WO2012023580A1 - Procédé de production d'une streptavidine présentant une faible immunogénicité liée à un anticorps - Google Patents

Procédé de production d'une streptavidine présentant une faible immunogénicité liée à un anticorps Download PDF

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WO2012023580A1
WO2012023580A1 PCT/JP2011/068656 JP2011068656W WO2012023580A1 WO 2012023580 A1 WO2012023580 A1 WO 2012023580A1 JP 2011068656 W JP2011068656 W JP 2011068656W WO 2012023580 A1 WO2012023580 A1 WO 2012023580A1
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amino acid
streptavidin
acid residue
substituted
mutation
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Japanese (ja)
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龍彦 児玉
浩平 津本
暁 杉山
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分子動力学抗体創薬技術研究組合
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to a method for producing a low immunogenic streptavidin conjugated with an antibody.
  • the avidin / streptavidin-biotin interaction is widely applied in the fields of biochemistry, molecular biology, and medicine (Green, (1975), Adv. Protein Chem., 29: 85-133; Green, (1990), Methods Enzymol., 184: 51-67).
  • Avidin is a basic glycoprotein derived from egg white and has an isoelectric point of more than 10.
  • streptavidin is derived from Streptomyces avidinii, has an isoelectric point near neutral and does not contain sugar chains. Both proteins form a tetramer and bind to one molecule of biotin per subunit.
  • the molecular weight is about 60 kDa.
  • the present inventors reduced the immunogenicity (antigenicity) of mammals possessed by streptavidin, a protein derived from Streptomyces avidinii, belonging to microorganisms, suppresses anti-streptavidin antibody production in the animal body, and binds to biotin Streptavidin variants (low immunogenic streptavidin) have been developed (PCT / JP2010 / 001100) that retain the ability to be used for a variety of purposes in medicine and other industries.
  • An object of the present invention is to provide a method for producing a low immunogenic streptavidin conjugated with an antibody in an active state with high efficiency.
  • the present inventor used as a recombinant protein a conjugate of a streptavidin mutant and an antibody having reduced immunogenicity compared to wild-type streptavidin using a host. After expressing in the insoluble fraction, by denatured and unwound the bound product in the insoluble fraction, it is possible to produce a low immunogenic streptavidin with an antibody bound in an active state with high efficiency.
  • the headline and the present invention were completed.
  • step (iii) The method according to [1], wherein the conjugate is modified with guanidine hydrochloride in step (iii).
  • step (iii) after the modification of the above conjugate using guanidine hydrochloride, stepwise reduction using a reduction treatment and an aqueous solution of guanidine hydrochloride whose concentration has been reduced stepwise
  • stepwise reduction using a reduction treatment and an aqueous solution of guanidine hydrochloride whose concentration has been reduced stepwise
  • the method according to [1] or [2] wherein the combined substance is rewound by performing a dialysis treatment.
  • the streptavidin variant has the amino acid sequence of core streptavidin described in SEQ ID NO: 2, wherein (a) the arginine at the 72nd amino acid residue is substituted with another amino acid, and (b) the 10th position Any one or more of tyrosine of the amino acid residue, tyrosine of the 71st amino acid residue, glutamic acid of the 89th amino acid residue, arginine of the 91st amino acid residue, and glutamic acid of the 104th amino acid residue Any one of [1] to [3], which is a streptavidin mutant comprising an amino acid sequence substituted with another amino acid and having reduced immunogenicity compared to wild-type streptavidin The method described.
  • the streptavidin variant has the amino acid sequence of core streptavidin shown in SEQ ID NO: 2 in which (a) the 71st amino acid residue tyrosine and the 72nd amino acid residue arginine are substituted with other amino acids. And (b) one or more of tyrosine at the 10th amino acid residue, glutamic acid at the 89th amino acid residue, arginine at the 91st amino acid residue, and glutamic acid at the 104th amino acid residue.
  • the method according to [4] comprising an amino acid sequence substituted with the amino acid.
  • a low immunogenic streptavidin conjugated with an antibody can be produced with high efficiency in an active state.
  • the low immunogenic streptavidin conjugated with the antibody obtained by the method of the present invention can be used for various purposes in medicine.
  • FIG. 1 shows a sensorgram in Biacore analysis.
  • FIG. 2 shows the reactivity of antisera to mutant streptavidin.
  • FIG. 3 shows the results of thermal shift assay of wild type (natural type) streptavidin, mcSA040, mcSA072, mcSA314, and mcSA414.
  • FIG. 4 shows the structure of the expression vector of B5209B mouse scFv-mcSA414 (SA).
  • FIG. 5 shows the purification of B5209B-mouse-ScFv-WT.
  • FIG. 6 shows the purification of B5209B-mouse-ScFv- mcSA001.
  • FIG. 7 shows the purification of B5209B-mouse-ScFv- mcSA072.
  • FIG. 8 shows the purification of B5209B-mouse-ScFv- mcSA314.
  • FIG. 9 shows the purification of B5209B-mouse-ScFv- mcSA414.
  • FIG. 10 shows the calculation results of the unwinding efficiency for wild type streptavidin (WT) and mutant streptavidin (mcSA001, mcSA072, mcSA314, mcSA414).
  • the method for producing a conjugate of a streptavidin variant and an antibody having reduced immunogenicity compared to wild-type streptavidin comprises (i) immunogenicity compared to wild-type streptavidin.
  • the streptavidin variant in the present invention is not particularly limited as long as it has reduced immunogenicity as compared to wild-type streptavidin, and any streptavidin variant can be used.
  • the streptavidin variant of the present invention has a predetermined amino acid mutation in the amino acid sequence of core streptavidin shown in SEQ ID NO: 2, and has reduced immunogenicity compared to wild-type streptavidin. preferable.
  • amino acid sequence of wild-type (natural) core streptavidin is shown in SEQ ID NO: 2 in the sequence listing, and the base sequence encoding this is shown in SEQ ID NO: 1 in the sequence listing.
  • the streptavidin variant used in the present invention is obtained by replacing (a) the arginine at the 72nd amino acid residue with another amino acid in the amino acid sequence of core streptavidin represented by SEQ ID NO: 2. And (b) tyrosine at the 10th amino acid residue, tyrosine at the 71st amino acid residue, glutamic acid at the 89th amino acid residue, arginine at the 91st amino acid residue, and 104th amino acid residue.
  • SEQ ID NO: 2 tyrosine at the 10th amino acid residue, tyrosine at the 71st amino acid residue, glutamic acid at the 89th amino acid residue, arginine at the 91st amino acid residue, and 104th amino acid residue
  • the streptavidin mutant used in the present invention comprises an amino acid sequence having any one or more of the following mutations in the amino acid sequence of core streptavidin described in SEQ ID NO: 2.
  • tyrosine of the 10th amino acid residue is substituted with another amino acid
  • specific examples of the other amino acid include glycine, serine, and threonine, and particularly preferably serine or threonine.
  • the 71st amino acid residue tyrosine is substituted with another amino acid
  • specific examples of the other amino acid include glycine, alanine, and serine, and particularly preferably alanine or serine.
  • arginine at the 72nd amino acid residue is substituted with another amino acid
  • specific examples of the other amino acid include glycine and lysine, and particularly preferably lysine.
  • glutamic acid at the 89th amino acid residue is substituted with another amino acid
  • specific examples of the other amino acid include glycine, alanine, and aspartic acid, and particularly preferably aspartic acid.
  • arginine at the 91st amino acid residue is substituted with another amino acid
  • specific examples of the other amino acid include glycine or lysine, and particularly preferably lysine.
  • glutamic acid at the 104th amino acid residue is substituted with another amino acid
  • specific examples of the other amino acid include serine, glutamine, and asparagine, and particularly preferably glutamine or asparagine.
  • the immunogenicity is reduced compared to wild-type streptavidin means that the immunogenicity is reduced when a streptavidin variant is administered to a mammal such as a human. .
  • the reduction in immunogenicity can be confirmed, for example, by the following method. That is, with respect to the mutant streptavidin of the present invention, the reactivity to the anti-streptavidin antiserum obtained by immunizing wild type streptavidin to cynomolgus monkeys was analyzed, and the reactivity to the above anti-streptavidin antiserum was If it is lower than that of the wild type streptavidin, it can be determined that the immunogenicity is lower than that of wild type streptavidin.
  • the streptavidin variant of the present invention preferably has an immunogenicity of 80% or less, more preferably 60% or less, compared to wild-type streptavidin. It is preferably 20% or less, more preferably 15% or less, further preferably 10% or less, and particularly preferably 5% or less.
  • streptavidin mutants include the following.
  • a streptavidin mutant comprising an amino acid sequence having any one or more of the following mutations in the amino acid sequence of core streptavidin described in SEQ ID NO: 2 and having reduced immunogenicity compared to wild-type streptavidin.
  • a streptavidin variant comprising an amino acid sequence having the following mutation in the amino acid sequence of core streptavidin described in SEQ ID NO: 2 and having reduced immunogenicity compared to wild-type streptavidin.
  • streptavidin variant which has the following mutations.
  • Streptavidin variant having all of the following mutations in the amino acid sequence of core streptavidin described in SEQ ID NO: 2 (1) Mutation in which tyrosine at the 10th amino acid residue is substituted with serine: (2) Mutation in which tyrosine at the 71st amino acid residue is substituted with serine: (3) Mutation in which arginine at the 72nd amino acid residue is substituted with lysine: (4) Mutation in which glutamic acid at the 89th amino acid residue is substituted with aspartic acid: (5) Mutation in which arginine at the 91st amino acid residue is substituted with lysine: (6) Mutation in which glutamic acid at the 104th amino acid residue is substituted with glutamine or asparagine:
  • the conjugate in the present invention is a conjugate of a streptavidin variant and an antibody that have reduced immunogenicity compared to wild-type streptavidin.
  • Various molecules can be used as the antibody to be bound to the streptavidin variant. Both polyclonal and monoclonal antibodies can be used. Although subclass of the antibody is not particularly limited, preferably IgG, particularly IgG 1 is suitably used.
  • Antibody includes all modified antibodies and antibody fragments.
  • Humanized antibodies, humanized antibodies, human antibodies, antibodies derived from various animals such as mice, rabbits, rats, guinea pigs, monkeys, chimeric antibodies of human antibodies and antibodies derived from various animals, diabody, scFv, Fd, Fab, Fab ′, F (ab) ′ 2 may be mentioned, but is not limited thereto.
  • a fusion protein can be obtained by ligating DNA encoding a streptavidin variant and DNA encoding an antibody and expressing them in a host cell using an expression vector or the like.
  • the DNA encoding the streptavidin variant and the DNA encoding the antibody may be linked via DNA encoding an appropriate peptide called a linker. It is desirable that the streptavidin variant-antibody conjugate is produced while leaving the specific binding force between the antibody and the target molecule.
  • the DNA encoding the streptavidin mutant used in the present invention can be prepared by site-directed mutagenesis on the DNA encoding wild-type (natural) streptavidin.
  • DNA encoding a conjugate of a streptavidin variant and an antibody that has reduced immunogenicity compared to wild-type streptavidin can be incorporated into a vector and used.
  • DNA encoding a conjugate of a streptavidin variant and an antibody that have reduced immunogenicity compared to wild-type streptavidin into an expression vector, and transforming the expression vector into a host, The conjugate can be expressed.
  • a vector used in the present invention has an origin of replication (ori) and a gene for selecting a transformed host (for example, ampicillin, tetracycline, kanamycin, or chlorampheny). It is preferable to have a drug resistance gene for a drug such as cole).
  • a promoter capable of efficiently expressing the streptavidin variant of the present invention in the host for example, a lacZ promoter or a T7 promoter.
  • vectors examples include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, or pET.
  • the host preferably uses BL21 expressing T7 RNA polymerase.
  • a signal sequence for increasing the yield of the streptavidin variant of the present invention can be added to the vector.
  • the introduction of the vector into the host cell can be performed using, for example, the calcium chloride method or the electroporation method.
  • a tag for improving solubility for example, a sequence encoding glutathione-S-transferase, thioredoxin, or maltose-binding protein may be added. It also encodes tags designed to facilitate purification, such as polyhistidine tags, Myc epitopes, hemagglutinin (HA) epitopes, T7 epitopes, Xpress tags and FLAG peptide tags, and other known tag sequences A sequence may be added.
  • expression vectors derived from mammals for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8), derived from insect cells
  • Expression vectors eg, “Bac-to-BAC baculovairus expression system” (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), an expression vector derived from a retrovirus (for example, pZIPneo), an expression vector derived from yeast (for example, “Pichia® Expression® Kit” (manufactured by Invitrogen), pNV11®, SP-Q01), an expression vector derived from Bacillus subtilis (for example, PPL608, p
  • promoters necessary for expression in cells such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have the MMLV-LTR promoter, EF1 ⁇ promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc., and genes for selecting transformation into cells (for example, More preferably, it has a drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
  • the host cell into which the vector is introduced is not particularly limited and may be either prokaryotic or eukaryotic.
  • E. coli and various animal cells can be used.
  • animal cells for example, animal cells, plant cells, and fungal cells can be used as the host.
  • animal cells mammalian cells such as CHO cells, COS cells, 3T3 cells, HeLa cells, Vero cells, or insect cells such as Sf9, Sf21, and Tn5 can be used.
  • CHO cells are particularly preferred for mass expression purposes.
  • Introduction of a vector into a host cell can be performed by, for example, a calcium phosphate method, a DEAE dextran method, a method using a cationic ribosome DOTAP (Boehringer Mannheim), an electroporation method, a lipofection method, or the like.
  • yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae, filamentous fungi such as the genus Aspergillus, such as Aspergillus niger, are known. .
  • E. coli for example, JM109, DH5 ⁇ , HB101, etc.
  • Bacillus subtilis is also known.
  • the culture can be performed according to a known method.
  • DMEM, MEM, RPMI1640, and IMDM can be used as the culture medium for animal cells.
  • a serum supplement such as fetal calf serum (FCS) can be used in combination, or serum-free culture may be performed.
  • FCS fetal calf serum
  • the pH during culture is preferably about 6-8.
  • the culture is usually performed at about 30 to 40 ° C. for about 15 to 200 hours, and medium exchange, aeration, and agitation are added as necessary.
  • a growth factor for promoting cell proliferation may be added.
  • a host is transformed with an expression vector containing a DNA encoding a conjugate of a streptavidin variant and an antibody that are less immunogenic than wild-type streptavidin.
  • Step (ii) culturing the host to express the bound product in the insoluble fraction, followed by (iii) denaturing and unwinding the bound product in the insoluble fraction. .
  • the bound product can be modified with guanidine hydrochloride. Further, in the step (iii), after the modification of the bound product using guanidine hydrochloride, a reduction treatment and a stepwise process using an aqueous solution of guanidine hydrochloride whose concentration is gradually reduced as an external solution. By performing a proper dialysis treatment, the combined substance can be unwound.
  • the streptavidin mutant By preparing a fusion of a cancer antigen-specific antibody molecule and a streptavidin mutant and administering it to a patient by the method of the present invention, the streptavidin mutant can be accumulated specifically in cancer cells. Next, by administering to the patient a diagnostic or therapeutic substance (radioisotope, low molecular weight compound, protein, etc.) conjugated to biotin having affinity for streptavidin or a derivative thereof, the substance can be accurately applied to cancer cells. Can be accumulated.
  • a diagnostic or therapeutic substance radioisotope, low molecular weight compound, protein, etc. conjugated to biotin having affinity for streptavidin or a derivative thereof.
  • Example 1 Design of low immunogenic streptavidin Based on the gene sequence and amino acid sequence of core streptavidin described in SEQ ID NOs: 1 and 2, the sequence of mutant streptavidin having a mutation that satisfies the following conditions: The mutant streptavidin having the mutations listed in Table 1 was designed. (1) The fusion protein with the antibody is a sequence that is predicted to minimize the immunogenicity in the human body as much as possible. (2) A sequence that maintains as much affinity as possible for biotin molecules.
  • Y22 in Table 1 corresponds to the tyrosine of the 10th amino acid residue in the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing.
  • Y22S in Table 1 represents the substitution from the above tyrosine to serine
  • Y22T in Table 1 represents the substitution from the above tyrosine to threonine.
  • Y83 in Table 1 corresponds to tyrosine at the 71st amino acid residue in the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing.
  • Y83A in Table 1 represents the substitution from tyrosine to alanine
  • Y83S in Table 1 represents the substitution from tyrosine to serine.
  • R84 in Table 1 corresponds to arginine at the 72nd amino acid residue in the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing.
  • R84K in Table 1 represents the above-mentioned substitution from arginine to lysine.
  • E101 in Table 1 corresponds to glutamic acid at the 89th amino acid residue in the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing.
  • E101D in Table 1 represents the substitution of glutamic acid to aspartic acid.
  • R103 in Table 1 corresponds to arginine at the 91st amino acid residue in the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing.
  • R103K in Table 1 represents the above-mentioned substitution from arginine to lysine.
  • E116 in Table 1 corresponds to glutamic acid at the 104th amino acid residue in the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing.
  • E116N in Table 1 shows the substitution from glutamic acid to asparagine
  • E116Q in Table 1 shows the substitution from glutamic acid to glutamine.
  • Example 2 Production of mutant streptavidin (1) Synthesis of base sequence of wild-type core streptavidin For the base sequence of the gene encoding core streptavidin shown in SEQ ID NO: 1 in the sequence listing, a service of artificial gene synthesis (Integrated DNA Technologies) was used.
  • Primer 1 GCTCTTCAAAGCTTTGGCCGAAGCTGGTATCACTG (SEQ ID NO: 3)
  • Primer 2 CTCGAGGAATTCTTAGCTAGCAGCAGAAGGCTTAAC (SEQ ID NO: 4)
  • the sample treated with the restriction enzyme was subjected to gel purification after electrophoresis.
  • the pPAL7 vector (manufactured by BIO-RAD) was subjected to enzyme treatment and gel purification.
  • the purified vector and PCR product were ligated by the designated method using 2 ⁇ Rapid Ligation Buffer and T4DNA Polymerase (Promega).
  • the transformation of E. coli was performed by adding 2 microliters of ligation product to 50 microliters of DH5 ⁇ competent cell (manufactured by TOYOBO). Plasmid extraction was performed using Miniprep Kit (manufactured by QIAGEN), and the sequence of the obtained plasmid was confirmed by sequence analysis.
  • a pPAL7 expression vector incorporating the wild-type streptavidin and mutant streptavidin gene sequences was transfected into Escherichia coli BL21 (BIO-RAD) according to a conventional method. Each protein was expressed as follows. That is, the cells were cultured at 37 degrees until the cell density of the E. coli culture solution reached OD (600 nm) 0.5-0.7, and IPTG (isopropyl- ⁇ -D-thiogalactocyanide) was added to a final concentration of 1 mM. Then, protein expression was induced and cultured at 20 degrees for 24 hours. After culturing for 24 hours, the cells were collected by centrifugation, and stored at minus 20 degrees until protein purification.
  • Example 3 Preparation of cynomolgus monkey anti-streptavidin antiserum To cynomolgus monkeys, 1 milligram of recombinant streptavidin (manufactured by PIERCE) was administered three times every two weeks. Blood collection before administration was set to Day 1 and carried out on Days 8, 15, 29, 36, 50, and 57 (Ina Research Co., Ltd.).
  • Example 4 Analysis of binding property between protein and biotin (1) Kinetics analysis of the interaction between protein and biotin using Biacore biosensor
  • the Biacore (registered trademark) biosensor ligand (substance to be attached to the sensor chip) is an anti-mouse IgG antibody (GE Healthcare Bioscience) Made by the company).
  • biotinylated mouse antibodies and various streptavidin mutants were prepared as analytes (substances that flow through the flow path system), and Biacore (registered trademark) 3000 (a biosensor based on surface plasmon resonance, GE Healthcare Bio Analysis of intermolecular interactions by Science).
  • Anti-mouse IgG antibody was immobilized on all the flow cells of the CM5 sensor chip by the amine coupling method. The immobilization amount of each flow cell was 8000 RU.
  • non-biotinylated mouse antibodies were captured in flow cells 1 and 3 for reference, and biotinylated mouse antibodies were captured in flow cells 2 and 4.
  • Various streptavidins were loaded into running buffer (HBS-EP, manufactured by GE Healthcare Biosciences) at 1 and 2 or 3 and 4 at a flow rate of 20 microliters / minute for 2 minutes. Sample dissociation was then monitored for 7 minutes.
  • Table 2 shows the results of kinetic analysis of intermolecular interactions using Biacore (registered trademark) 3000 (biosensor based on surface plasmon resonance, manufactured by GE Healthcare Biosciences) between recombinant streptavidin and biotin.
  • Biacore registered trademark
  • the dissociation constant of the obtained streptavidin variant was on the order of 10 ⁇ 10 M, which was the same order as the dissociation constant of wild-type streptavidin we measured this time. From this result, it was revealed that the streptavidin variant is a protein having a very high affinity with biotin as in the wild type. It can be applied to streptavidin / biotin technology, which is currently widely applied.
  • Biacore registered trademark
  • Amine-PEG 3 -Biotin Thermo SCIENTIFIC
  • a cynomolgus monkey antiserum diluted 20-fold with running buffer (HBS-EP, manufactured by GE Healthcare Biosciences) was prepared as an analyte (substance flowing through the flow path system), and Biacore (registered trademark) 3000 (surface)
  • HBS-EP running buffer
  • Biacore registered trademark
  • Amine-PEG 3 -Biotin was immobilized on all the flow cells of the CM5 sensor chip by the amine coupling method. The average immobilized amount of each flow cell was 160 RU.
  • two types of various streptavidin variants were passed through the wild type streptavidin and the flow cells 3 and 4, respectively, and immobilized by a binding reaction with biotin.
  • the flow cell 1 was used as a reference.
  • the diluted cynomolgus monkey antiserum was set at a measurement temperature of 37 ° C. and loaded into a running buffer (HBS-EP, manufactured by GE Healthcare Biosciences) at 5 microliters / minute for 2 minutes. Sample dissociation was then monitored for 7 minutes. Thereafter, the regeneration operation was repeated with 10 mM glycine hydrochloride buffer, pH 1.7 (manufactured by GE Healthcare Bioscience), and repeated measurements were performed. From the obtained sensorgram (FIG. 1), analysis software BIAevaluation ver.
  • the binding amount of the streptavidin variant and the reaction amount of the antiserum were derived, standardized by the amount of streptavidin bound to each flow cell, and the reaction of the antiserum was compared.
  • a numerical value was calculated by the following formula: (value after antiserum reaction ⁇ value before reaction) / streptavidin binding amount (FIG. 2).
  • Example 5 Analysis of Streptavidin Immunogenicity in Silico Using the Epibase T-cell epitope profiling service (Algonomics), wild-type streptavidin, mcSA072, mcSA040, mcSA314, and mcSA414 are immunogenic in silico ( Immunogenicity was analyzed (Desmet, (2005), Proteins, 58, 53-69; ES126528). Allotypes used in the prediction were selected from Caucasian, Oriental, Indo-European, Afro-American plus West African, Australian, and Mestizo with an appearance frequency of 30% or more.
  • the crystal structure or the closest structure modeled based on the crystal structure was used, and a method including an original side chain arrangement method was used (Desmet, (2002), Proteins, 48, 31-34).
  • the binding free energy between the receptor and the target peptide was calculated, and the antigenic strength was classified based on the strength of the binding force (Kapoerchan, (2009), Mol. Immunol. 47 (5 ), 1091-1097).
  • Tables 3 and 4 show the results of profiling at the allotype level for 37 types of DRB1, 8 types of DRB3 / 4/5, 23 types of DQ, 10 types of DP, and a total of 78 types of HLA class II receptor. From Table 3, which shows the number of critical epitopes, wild-type streptavidin has the least DBR1 epitope, but the most DRB3 / 4/5 epitope, while mcSA314 and mcSA414 have DP epitopes lost. It was shown that the DQ epitope is increasing. Table 4 that summarizes the allotypes that affect them showed that mcSA314 and mcSA414 have a reduced number of epitopes compared to other proteins. From these results, the immunogenicity prediction results were in the order of mcSA314 ⁇ mcSA414 ⁇ wild-type streptavidin (the left is less immunogenic).
  • Example 6 Evaluation of thermal stability of streptavidin protein Thermal shift assays were performed on the following five proteins purified according to Example 2, natural streptavidin, mcSA040, mcSA072, mcSA314, and mcSA414 (Vedadi, ( 2006), Proc Natl Sci USA., 103 (43), 15835-15840). Samples were prepared in real-time PCR tubes (PCR Tube Strip, Flat Cap Strip, manufactured by BIO-RAD) so that the final concentration of each sample was as follows. SYPRO Orange was diluted 5000 times, each protein was 10 ⁇ M, and the buffer was 1 ⁇ PBS.
  • the guanidine hydrochloride solution was prepared so that the final concentration was 0M, 0.5M, 1M, 2M.
  • the reaction volume was 20 ⁇ l.
  • the measuring device used was a CFX96 real-time PCR detection system (manufactured by BIO-RAD).
  • the program mode of the CFX96 real-time PCR detection system was for FRET detection, and the reaction and detection were performed with a program that raised the temperature by 0.5 ° C every 10 seconds.
  • the modified streptavidin, mcSA040, mcSA072, mcSA314, and mcSA414 showed the same thermal stability as that of natural streptavidin at 100 ° C. (FIG. 3). This suggests that the mutation described above reduces immunogenicity but does not affect thermal stability.
  • Example 7 Production of modified monoclonal antibodies (1) Preparation of total RNA from hybridoma cells As a monoclonal antibody B5209B (IgG2b) -producing hybridoma cell, a hybridoma producing the monoclonal antibody B5209B described in JP-A-2008-290996 was used.
  • the hybridoma producing this monoclonal antibody B5209B has the accession number FERM P-21238 on March 2, 2007 (National Institute of Advanced Industrial Science and Technology), Biological Center for Biological Biology (Tsukuba, Ibaraki, Japan). 1-chome, 1-shi-shi, Chuo No. 6 (postal code 305-8656)), and was transferred to the international deposit on October 16, 2007 as deposit number FERM BP-10921 .
  • the above monoclonal antibody B5209B (IgG2b) -producing hybridoma cells 1 ⁇ 10 7 were washed once with phosphate buffered saline (PBS), and solubilized by adding 1 ml of Trizol solution (manufactured by Invitrogen) to the cell precipitate. After passing the extract twice through a 20G injection needle and shearing the DNA, total RNA was purified by chloroform extraction, isopropanol precipitation, and 80% ethanol washing according to the instructions attached to the Trizol solution, and dissolved in sterilized distilled water containing diethyl pyrocarbonate. . It was confirmed that the obtained total RNA was not degraded by agarose gel electrophoresis.
  • MuIgVH5′-A primer of Novagen Mouse Ig-Primer Set was added to the obtained 1st strand cDNA, and double-stranded cDNA was amplified using Expand High Fidelity PCR System (Roche Diagnostics).
  • the obtained double-stranded cDNA was subcloned into pGEM-T vector (Promega) by TA cloning and introduced into E. coli DH5 ⁇ to obtain a plasmid-containing vector.
  • Plasmid DNA of 6 clones was purified with Qiagen Plasmid Midi Kit (manufactured by Qiagen), and the DNA base sequence was determined according to a conventional method.
  • the amino acid sequence of the heavy chain variable region (VH) of the antibody was found to be the amino acid sequence from the first to the 122nd amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 16.
  • B5209B mouse-scFv-mcSA414 expression vector (Fig. 4) An expression vector for B5209B mouse-scFv-mcSA414 having the structure shown in FIG. 4 was constructed.
  • the base sequence of B5209B mouse-scFv-SA in the expression vector is described in SEQ ID NO: 15, and the amino acid sequence is described in SEQ ID NO: 16.
  • the amino acid sequence of the antibody heavy chain variable region (VH) corresponds to the 1st to 122nd amino acid sequence of SEQ ID NO: 16
  • the amino acid sequence of the antibody light chain variable region (VL) is SEQ ID NO: 16. It corresponds to the 142nd to 248th amino acid sequences of the described amino acid sequences.
  • Example 8 Preparation of fusion protein of ScFv and various streptavidin mutants (1) Expression of scFv-SA protein For wild type (WT) streptavidin and modified streptavidin (mcSA001, mcSA072, mcSA314, mcSA414), expression vectors similar to those described in Example 7 (4) were used. Then, Escherichia coli was transformed and cloned according to a conventional method. As Escherichia coli, Rosetta2 (DE3) (Novagen) was used. The cloned clone was inoculated into 2 mL of LB and pre-cultured at 28 ° C.
  • dialysis was carried out for 6 hours using 500 ml of 2M guanidine hydrochloride aqueous solution (50 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 8.0) as an external solution. Thereafter, dialysis was performed for 12 hours against a 1 M guanidine hydrochloride aqueous solution (50 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 8.0) containing 0.4 M of arginine hydrochloride having a protein aggregation inhibitory effect.
  • 2M guanidine hydrochloride aqueous solution 50 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 8.0
  • dialysis was performed for 12 hours against a 1 M guanidine hydrochloride aqueous solution (50 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 8.0) containing 0.4 M of argin
  • dialysis was performed for 12 hours against a 0.5 M guanidine hydrochloride aqueous solution (50 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 8.0) containing 0.4 M arginine hydrochloride. Then, dialysis was performed 3 times (6 hours each) against an aqueous tris buffer solution containing no guanidine / arginine to completely remove guanidine / arginine. Finally, the dialyzed solution was centrifuged (5,800 ⁇ g, 30 minutes), and the supernatant was subjected to centrifugal concentration using an ultrafiltration membrane.
  • a guanidine hydrochloride aqueous solution 50 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 8.0
  • dialysis was performed 3 times (6 hours each) against an aqueous tris buffer solution containing no guanidine / arginine to completely remove guanidine /
  • Size exclusion chromatography The sample that had been concentrated was subjected to size exclusion chromatography to purify the target protein.
  • a column capable of fractionating the target molecular weight of about 160 kDa was used (for example, GE Healthcare, HiLoad 26/60 Superdex 200 pg).
  • a column was connected to the HPLC, and size exclusion chromatography was performed using a Tris buffer aqueous solution (50 mM Tris, 200 mM NaCl, pH 8.0) at a flow rate of 0.5 ml / min to fractionate a protein with the desired molecular weight.
  • FIGS. 5C, 6C, 7C, 8C, 9C CBB stained images

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Abstract

La présente invention concerne un procédé de production hautement efficace d'une streptavidine présentant une faible immunogénicité liée à un anticorps et l'état actif. L'invention concerne également un procédé de production d'un conjugué consistant en une variante de la streptavidine et d'un anticorps, ladite variante de la streptavidine présentant une immunogénicité plus faible que la streptavidine de type sauvage, qui comprend : (i) une étape destinée à transformer un hôte en utilisant un vecteur d'expression contenant un ADN codant pour le conjugué de la variante de la streptavidine et l'anticorps, ladite variante de la streptavidine présentant une immunogénicité plus faible que la streptavidine de type sauvage ; (ii) une étape destinée à cultiver l'hôte et ainsi à exprimer le conjugué dans une fraction insoluble ; et (iii) une étape destinée à dénaturer et à dérouler le conjugué dans la fraction insoluble.
PCT/JP2011/068656 2010-08-19 2011-08-18 Procédé de production d'une streptavidine présentant une faible immunogénicité liée à un anticorps WO2012023580A1 (fr)

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WO2015125820A1 (fr) * 2014-02-18 2015-08-27 サヴィッド・セラピューティックス株式会社 Variant de biotine, mutant de streptavidine, et leurs utilisations

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015125820A1 (fr) * 2014-02-18 2015-08-27 サヴィッド・セラピューティックス株式会社 Variant de biotine, mutant de streptavidine, et leurs utilisations
JP6096371B2 (ja) * 2014-02-18 2017-03-15 サヴィッド・セラピューティックス株式会社 ビオチン改変体、ストレプトアビジン変異体およびそれらの利用
JPWO2015125820A1 (ja) * 2014-02-18 2017-03-30 サヴィッド・セラピューティックス株式会社 ビオチン改変体、ストレプトアビジン変異体およびそれらの利用
JP2017066155A (ja) * 2014-02-18 2017-04-06 サヴィッド・セラピューティックス株式会社 ビオチン改変体、ストレプトアビジン変異体およびそれらの利用
US11161884B2 (en) 2014-02-18 2021-11-02 Savid Therapeutics Inc. Modified biotin, streptavidin mutant, and usage of them
US11286285B2 (en) 2014-02-18 2022-03-29 Savid Therapeutics Inc. Modified biotin, streptavidin mutant, and usage of them

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