cells are available! /, Is an MSL-deleted L protein [MSL (105-154) / PD-d54] -expressing COS7 cells solubilized with l% TritonX-100-PBS It was diluted with 1% FBS-TBS (150 mM NaCl, 50 mM Tris_HCl buffer ⁇ 5 ⁇ 5). This was measured by an enzyme immunoassay using an anti-FLAG antibody as a solid phase and a human anti-HBsAg antibody (Hevesbulin) and an HRP-labeled anti-HgG antibody (Prozyme) in the liquid phase.
  • WT -d54-3xFLAG showed a binding reaction with an absorbance of 0.051 when 9 ng particles were added, but MSL (105_154) / PD-d54 had a detection limit. It was below the boundary (negative control Cos7 cell extract).
  • Anti-HBs antigenic determinant d Monoclonal antibody (Special Immunology Laboratories) or anti-HBs antigenic determinant r Monoclonal antibody (Special Immunology Laboratories) in solid phase, biotinylated anti-FLAG M2 antibody and HRP-labeled streptavidin MSL-deficient L protein-expressing Cos7 cell culture supernatants were measured using the enzyme immunoassay used for the phases (subtype d-ELISA and subtype r-ELISA, respectively).
  • Anti-subtype d monoclonal antibody reactivity of MSL (105_152) / PD-d54 particles and MSL (105_154) / PD-d54 particles is 1-3% of WT-d54-3xFLAG particles, anti-subtype r Monoclonal antibody reaction Sex dropped to 13-14%.
  • HBsAg diagnostic kit _EIA b 1.0 0 0. 2 1. 7 Sat 0.3 subtype d -ELISA c) 1.3 3 0. 9 3. 2 Sat 2. 0 subtype r-ELISA d) 12. 8 people 2. 6 14. 4 ⁇ 2. 9
  • MSL (105_154 / PD) -d54 sample was measured by each ELISA, and the reactivity to each antibody (reaction value per particle amount (absorbance)) was calculated. The relative reactivity of each mutant to the reactivity of WT-d54-3xFLAG is shown.
  • Dynabot Mx-HBsAg kit mainly recognizes common antigenic determinant a.
  • All of the MSL-deleted L protein expression vectors constructed in Example 1 are the L protein C A FLAG-tag for detecting the expressed protein is added to the end. Therefore, first, FLAGtag added to the C-terminus of MSL-deleted L protein by site-directed mutagenesis using MSL-deleted L protein [MSL (105_154) / PD-d54] expression vector and synthetic DNA SEQ ID NO: 25 & 26 A stop codon is inserted in front of the sequence to construct a vector pBO1077 for expressing the absence of the C-terminal FLAGtag sequence and the MSL-deleted L protein [MSL (105_154) / PD-d54].
  • the L protein [MSL (105_154) / PD-d54] expression vector pBO1077 is constructed. This was introduced into the Sac charomyces serevisiae AH22R strain, and transformant colonies were selected according to leu requirements. Among the obtained transformant clones, select a clone with high expression of MSL-deleted L particles.
  • the obtained high expression strain was mass-cultured in the same manner as wild-type HBsL particles, and the ultracentrifugation method described in J. Biol. Chem. Vol. 267 (3) pl953-1961 (1992) Purify the MSL-deficient L particles using.
  • Reference Example 1 Enzyme-linked immunosorbent assay for antibodies to wild-type L particles and MSL-deficient L particles
  • the purified wild-type L particle was dissolved in PBS to a concentration of 20 g / ml, and then dispensed into a 96-well plate at 100 1 / well, at 4 ° C Soot was adsorbed. After washing with PBS containing 0.05% Tween 80 (PBST), 150% PBS containing 5% Block Ace (snow mark) was dispensed and stored at 4 ° C.
  • Mouse antiserum for dilution (containing 10% FBS) Diluted 20 times to 20,000 times with PBST), added to each well, and allowed to react at room temperature for 2 hours.
  • MSL deletion mutants MSL ([deletion region]) / [linker sequence] -d [number of C-terminal deletion residues]

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Abstract

L'invention porte: sur une nanoparticule creuse présentant une antigénicité/immunogénicité faible; sur une protéine constitutive de la nanoparticule creuse; sur une particule faite de ladite protéine; et sur un procédé de préparation de ladite particule. L'invention porte spécifiquement sur la protéine mutante HbsAg (de l'antigène de surface du virus humain de l'hépatite B) qui présente une délétion d'au moins un résidu d'acide aminé entre la position-105 et la position-148 d'un segment du polypeptide S d'une protéine HBsAg contenant ledit segment.
PCT/JP2007/065646 2006-08-11 2007-08-09 PARTICULE HBsAg À FAIBLE ANTIGÉNICITÉ ET SON PROCÉDÉ DE PRODUCTION WO2008018555A1 (fr)

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JP2006-220346 2006-08-11

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WO2008018555A1 true WO2008018555A1 (fr) 2008-02-14

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JP (1) JP5147697B2 (fr)
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064930A1 (fr) * 2000-02-28 2001-09-07 Japan Science And Technology Corporation Nanoparticules creuses proteiques, transporteur associe a l'utilisation de ces nanoparticules et procede d'introduction d'une substance dans des cellules
WO2003082330A1 (fr) * 2002-03-29 2003-10-09 Japan Science And Technology Agency Medicament therapeutique comprenant des nanoparticules proteiques creuses presentant un anticorps et nanoparticules proteiques creuses

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064930A1 (fr) * 2000-02-28 2001-09-07 Japan Science And Technology Corporation Nanoparticules creuses proteiques, transporteur associe a l'utilisation de ces nanoparticules et procede d'introduction d'une substance dans des cellules
WO2003082330A1 (fr) * 2002-03-29 2003-10-09 Japan Science And Technology Agency Medicament therapeutique comprenant des nanoparticules proteiques creuses presentant un anticorps et nanoparticules proteiques creuses

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KONDO A. ET AL.: "Chuku Bio Nano Ryushi o Mochiita DDS no Kaihatsu to Sono Sangyoka", DRUG DELIVERY SYSTEM, vol. 21, no. 4, 31 July 2006 (2006-07-31), pages 435 - 443, XP003020247 *
O'MALLEY B. AND LAZINSKI D.: "A Hepatitis B Surface Antigen Mutant That Lacks the Antigenic Loop Region Can Self-Assemble and Interact with the Large Hepatitis Delta Antigen", J. VIROL., vol. 76, no. 19, 2002, pages 10060 - 10063, XP003020248 *
TADA H. ET AL.: "Ryushi Keisei ni Hitsuyo na Hito B-gata Kan'en Virus Envelope Tanpakushitsu Hairetsu no Tokutei", PROTEIN SCIENCE SOCIETY OF JAPAN NENKAI PROGRAM YOSHISHU, vol. 7TH, 7 May 2007 (2007-05-07), pages 135, XP003020249 *

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JP5147697B2 (ja) 2013-02-20

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