WO2009113742A1 - Procédé de fabrication d'un anticorps produit par génie génétique - Google Patents

Procédé de fabrication d'un anticorps produit par génie génétique Download PDF

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Publication number
WO2009113742A1
WO2009113742A1 PCT/JP2009/055555 JP2009055555W WO2009113742A1 WO 2009113742 A1 WO2009113742 A1 WO 2009113742A1 JP 2009055555 W JP2009055555 W JP 2009055555W WO 2009113742 A1 WO2009113742 A1 WO 2009113742A1
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antibody
cells
producing
gene
cell
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PCT/JP2009/055555
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Japanese (ja)
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松葉隆雄
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東ソー株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates to a method for producing a recombinant rabbit monoclonal antibody.
  • Monoclonal antibodies are widely used not only as experimental reagents for detecting specific substances, but also as core materials for cancer therapeutics and immunodiagnostics because of their high specificity and affinity.
  • mouse-derived monoclonal antibodies are generally recognized.
  • the degree of affinity of mouse-derived monoclonal antibodies is often sufficient.
  • mouse-derived monoclonal antibodies may have insufficient affinity in areas where measurement sensitivity has become increasingly demanded in recent years, such as immunodiagnostic drugs.
  • the competition method it can be theoretically proved that the higher the affinity of the antibody, the higher the detection sensitivity.
  • an antibody with high affinity is required. Become.
  • even with the sandwich method it becomes possible to detect a small amount of antigen by using an antibody with high affinity, and a highly sensitive measurement system can be constructed.
  • estradiol E 2
  • E 2 requires a highly sensitive measurement system, and there is a strong demand for antibodies with high affinity.
  • Non-patent Document 1 antibodies with high affinity tend to be isolated from antibodies of large animal species .
  • the effect of individual differences is great, and the antisera obtained are also different.
  • at least several animals must be isolated in order to isolate antibodies with high affinity. It is necessary to immunize an individual with an antigen and isolate a monoclonal antibody from an individual with a high immune response. Therefore, in reality, Usagi is chosen as an immunized animal.
  • Rabbits are also considered to be suitable as immunized animal species, because Rabbit's serum is often used as an antiserum for immunodiagnostics.
  • a method for producing a mouse monoclonal antibody has been established and is produced by the method shown below.
  • spleen cells antibody-producing cells
  • mouse myeloma cells myeloma cells
  • the antibody produced by culturing the cells is purified to produce a mouse monoclonal antibody.
  • Usagi monoclonal antibodies can also be produced theoretically in the same manner as mouse monoclonal antibodies.
  • Usagimyeloma cells used for cell fusion with antibody-producing cells Proc. Nat. A cad. Sci. USA, 9 2, 9 3 4 8; 1 9 9 5: Non-patent document 2
  • Heron monoclonal antibodies cannot be produced. For this reason, several methods have been investigated for producing rabbit antibody monoclonal antibodies without using rabbit cells. First, there is a method of fusing rabbit antibody-producing cells with mouse myeloma cells. However, this method is fusion between different animals, and the fusion efficiency is low. Therefore, the number of hybridomas obtained is small, and the obtained hybridomas lose their antibody-producing ability within a short period of time (
  • a rabbit antibody library was prepared, and the rabbit antibody antibody was obtained by the phage display method.
  • J. Biol. Chem., 2 75, 1 3 6 6 8; 2 0 0: Non-patent document 6; Gene, 1 7 2, 2 9 5; 1 996: Non-patent document 7; J. Immunol. Method., 2 13, 20.1; 1 998: Non-patent document 8 an operation called “banning” is essential in the process of selecting antibodies from the library.
  • a water-insoluble carrier on which an antigen is immobilized is brought into contact with a library, and an antibody bound to the antigen is recovered. Then, the antibody bound to the antigen is amplified by some method to create a new library, and the reaction of the antigen with the water-insoluble carrier is repeated. By this operation, the antibody that binds to the antigen is concentrated, and the antibody that finally binds to the antigen can be isolated.
  • the antigen-antibody reaction is an equilibrium reaction, the binding between the antigen and the antibody is affected by the concentration.
  • the library In principle, it is impossible to selectively concentrate high-affinity antibodies that are present in only amounts. In order to obtain antibodies with high affinity in general,
  • the antibody needs to be a combination of the H and L chains originally produced by the Usagi.
  • the combination of the H and L chains originally produced by the Usagi may be reproduced. Since it is extremely low, it is extremely difficult to recover an antibody with improved affinity by this operation.
  • Non-Patent Document 9 may be used as long as a rabbit monoclonal antibody that binds to an antigen is isolated, but thousands of high-performance monoclonal antibodies that can be used in immunodiagnostics are isolated. Even if a recombinant antibody of tens of thousands of clones is prepared, there is no guarantee that the target antibody can be isolated. Furthermore, since the above work requires a large number of experimental man-hours, it is practically difficult to implement. In addition, since the gene is isolated from a single cell, the efficiency of antibody gene amplification is low even if an antibody-expressing cell is found, and the antibody gene amplification probability reported in Non-Patent Document 9 is H chain. 27%, about 55% for L chain.
  • the problem to be solved by the present invention is that it is possible to produce a Usagi monoclonal antibody that could only be produced by a specific institution having Usagimie mouth cells with knowledge of normal laboratory facilities and molecular biology. It is to provide technology.
  • the present invention made in view of the above problems includes the following inventions.
  • the first invention is to isolate antibody-producing cells from rabbits immunized with antigen, to select cells carrying the desired antibody gene, and to isolate and amplify heavy and light chain antibody genes from the selected cells
  • a cell having the target antibody gene is selected.
  • the second invention is characterized in that the operation of isolating heavy and light chain antibody genes from the selected cells is carried out within a period in which the antibody genes are not dropped from the selected cells. This is a method for producing the genetically engineered antibody described in the invention.
  • the third invention is characterized in that the operation of isolating heavy and light chain antibody genes from the selected cells is carried out within 14 days after the start of proliferation of antibody-producing cells. It is a manufacturing method of the gene recombinant antibody of description.
  • the fourth invention is characterized in that the antibody-producing cells are any of spleen cells, lymph node cells, and peripheral blood cells of a rabbit immunized with an antigen.
  • the method for producing a recombinant antibody according to any one of the first to third inventions is characterized in that the method for growing antibody-producing cells is a method for culturing antibody-producing cells in a medium.
  • the sixth invention is characterized in that the method of growing antibody-producing cells is a method of fusing and culturing cells capable of imparting immortalizing ability, as described in the first to fourth inventions This is a method for producing a recombinant antibody.
  • a seventh invention is the method for producing a genetically engineered antibody according to the first to sixth inventions, characterized in that the antigen to immunize the rabbit is a hapten or a peptide.
  • the eighth invention is the method for producing a recombinant antibody according to any one of the first to seventh inventions, wherein the host cell is an animal cell.
  • Fig. 1 shows the results of culturing cells derived from the spleen and evaluating the antigen-binding ability of the culture supernatant of 192 clones.
  • Figure 2 shows the results of evaluation of antibodies in the culture supernatant after cell fusion using lymphocytes derived from peripheral blood by ELISA.
  • FIG. 3 shows the results of screening the culture supernatant of 3840 clones.
  • the vertical axis shows competitive characteristics
  • the horizontal axis shows specificity
  • the size of each plot shows reactivity.
  • the antibody indicated by the black circle plot in the figure is a clone with high competitive performance, high specificity, and high reactivity, and the antibody gene was isolated from the clone.
  • FIG. 4 shows the structure of the expression vector p ECE dhfr.
  • SV 40 P in the figure is the SV 40 promoter, dhfr is dihydrophore * Reductorase gene, poly A indicates polyadenylate displacement signal, Amp indicates ampicillin resistance gene, and MCS indicates multiple cloning site.
  • FIG. 5 shows the results of comparison of competition characteristics using mouse monoclonal antibody, rat monoclonal antibody, and rabbit monoclonal antibody produced in the present invention.
  • the vertical axis shows the BZB 0 value when measuring E 2 at the concentration of l O O O p gZmL, and the horizontal axis shows the dissociation constant.
  • Figure 6 shows the results of evaluating the performance of each antibody by fluorescence depolarization.
  • the vertical axis indicates the degree of polarization, and the horizontal axis indicates the antibody concentration.
  • the degree of polarization of the fluorescent substance label E 2 used as the antigen was set to 20.
  • the black circles in the figure indicate the results when the mouse monoclonal antibody Ma-1 is used, the white circles indicate the rat monoclonal antibody Ra-1 and the black squares indicate the rabbit monoclonal antibody U-1 prepared according to the present invention. It is.
  • FIG. 7 shows the result of preparing a calibration curve by preparing a reagent for a fully automatic immunodiagnostic system manufactured by Tosoh Corporation.
  • the white circles in the figure are immune reaction reagents using polyclonal antibodies (E test “TOS OH” IIE 2, manufactured by Tosoh Corp.), and the black circles are immunizations using Usagi monoclonal antibody U-1 produced in the present invention. The results are obtained when each reaction reagent is used.
  • Figure 8 shows an immunoreaction reagent using a polyclonal antibody (E-test “TOS OH” IIE 2) and an immunoreaction reagent using Usagi Monoclonal Antibody U-1 produced in the present invention. The result of measuring the sample and correlating it is shown.
  • FIG. 9 shows the results of screening the culture supernatant of 3840 clones.
  • the vertical axis shows competitive characteristics
  • the horizontal axis shows specificity
  • the size of each plot shows reactivity.
  • Fig. 10 shows the results of preparing a calibration curve by preparing a reagent for a fully automatic immunodiagnostic system manufactured by Tosoh Corporation.
  • the black circles in the figure are the results of using the immune reaction reagent using the rabbit monoclonal antibody UT 1 D-5 produced in the present invention, and the white squares are the results of using the immune reaction reagent using the mouse monoclonal antibody. It is.
  • Figure 11 shows the results of evaluating cross-reactivity.
  • the horizontal axis shows the concentration of T3 or T4, and the vertical axis shows the BZB 0 value.
  • Figure 12 shows the results of screening of the culture supernatant of 3840 clones.
  • the vertical axis represents fluorescence intensity, and the horizontal axis represents clones.
  • Figure 13 shows the results of a sandwich assay of BNP with anti-BNP antibody (C-terminal recognition) obtained by transient expression and an antibody that recognizes the cyclic site of BNP.
  • Figure 14 shows the results of ELISA evaluation of the change in the amount of antibody in the culture supernatant after cell fusion.
  • the antigen to immunize the rabbit is not particularly limited and can be arbitrarily selected from haptens, peptides, proteins, nucleic acids, cells, lipids, sugar chains, bacteria, fluorescent substances, and biamines.
  • the present invention is preferably applied to an antigen that requires high antibody performance in terms of affinity and specificity.
  • antigens for competitive methods that require high affinity in order to construct a highly sensitive measurement system based on the measurement principle, such as estrone (E 1), estradiol (E 2), Triol (E 3), steroids such as steroid hormones such as progesterone and cortisol, and thyroid hormones such as thyronine, thyroxine, and lysine thyronine.
  • application to peptides that require high sensitivity such as C peptide and brain natriuretic peptide (BNP) is also a preferable example.
  • Usagi lines include, but are not limited to, Japanese white varieties that are generally available and New Zealand land moth.
  • antibody-producing cells derived from Usagi having a high immune response by immunizing several to about 10 individuals.
  • immunization there are no particular restrictions on the method of immunization to the rabbits, and immunization may be carried out by a commonly used method.
  • a method of immunizing by making an adjuvant and emulsion and a method of immunizing DNA encoding a protein or peptide can be mentioned.
  • the place of immunization is not particularly limited. It is sufficient to immunize the rabbit at the usual place, such as subcutaneous, abdominal cavity, or footpad.
  • the antibody titer of the serum is analyzed by measuring by ELISA or BIA core (trade name) (Biacore), and It is preferable to determine by observing the progress. Usually four immunizations are enough, but in some cases, multiple immunizations (from several months to one year) may be required.
  • the antibody-producing cell to be isolated is not particularly limited as long as it is an antibody-producing cell, but it is preferable to use any one of spleen-derived cells, lymph node-derived cells, and terminal blood-derived cells. .
  • isolation of antibody-producing cells may be performed by a conventional method. Although isolated cells are preferred for immediate use, they can be stored in liquid nitrogen or deep freezer, dispersed in commonly available cell storage solutions, as needed Can be used.
  • Non-Patent Document 9 there is a method of selecting one cell from antibody-producing cells and isolating and amplifying an antibody gene from the selected cell. Since only the antigen-binding ability can be evaluated, it is difficult to obtain an antibody having the target ability. Therefore, in the present invention, the antibody-producing cells isolated in (3) are proliferated, and the performance such as competitive characteristics, specificity, and reactivity of the antibody expressed in the culture supernatant is evaluated, and the performance is high. It is characterized by isolating the antibody gene after selecting cells expressing the antibody.
  • the following method can be exemplified as a method for growing antibody-producing cells.
  • a) A method in which antibody-producing cells are cultured in a growth medium until the antibody performance in the culture supernatant can be evaluated in a culture medium in a microwell plate.
  • c) A method of selecting cells for antibody gene isolation by immortalizing and culturing antibody-producing cells with a virus such as Epstein-Barr virus (EBV).
  • EBV Epstein-Barr virus
  • the method of growing using the method a) or b) is preferred.
  • a medium used in the growth method of a a medium for animal cells or a medium supplemented with well-known cytokines such as IL-4 and LPS can be used.
  • the cell that provides the immortalizing ability used in the proliferation method of b) is a cell that can obtain an antibody-secreted antibody after fusion, and after the growth, the antibody performance is evaluated in the fused cell.
  • the cell is not particularly limited as long as the antibody gene remains in the cell, but a cell from which a fused cell capable of maintaining the antibody-producing ability for a long time can be obtained is particularly preferable.
  • Mouse myeloma related such as 0 0 2 9), NSO / (RCB 0 2 1 3), S 1 9 4 X 5 XX ⁇ , BUI (AT CCCRL-8 8 3 7), FO (AT CCCRL-1 6 4 6) , Human myeloma cell lines such as SK 0-0 0 7 and HO — 3 2 3, human B cell lines such as GM 1 5 0 0 6 GT-A 1 2, Y 3 — A gl ⁇ 2 ⁇ Examples include rat myeloma cell lines such as 3, rabbit-transformed cell lines of TP-3, and rabbit cancer cells such as VX-2.
  • a cell line starting from the mouse myeloma cell line (for example, 2-3A described in JP-A No. 10 1 2 6 2 6 5 7 (Patent Document 1)) is also preferred immortalizing ability in the present invention. It is an example of the cell which gives.
  • the cell fusion method used in the propagation method of b) may be carried out by a commonly used method. Examples thereof include a polyethylene glycol method, an electrofusion method, and a method using Sendai virus.
  • Proliferation method is well known to those skilled in the art, infecting a human lymphocyte with a virus such as EBV (C o 1 eeta 1., “T he EBV-Hybridoma T echniqueand I ts A pplicationto Human Lung C ancer "M onoclonal A ntibodiesand C ancer T herapy, Reisfeldeta 1., N ew Y ork: A 1 an R. L iss I nc. It is a method in which a rabbit such as EBV is infected with a virus such as EBV and immortalized temporarily by a technique similar to that of Patent Document 10).
  • the virus that infects rabbit B cells is not particularly limited to E B V as long as it can achieve the above-mentioned purpose.
  • the antibody performance evaluation method for selecting cells having the antibody gene may be appropriately selected according to the evaluation contents of the target antibody.
  • the contents of evaluation include antigen binding ability, competitive characteristics, specificity, and antibody productivity
  • examples of evaluation methods include the ELISA method, the RIA method, and the fluorescence polarization method.
  • the optimal time for isolating the heavy chain (H chain) and light chain (L chain) antibody genes from the cells selected in (4) above differs depending on the antigen and the rabbit strain. There is no particular limitation as long as a sufficient amount of antibody for evaluation can be secured and the antibody gene is not lost. However, as in Example 1 2 Isolation of antibody genes is not possible because isolation of antibody genes 16 days after the start of production cell growth reduces the efficiency of antibody gene amplification and the availability of expression vectors with antigen-binding activity. It is preferably performed within 14 days after the start of production cell growth.
  • RT-PCR As a method for amplifying the antibody gene, a commonly used RT-PCR method can be used.
  • commercially available enzymes can be used for reverse transcriptase and DNA polymerase used in RT-PCR reactions.
  • DNA polymerase avoids introducing mutations during DNA amplification. Therefore, Phismion High Fidelity DNA ⁇ ⁇ l ym erase (trade name) (Daiichi Chemicals Co., Ltd.) and P rimes TAR HS DNA pol ym erase (trade name) (Yukara Bio Inc.) It is preferable to use the enzyme with the highest fidelity.
  • the primer for amplifying the antibody gene is not particularly limited as long as it is designed at a position where the region encoding the variable region of the antibody is amplified.
  • Primer combinations used to amplify antibody genes include primer combinations designed in the untranslated region upstream from the start codon and downstream from the codon for the purpose of amplifying the entire length of the antibody gene, antibody Examples include a combination of primers designed inside the antibody gene for the purpose of amplifying the variable region of the gene, or a system in which the above primers are appropriately combined. If the amplification efficiency of the antibody gene is low in a single PCR reaction, the antibody gene can be efficiently amplified by performing the second PCR reaction using the first PCR reaction product as a saddle. In this case, it is often possible to amplify the gene more efficiently by creating a new primer slightly inside the first amplification region. Is not something
  • the Hagi chain is 80% or more of VH 1 (J. Immu nol., 1 5 2, 3 9 3 5; 1 9 94: Non-patent document 1 1), and L chain Are reported to be 80% or more of ⁇ chain (J. Mo 1.
  • Amplification is preferably carried out using a primer set specific for the gene of interest.
  • Non-Patent Document 10; Biotechno, 1 3, 6 1 7 1; 1 995 Non-Patent Document 13
  • Nuc 1 Nuc 1.
  • a cids R es., 1 5, 6 1 7 1; 1 9 8 7 (Non-Patent Document 1 4); N uc 1.
  • a cids R es., 1 0, 1 5 3 5; 1 9 8 2 (Non-patent The primer sequence described in Reference 15) and the primer sequence (SEQ ID NO: 1 to 1 1) designed with reference to Gen Bank are disclosed. You can use.
  • the expression vector is not limited as long as it is a generally reported vector, but in particular, p ECE dhfr (J. Bioche m., 1 0 8, 6 7 3; 1 9 90: Non-patent document 1 6, Figure 4) is preferably used as an expression vector.
  • the region of the antibody to be expressed may be expressed so as to include the variable region of the antibody, and the expression forms include Ig G, F (ab ') 2, Fab, FV, and sc FV. It can be illustrated.
  • Expression vectors can be introduced into appropriate host cells to Recombinant antibodies can be produced.
  • SV40 large T antigen protein-expressing cells are preferably used as host cells for transiently high expression of recombinant antibodies, and examples include Examples 7, 1 3, and 14 C o S 1 cells used in Biotechno 1. Lett., 1 7, 1 3 5; 1 9 95: Non-patent literature 1 7; J. Ferm. B ioeng., 7 9, 40 5; 1 9 9 5: Non-patent literature 1 8), 2 9 3 T cells and COS 7 cells can be used.
  • animal cells and microorganisms typified by yeast and Escherichia coli are preferably used as phosphine cells, and animal cells, particularly CH 0 cells are preferably used.
  • a more preferred stable production strain is a phosphine vector system in which a vector incorporating a dhfr gene augmentation system capable of amplifying an antibody gene is introduced into CH cells.
  • the method for producing a rabbit monoclonal antibody according to the present invention allows an antibody-producing cell isolated from a rabbit immunized with an antigen to proliferate without using a generally non-circular rabbit-myeloma cell, and then possesses an antibody gene.
  • This method is characterized in that the antibody gene is isolated and amplified from the selected cell, and it is possible to produce a rabbit antibody with the knowledge of normal laboratory facilities and molecular biology. Is.
  • a monoclonal antibody having a combination of heavy chain and light chain originally produced by Usagi can be easily produced.
  • Usagi Monoclonal Antibodies with high affinity that can only be handled in Japan can be easily handled at the normal laboratory level.
  • E 2 —B S A (trade name) (manufactured by Sigma) was used as an immunizing antigen.
  • an antigen for screening with ELIISA was prepared by the following method.
  • BCP Blue Carrier Protein: manufactured by Pierce
  • 10 O mg of PBS was added to 1 O mg ZmL, and the 6th position was activated with N-hydroxysuccinimide.
  • Tradiol (E 2) 1. 2 5 mg was mixed with DM F 1 mL.
  • the immunized animal As the immunized animal, a 12-week-old female of Usagi (Japanese white species) was used. Immediately after the first immunization, Freund's complete adjuvant is used. After the second immunization, Freund's incomplete adjuvant is used. After mixing the antigen solution (500 gZmL) with the same amount of adjuvant, immunization is performed at intervals of 2 weeks. did. The amount of immunizing anti-antigen is 50 g at the first time and 25 g after the second immunization.
  • E 2—B C P (0. b g / m L) was immobilized on an E L I S A plate and blocked with 1% skim milk.
  • the spleen of a rabbit with a sufficiently increased antibody titer was removed, and spleen cells were isolated according to a conventional method.
  • Spleen cells were suspended in GIT medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% F C S, and cultured on a microplate.
  • GIT medium manufactured by Nissui Pharmaceutical Co., Ltd.
  • F C S 10% F C S
  • the culture supernatant was evaluated by the method described in Example 2 for the antibody in the culture supernatant of 192 clones. The results are shown in FIG. Thus, the production of antibodies could be confirmed in the supernatant of the microwell plate.
  • Example 4 Proliferation of antibody-producing cells using lymphocytes derived from peripheral blood cells (culture using cell fusion)
  • Peripheral blood cell-derived lymphocytes were fused with immortalizing cells by the method described below.
  • the antibodies in the culture supernatant of 3 8 4 clones were evaluated by the method described in Example 2. The calculated results are shown in FIG. In this way, antibody production could be confirmed in the supernatant of the microwell plate.
  • Spleen cells were fused with immortalizing cells by the method described below.
  • E 2 — B C P (0.5 g ZmL) was immobilized on an ELIISA plate and then blocked with 1% skim milk. Thereafter, the culture supernatant after cell fusion was reacted.
  • Anti-E 2 Usagi monoclonal antibody bound to E 2 binds alkaline phosphatase-labeled anti-rabbit Ig G antibody, B / F separation of unreacted enzyme-labeled antibody, and 4-methylumbelliferous enzyme substrate.
  • Ferryl phosphate (41-MU P) was dispensed and detected by measuring fluorescence intensity.
  • Example 6 Isolation / Amplification of antibody gene and preparation of expression vector As a result of Example 5, after selecting clones filled with black circles in Fig. 3, antibody genes were isolated from the clones by the following method, amplified and expressed A vector was fabricated.
  • the buffer solution supplied with the kit was used with d N T P 0.2 M and the primer 0.5.
  • H-chain and L-chain PCR fragments were treated with restriction enzymes BglII and XbaI, and then the animal cell expression vector (pECE dhfr: Fig. 4) BglIiZXbal Introduced into the site, expression vectors for H and L chains were prepared.
  • Example 6 Using the expression vector for H chain and L chain obtained in Example 6, recombinant antibodies were produced in COS 1 cells (Non-patent Documents 17 and 18). The antibody gene isolation procedure in Example 6 was performed 8 days after cell fusion.
  • the anti-E2 antibody was purified from the culture supernatant using a Protein ⁇ G. column (manufactured by GE HEALTHEARE) according to a conventional method. The performance of the antibody was evaluated, and the antibody with the highest performance in terms of competitive characteristics, specificity, and productivity was selected.
  • An antibody expression vector selected in Example 7 was introduced into CHO cells to produce a stable antibody-producing strain.
  • C HO cells (DXB 11: dhfr-deficient strain) were transfected with a gene under the same conditions as in Example 7, and alpha 1 ME M (—) was added with 10% dialyzed serum. Cells were cultured. Thereafter, the concentration of MTX (mesotrexate) was gradually increased from 5 nM to 500 nM to obtain rabbit monoclonal antibody U-1-expressing CHO cells.
  • MTX mesotrexate
  • Dissociation constant (K d) on the horizontal axis and vertical axis on the competitive property value obtained when E 2 of l OOO pg Zm L coexists with the value when E 2 does not exist.
  • K d Dissociation constant
  • the monoclonal antibody U-1 has an increased degree of polarization at a concentration that is at least 100 times lower than that of the mouse monoclonal antibody Ma-1 and the rat monoclonal antibody Ra-1. This indicates that U-1 has a much higher affinity than M a-1 and R a _ 1.
  • Example 1 1 Evaluation of Monoclonal Antibody in AIA Reagent Form
  • the immune reaction reagent using the rabbit antibody monoclonal antibody U-1 produced in the present invention was changed to the immunoreaction reagent using the polyclonal antibody (E test “T ⁇ ”).
  • S ⁇ H ”IIE 2 manufactured by Tosohichi Co., Ltd.
  • Fig. 7 shows the calibration curves for both immune reaction reagents.
  • the detection limit for E 2 is 25.2 pg ZmL for immunoreactive reagents using polyclonal antibodies, whereas 2 2.4 pg ZmL for immunoreactive reagents using U-1 is It has been clarified that a measurement system with higher sensitivity than an immunoreaction reagent using a null antibody can be constructed.
  • FIG. 8 shows the results of examining the correlation between both measurement systems using 40 actual samples.
  • Example 1 2 Gene isolation time and percentage of isolated genes
  • the present inventors also used the culture supernatant after cell fusion as EL. After evaluating by ISA and confirming that the antibody was expressed in the culture supernatant, even when attempting to amplify the antibody gene from the cell, the antibody gene could not be amplified. As a result of investigating why the gene cannot be amplified from various viewpoints such as PCR conditions and primer sequences, it is the same as handling normal mouse monoclonal antibodies when subculturing heterohybridomas obtained after cell fusion. When it is handled with a sense, it was found that it is difficult to specify when the antibody is not expressed.
  • the cell suspension is transferred to a new medium of about 10 times volume and the culture is continued, so the first culture supernatant is diluted by transferring to a new medium. Go.
  • the culture supernatant is diluted, and the signal of the culture supernatant after subculture is drastically reduced.
  • this theory did not hold for the rabbit monoclonal antibody.
  • Usagi monoclonal antibody has extremely high affinity compared to mouse monoclonal antibody, and sufficient signal can be obtained by ELISA even if the culture supernatant is diluted about 10 times or 100 times by subculture.
  • the antibody gene has already dropped from the heterohybridoma, and the ELISA is positive even though the antibody is not expressed.
  • the binding properties of the rabbit monoclonal antibody isolated in this patent and the mouse monoclonal antibody were compared.
  • the rabbit monoclonal antibody was more than 100 times lower than the mouse monoclonal antibody. This is supported by the fact that antigen-binding ability was observed even at concentrations.
  • Example 6 the time for isolating the antibody gene after cell fusion was changed, and the optimum time for isolation was examined. The results are shown in Table 1. table 1
  • the H chain gene was amplified with a probability of 86.1% and the L chain gene with a probability of 94.4% on average of three experiments. Then, when an expression vector was prepared from the amplified gene and the antibody expressed in COS cells was evaluated, 75.0% of the antibodies had antigen-binding ability. In addition, a stable amplification rate and antibody acquisition rate were demonstrated through three experiments, indicating that a vector with antibody binding activity can be stably acquired by amplification during this period.
  • Example 1 Isolation of Anti-Triyoidothyronine (T 3) Antibody Anti-triyodothyronine (T 3) Usagi monoclonal antibody was isolated by the following method.
  • BCP Blue Carrier Protein: manufactured by Pierce
  • Boric acid buffer 0.05 M, pH 8.5
  • 5 mg of BCP was 1 O mg ZmL.
  • N-hydroxysucci A solution obtained by dissolving 7 mg of T 3 activated with medium in DMSOIOOL was mixed. After stirring at room temperature for 30 minutes, dialysis against PBS (50 mL ⁇ 3 times) yielded ELISA screening antigen T 3 —BCP.
  • the immunized animal As the immunized animal, a 12-week-old female of Usagi (Japanese white species) was used. Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for the second and subsequent immunizations. After mixing the antigen solution (500 / g mL) with the same amount of adjuvant, the emulsion is prepared and separated for 2 weeks. Immunized with. The amount of immunizing anti-antigen is 1 mg for the first time and 500 g after the second immunization.
  • H chain Sequences 2 and 5 for the first PCR, sequences 1 and 6 for the second PCR
  • H-chain and L-chain PCR fragments were treated with restriction enzymes BglII and XbaI, and then the Bgl of the expression vector for animal cells (p ECE dhfr: Fig. 4) was used. Introduced into the II / Xbal site, expression vectors for H chain and L chain were prepared.
  • a I A is a fully automated immunodiagnostic system sold by Tosoh Corporation. Using the antibody prepared this time, an immune reaction reagent for A I A was prepared and evaluated.
  • Fig. 10 shows the calibration curves of the immune reaction reagent using the isolated rabbit antibody UT 1 D-5 and the immune reaction reagent using the mouse-derived anti-T 3 monoclonal antibody. As shown in Fig. 10, it became clear that the use of the isolated antibody makes it possible to construct a measurement system that is more sensitive than mouse-derived reagents.
  • Figure 11 shows the results of cross-reactivity evaluation using an immunoreaction reagent using UT 1 D-5 prepared this time.
  • Figure 11 shows that the antibody isolated this time has no cross-reactivity to the thyroxine (T 4), a similar compound of T 3, and has a very high specificity for T 3. Being I understand.
  • Example 14 4 Isolation of anti-brain natriuretic peptide (BNP) antibody
  • Anti-brain natriuretic peptide (BNP) Usagi monoclonal antibody was isolated by the following method.
  • a peptide corresponding to 7 amino acids on the C-terminal side of BNP (C KV L R R H, SEQ ID NO: 12) was synthesized. Using the cysteine at the N-terminus of the synthesized peptide, it was reacted with maleimide K L H (manufactured by PIERC) and used as an immunizing antigen. The reaction method was according to the attached protocol, and 1 mg of peptide was reacted with 1 mg of maleimide K L H.
  • a peptide with a linker sequence (GGGSGGGS, SEQ ID NO: 13) added to the N terminus of the peptide corresponding to the C-terminal 7 amino acids of BNP (C KV LRRH, SEQ ID NO: 12), and then the N-terminus is piotinized.
  • Bio-BNC Biotin-GGGS GG GSC KV LRRH, SEQ ID NO: 14; hereinafter referred to as Bio-BNC
  • the immunized animal As the immunized animal, a 12-week-old female of Usagi (Japanese white species) was used. The first immunization is Freund's complete adjuvant, the second and subsequent immunizations are Freund's incomplete adjuvant, mixed with an antigen solution (500 ⁇ gZmL) and the same amount of adjuvant, and then immunized at intervals of 2 weeks after creating the emulsion. did. The amount of immunizing anti-antigen is 50 g at the first time and 25 z after the second immunization.
  • BIO—BNC (0.5 g / ml) was reacted with an ELISA plate. After that, the rabbit serum immunized with the antigen was reacted.
  • Bio—Anti-BNP Usagi monoclonal antibody conjugated to BNC binds alkaline phosphatase-labeled anti-rabbit Ig G antibody, and unreacted enzyme-labeled antibody is separated by BZF, followed by the enzyme substrate 4-methylumbelliferyl phosphorus. It was detected by dispensing acid (4-MU P) and measuring fluorescence intensity.
  • Anti-BNP Usagi monoclonal antibody bound to Bio_BNC binds alkaline phosphatase-labeled anti-rabbit Ig G antibody, and unreacted enzyme-labeled antibody is separated by B / F, followed by enzyme substrate 4-methylun It was detected by dispensing berylferyl phosphate (4-MU P) and measuring the fluorescence intensity.
  • the antibody gene was isolated from the clone selected in (4) by the following method.
  • the buffer solution supplied with the kit was used with d N T P 0.2 M and the primer 0.5 L each.
  • H chain Sequences 2 and 6 for the first PCR, sequences 8 and 9 for the second PCR
  • Usagi-derived anti-BNP mono obtained from the clone obtained in (6) The clonal antibody (27, 45) was evaluated by ELISA according to the following method.
  • the experiment shown below was conducted using the frozen spleen cells of rabbits immunized with E 2 — B SA.
  • Example 5 cell fusion was performed, and the fused cells were spread on a microtiter plate.
  • a rabbit antibody having high affinity characteristics can be produced with knowledge of ordinary laboratory facilities and molecular biology. Furthermore, by using the rabbit monoclonal antibody produced by the method of the present invention as an immunodiagnostic drug, a highly sensitive measurement system that is not affected by foreign substances can be constructed.

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Abstract

Selon l'invention, un anticorps produit par génie génétique est obtenu par des opérations consistant à isoler des cellules productrices d'anticorps provenant d'un lapin qui a été immunisé par un antigène, à faire proliférer des cellules portant un gène d'anticorps cible puis à les cribler, à isoler des gènes d'anticorps à chaîne lourde et à chaîne légère à partir des cellules criblées et à les amplifier, à transférer ceux-ci dans un vecteur d'expression, puis à les transférer dans des cellules hôtes pour exprimer de cette manière un anticorps monoclonal de lapin produit par génie génétique.
PCT/JP2009/055555 2008-03-14 2009-03-13 Procédé de fabrication d'un anticorps produit par génie génétique WO2009113742A1 (fr)

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