WO2008047480A1 - Procédé de régulation de la distribution de sites de mutation dans la région variable d'un gène d'anticorps - Google Patents

Procédé de régulation de la distribution de sites de mutation dans la région variable d'un gène d'anticorps Download PDF

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Publication number
WO2008047480A1
WO2008047480A1 PCT/JP2007/001139 JP2007001139W WO2008047480A1 WO 2008047480 A1 WO2008047480 A1 WO 2008047480A1 JP 2007001139 W JP2007001139 W JP 2007001139W WO 2008047480 A1 WO2008047480 A1 WO 2008047480A1
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Prior art keywords
gene
seq
antibody
function
variable region
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PCT/JP2007/001139
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English (en)
Japanese (ja)
Inventor
Kunihiro Ohta
Waka Lin
Hidetaka Seo
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Riken
Japan Science And Technology Agency
Saitama Small Enterprise Promotion Corporation
Chiome Bioscience Inc.
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Publication of WO2008047480A1 publication Critical patent/WO2008047480A1/fr

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    • 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

Definitions

  • the present invention relates to a method for introducing a mutation into an antibody gene region. More specifically
  • the present invention relates to a method for obtaining various antibody molecules by introducing various mutations into the antibody gene region.
  • Somatic cell homologous recombination plays an important role in vivo as one of the factors generating gene diversity.
  • antibody genes will be further rearranged in the variable regions generated by the VDJ recombination, and will code for antibody molecules with very many different specificities. If it becomes possible to freely induce such a series of phenomena in the cell, especially the reorganization of the variable region, it is possible to obtain an antibody having a desired characteristic.
  • somatic mutation is one of the phenomena that brings diversity to antibodies.
  • the frequency of somatic mutations increases.
  • Non-patent document 1 Non-patent document 1
  • attempts have been made to obtain antibodies having various characteristics using DT 40 cells knocked with XRCC2 or XRCC3 (patent document 1, non-patent document 2).
  • AID activation induced and ytidine deaminase involved in both somatic gene conversion and somatic mutation is temporarily stopped by the Cre-I ox P system
  • Techniques for isolating antibody-producing clones have also been reported (Patent Document 2, Non-Patent Document 3).
  • the antibodies obtained by these techniques still have insufficient points in terms of antigen recognition specificity and affinity.
  • Non-Patent Document 1 S ale et al., Nature 4 1 2: 92 1 —926, 200
  • Non-Patent Document 2 Cum bers et al., Natre Biot c h. 20: 1 1 29- 1 1 34, 2002
  • Non-licensed document J K a na y ama, Nu c l e i c A c i d s R e s. 34: e 10.
  • Non-Patent Document 4 Seo et al., Natre Biot c h. 23:73 1 —7 35, 2005
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2003_503750
  • Patent Document 2 JP-A-2004-298072
  • Patent Document 3 International Publication WO 2004/0 1 1 644
  • H DAC 2 histone deacetylase 2
  • an object of the present invention is to provide a method for introducing a mutation into the entire region of the antibody gene variable region.
  • the present invention also introduces mutations into the entire antibody gene variable region. And to provide a method for producing antibody molecules having various characteristics.
  • the present invention relates to the following (1) to (12).
  • the coding region selectively reduces or loses the function of a gene consisting of the base sequence represented by (a) or (b) below: A method for introducing various mutations into the variable region of an antibody gene on a chromosome.
  • a protein that hybridizes with a polynucleotide comprising a complementary nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 under highly stringent conditions and has a histone deacetylating activity.
  • the nucleotide sequence of the polynucleotide ”.
  • the above-mentioned decrease in function or loss of function is achieved by introducing a mutation or deletion into a gene comprising the base sequence represented by (a) or (b) above”.
  • the method according to (1) above which is characterized in that
  • the third aspect of the present invention is characterized in that “the function decrease or function loss is achieved by disrupting the entire gene comprising the base sequence represented by the above (a) or (b). The method described in (1) above.
  • the activity or function of the protein shown in the following (a) or (b) is selectively reduced or lost and is present on the chromosome of an immune cell.
  • amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, consisting of an amino acid sequence having one or several amino acid substitutions, deletions or insertions, and histone deacetylation A protein having activity.
  • a fifth aspect of the present invention is as follows: “The above immune cells are additionally added as HD AC inhibitors. The method according to any one of the above (1) to (4), wherein the treatment is conducted by contacting with the other.
  • a sixth aspect of the present invention is “the method according to (5) above, wherein the HDA inhibitor is trichostatin A”.
  • a seventh aspect of the present invention is “the method according to (6) above, wherein the immune cell is a DT40 cell”.
  • the eighth aspect of the present invention is “the method according to any one of (1) to (7) above, wherein the antibody gene is an antibody heavy chain gene”.
  • a ninth aspect of the present invention is “the method according to any one of (1) to (7) above, wherein the antibody gene is an antibody light chain gene”.
  • a tenth aspect of the present invention is “an immune cell having a mutation introduced into an antibody gene variable region by the method described in any one of (1) to (9) above”.
  • the first aspect of the present invention is as follows: “There are few polypeptides encoded by the antibody gene into which a mutation has been introduced by the method described in any of (1) to (9) above. An antibody molecule containing at least one.
  • the first and second aspects of the present invention are “the antibody molecule according to (1 1) above, wherein the antibody molecule is IgM”.
  • mutations can be introduced in the entire region of the antibody gene variable region.
  • antibody molecules having various characteristics can be prepared by introducing mutations into the entire antibody gene variable region.
  • Fig. 1 shows a schematic diagram of the HDAC2 gene knockout construct.
  • FIG. 2 shows the results of RT_PCR confirming that the cHDAC2 gene was destroyed.
  • WT is the wild type strain
  • (+ / _) is the heterozygous gene disruption strain
  • -) is the homozygous gene disruption strain.
  • FIG. 3 shows the results of gene conversion frequency of wild type and ch HDAC2— cells. side The axis represents the culture time (days), and the vertical axis represents the percentage of IgM + cells. The average of three culture results is shown.
  • FIG. 4 shows the results of the sequence diversity of the antibody light chain variable region of the c h HDAC2 — / — 2-month-old cell population.
  • FIG. 5 shows the results of sequence diversity of antibody light chain variable regions of wild type + trichostatin A (5 nM) 2-month-old cell population.
  • FIG. 6 shows the ratios of sequence diversification sites in each hypervariable region in the c h HDAC2, c h HDAC 2 + TSA (trichostatin A) and TSA (trichostatin A) cell populations.
  • FIG. 7 shows the results of the sequence diversity of the antibody light chain variable region of c h HDAC2 ⁇ / ⁇ ) + trichostatin A (5 nM) 1-month-old cell population.
  • FIG. 8 shows the results of the ELISA of antibody clones prepared against Usagi IgG.
  • FIG. 9 shows the results of the sequence diversity of the antibody heavy chain variable region in the c h HDAC2 ⁇ -/-) 2-month-old cell population.
  • histone deacetylase (hereinafter referred to as HDAC) 2 gene function is selectively reduced or lost, or HDAC2 protein activity or function is selectively reduced.
  • HDAC histone deacetylase
  • it is a method in which various mutations are introduced into the antibody gene region existing on the immune cell staining body.
  • Histone deacetylase is a metalloenzyme containing zinc at the active center, and catalyzes the reaction of eliminating the acetyl group from the acetylated histone.
  • HDAC Histone deacetylase
  • HD AC is roughly divided into several classes, for example, in the case of humans, class I (HDAC 1, HDAC2, HDAC3, HDAC8) and class II (HDAC4, HD AC5, HDAC6, HDAC7). These isotopes form large complexes by interacting with many other proteins.
  • HDAC 1 and HDAC2 are SIN 3 complex or NURD / Present in the M i 2 complex.
  • HDAC2 gene means that the coding region is SEQ ID NO:
  • DNA consisting of the base sequence represented by 1, 3 or 5
  • DNA comprising the base sequence complementary to the DNA consisting of the base sequence represented by SEQ ID NO: 1, 3 or 5
  • genes consisting of DNA that hybridizes under highly stringent conditions and encodes a protein having histone deacetylation activity.
  • gene includes c DN A and genomic DN A. In the case of genomic DN A, not only exons and introns but also transcription regulatory regions such as promoters and enhancers are included.
  • the “antibody gene” in the present invention is synonymous with an immunoglobulin gene and includes both a heavy chain (H chain) antibody gene and a light chain (L chain) antibody gene.
  • the DNA capable of hybridizing under stringent conditions with the polynucleotide comprising the base sequence represented by SEQ ID NO: 1, 3 or 5 is preferably about the base sequence represented by SEQ ID NO: 1, 3 or 5. More than 70%, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97 ⁇ 1 ⁇ 2, 98%, most preferably about 99% polynucleotide sequence DNA having a nucleotide sequence homology.
  • stringent conditions are hybridization conditions that are easily determined by those skilled in the art, and are generally empirical conditions that depend on probe length, washing temperature, and salt concentration. is there. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally depends on the ability of the denatured DNA to re-anneal when the complementary strand is in an environment close to or below its melting point.
  • a temperature condition of 37 ° C. to 42 ° C., 0.1 X SSC, 0.1% SDS Cleaning in solution can be raised .
  • a high stringent condition for example, washing in 65 ° C., 5 ⁇ SSC and 0.1% SDS in the washing step may be mentioned.
  • the “HDAC2 protein” in the present invention is a polypeptide containing an amino acid sequence that is the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 2, 4, or 6.
  • the “polypeptide containing substantially the same amino acid sequence” means about 60% or more, preferably about 70% or more, more preferably about 80%, with the amino acid sequence represented by SEQ ID NO: 2, 4, or 6. %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96% 97%, 98%, most preferably about 99% amino acid identity and a protein having histone deacetylation activity.
  • the polypeptide containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 includes one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6. (Preferably about 1 to 30, more preferably about 1 to 10 and even more preferably 1 to 5) amino acid sequence deleted, substituted or added, and histone deacetylating activity It is protein which has.
  • protein and “polypeptide” are used interchangeably unless otherwise specified.
  • the "immune cell” in the present invention refers to a B cell having antibody-producing ability, and for example, cells derived from human, mouse, rat, hidge, chicken and the like can be used. Preferably, established cells are used, and DT40 cells are particularly preferably used as immune cells.
  • a “DT40 cell” is a cultured cell of a B cell derived from a chicken, and is a derivative that has been modified in some way (for example, recombination, insertion, deletion, etc. of a specific gene). Includes stocks and sublines.
  • the culture conditions for the cells used in the present invention are determined by methods well known in the art.
  • the culture medium and culture conditions (culture temperature, c) suitable for the selected immune cells are used.
  • the immune cells selected are DT 40 cells, for example, the medium is IMDM (Invitrogen), the culture temperature is 39.5 ° C, and the CO 2 concentration condition is 5%. Do this below. Culturing is carried out while keeping the cell concentration constant, and the presence or absence of somatic homologous recombination at the antibody gene locus of the target cell is confirmed at appropriate intervals (for example, every day or every week).
  • the function of the HDAC2 gene can be altered in order to reduce or lose the function of the HD AC 2 gene.
  • the method of modifying the function of the gene is not limited, but, for example, a method of introducing a mutation into the HD AC 2 gene present in the cell, a method of destroying the entire HD AC 2 gene, transcription of the HD AC 2 gene
  • Methods well known to those skilled in the art can be used, such as a method for controlling the expression level by modifying the promoter, a method using RNA interference (RNA i), a method for introducing antisense to the HDAC2 gene into cells.
  • RNAi RNA interference
  • Preferred are a method for introducing a mutation into the HDAC2 gene, a method for destroying the entire HDAC2 gene, or a method using RNA interference (RNAi), and a method for destroying the entire HDAC2 gene.
  • DN A which has a marker gene inserted into the essential region of the cloned target gene (HDAC2 gene), the DN A sequence (the region to be disrupted).
  • HDAC2 gene the essential region of the cloned target gene
  • the DNA introduced into the cell induces homologous recombination via both flanking sequences of the HD AC 2 gene, so that the HDAC 2 gene on the chromosome can be replaced with a marker gene.
  • the HD AC 2 gene can be destroyed.
  • RNA interference can be used to lose gene function.
  • RNA i RNA interference
  • a short RNA duplex or a vector that produces the RNA is introduced into the cell, and the HD AC 2 gene Can result in loss of function or loss of function.
  • an inducible transcription promoter can be placed upstream of the HDAC 2 gene to conditionally control HDAC 2 gene expression, or a site-specific recombinase system such as Cre_IoXP There are methods to control the expression by changing the relative positions of the two genes and the transcription promoter.
  • a method for introducing a mutation in the in vitro mouth a method known in the art such as site-directed mutagenesis can be used.
  • site-directed mutagenesis When functional changes are made by introducing mutations into the HDAC 2 gene, it is necessary to introduce mutations so that the activity of the HDAC 2 protein is lost, and to interact with the protein and other proteins. It is desirable to introduce a mutation that introduces a mutation at the site and eliminates the interaction.
  • HDAC 2 protein In order to reduce or lose the activity or function of HDAC 2 protein, in addition to the methods that can be used to reduce or lose the function of HDAC 2 gene described above, methods well known to those skilled in the art can be used. it can. Without limitation, for example, a method of introducing an antibody that decreases or loses the activity or function of HDAC 2 protein into the target cell, or an inactive form of HDAC 2 protein (dominant negative) into the target cell. Examples thereof include a method of introducing or expressing in a target cell, and a method of introducing a low molecular weight inhibitor that inhibits the activity of the HDAC 2 protein into the target cell.
  • a cell membrane permeable peptide may be linked to an inactive form of an antibody or HDAC 2 protein and introduced into the cell.
  • a commercially available kit can be used
  • HDAC 2 protein In order to express an inactive form of HDAC 2 protein in a cell, it can be easily carried out according to the common general technical knowledge of those skilled in the art. Depending on the target cell, select an expression vector (having a component necessary for expression induction, such as a promoter appropriate for the target cell), and select the HDAC 2 protein under appropriate culture conditions. Inactive forms of can be expressed. [0019] In another embodiment of the present invention, the function of the HD AC 2 gene is reduced or lost, or the activity or function of the HD AC 2 protein is reduced or lost, and the HD AC 2 gene, HD AC 2 In this method, immune cells in which proteins are present are treated by contacting them with HDA C inhibitors, and various mutations are introduced into antibody gene regions present on the chromosomes of the immune cells.
  • HDAC inhibitor examples include protein factors such as an antibody having an activity to suppress the activity of HD AC, and small molecule compounds such as trichostatin A, butyric acid and valproic acid, as long as they are known to those skilled in the art. Although any could be used, trichostatin A is most preferably used.
  • the treatment concentration and treatment time with an HDAC inhibitor can be used as long as the cells to be contacted do not die. Specifically, in the case of trichostatin A, the treatment concentration is preferably 1 to 20 nM, and the treatment time is preferably 2 weeks to 4 months.
  • Example 1 Preparation of c h HDAC2 _ / _ strain and diversification of variable region of antibody gene (heavy chain light chain) by continuous culture
  • a DT 40 genomic DNA was used as a saddle, and the BamHI (6949) -Sac I (1 31 40), 6.2 kbp fragment of the ch HDAC2 gene was amplified by PCR.
  • the pBI uescript SK (-) vector I was inserted into the BamHI_SacI site and cloned.
  • Stu I (7543) —Stu I (1 01 29), 2.6 kbp fragment (including part of 3rd exon, 4th and 5th exons) was cut out from this plasmid.
  • I o XP flanked blasticidin R (3 ⁇ 4 ⁇ or puromy cin R) expressed by the S-actin promoter was inserted as a single selection sequence (Fig. 1). This Then, 30 g of recombinant DNA was cleaved with Sac I, and electroporation (Bio-Rad Gene Pulser, 550V, 25 u FD) was performed. Transformation was performed. First, select the heterozygous gene disruption strains (ch HDAC2 + / _) from the blasticidin-resistant colonies, and further transform them into gene disruptions including the puromycin resistance. Two homozygous transformants (ch HDAC2 _ /-) were isolated.
  • RT PCR reaction was performed using “Superscriptlll One—Step RT—PCR with Platinium Taq Polymerase” (Invitrogen) and 200 ng vertical RNA, 10 pmol ch HDA PCR primer sets for C2 and S-actin (control) were added, and the reaction was performed in a 50 I reaction solution.
  • Reaction conditions were 55 ° C for 30 minutes and 94 ° C for 2 minutes, followed by 25 cycles of 94 ° C for 15 seconds / 60 ° C for 30 seconds / 68 ° C for 1 minute, and finally 68 minutes at 68 ° C for 5 minutes. Reacted.
  • the above WL 3.1 was cultured in a CO 2 constant temperature bath in the presence of 5% CO 2 at 39.5 ° C for the indicated period (3 weeks to 2 months).
  • I MDM medium In Vitrogen
  • Trichostatin A (Wako Pure Chemical Industries, Ltd.) should be diluted in DMSO 2 mg / m I as stock, and diluted appropriately in the medium so that the final concentration is 2.5, 5, 10 nM. It was.
  • the culture was continued while maintaining the cell concentration at 10 5 to 2 ⁇ 10 6 cells / I.
  • the cells were suspended in 100 I staining buffer containing 5 g / m I Providium iodide (Nacalai). I% of cells expressing gM In this case, about 10,000 cells were measured and calculated by EPICSELITEESP (Bec km an Coulter). At that time, the cells stained with fluorinated protease were gated as dead cells.
  • PCR Perkin E lmer 9600
  • Genomic DNA solution 1 I corresponding to 500,000 cells
  • 1 O pmoI was used for each of the following primers.
  • PCR reaction was performed in 50 I reaction solution using Pyrobest DNA Polymerase (Takara Shuzo). The reaction conditions were 98 ° C after 2 minutes, 9 8 ° C for 30 seconds, 57 ° C for 30 seconds, 7 2 ° C for 1 minute, 2 7 cycles, and finally 7 2 ° C for 5 minutes. Then, 1 I of Ex Taq DNA Polymerase (Takara Shuzo) was added and reacted at 72 ° C for 15 minutes, and then 20 I of the total reaction mixture was separated by agarose gel electrophoresis.
  • a band corresponding to the light chain gene variable region was excised, DNA was collected by Geltxtractionkit (Qiagen), and incorporated into pCR2.1_TOPO vector by TOPO—TAC loningkit (Invitrogen). Transformation into fungus. Thereafter, the plasmid was extracted, and the sequence was analyzed with ABIPRI SM 37 7 DNA Sequencer (Perkin Elmer) or ABI 37 30 xl (Applied Biosystems).
  • Buffer 1 D (0.2 MT ris-HCI pH 8.5, 0.1% BSA) 200 UI was added, and the mixture was reacted by stirring at 37 ° C for 4 hours with rotation, followed by blocking. . Thereafter, the plate was washed twice with 500 I buffer C, and then suspended in 0.02 ⁇ 1 ⁇ 2 sodium azide buffer_C 200 I.
  • the cells bound to the magnetic beads were suspended in 500 I, added to 3 Om I medium, dispensed 300 I into a 96-well plate, and cultured at 39.5 ° C.
  • the diversification pattern is also more complicated than wild type + trichostatin A (16 types for 31 clones, Fig. 5), totaling 38 types for 43 clones or 22 types for 30 clones. It had a different sequence.
  • FIG. 8 shows the ELISA (E L I SA) data of an example of antibody production against rabbit IgG (clone H 11 is a specific antibody).
  • the present invention provides a method for introducing a mutation into the variable region of an antibody gene. Furthermore, when this method is used, desired antibodies having various properties (specificity to antigen, affinity to antigen, etc.) can be produced. Therefore, a desired antibody that has been difficult to produce can be obtained by the method of the present invention, and can be used as an effective means for treatment, drug discovery, etc. in the medical field.

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Abstract

La présente invention concerne un procédé permettant d'introduire une mutation dans toute la partie de la région variable d'un gène d'anticorps. L'invention concerne spécifiquement un procédé permettant d'introduire une variété de mutations dans la région du gène d'anticorps située sur le chromosome d'un immunocyte grâce à la réduction sélective ou la perte de la fonction du gène de l'histone désacétylase (HDAC) 2, ou grâce à la réduction sélective ou la perte de l'activité ou de la fonction, de la protéine HDAC 2.
PCT/JP2007/001139 2006-10-19 2007-10-19 Procédé de régulation de la distribution de sites de mutation dans la région variable d'un gène d'anticorps WO2008047480A1 (fr)

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US8679845B2 (en) 2007-05-31 2014-03-25 University Of Washington B cells modified to reversibly induce accelerated mutagenesis of target genes
EP3138903A4 (fr) * 2014-05-02 2017-11-01 Chiome Bioscience, Inc. Cellules pour la production d'anticorps humain
WO2018147432A1 (fr) 2017-02-10 2018-08-16 株式会社カイオム・バイオサイエンス Procédé favorisant la diversification d'une région variable d'anticorps
CN109890842A (zh) * 2016-09-30 2019-06-14 凯奥目生物科学株式会社 抗体的取得方法

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8679845B2 (en) 2007-05-31 2014-03-25 University Of Washington B cells modified to reversibly induce accelerated mutagenesis of target genes
US9273119B2 (en) 2007-05-31 2016-03-01 University Of Washington Inducible mutagenesis of target genes
US9815885B2 (en) 2007-05-31 2017-11-14 University Of Washington Inducible mutagenesis of target genes
EP3138903A4 (fr) * 2014-05-02 2017-11-01 Chiome Bioscience, Inc. Cellules pour la production d'anticorps humain
CN112877292A (zh) * 2014-05-02 2021-06-01 凯奥目生物科学株式会社 产生人抗体的细胞
EP3851453A1 (fr) 2014-05-02 2021-07-21 Chiome Bioscience Inc. Cellules pour la production d'anticorps humain
CN109890842A (zh) * 2016-09-30 2019-06-14 凯奥目生物科学株式会社 抗体的取得方法
US11008566B2 (en) 2016-09-30 2021-05-18 Chiome Bioscience Inc. Method for obtaining antibody
CN109890842B (zh) * 2016-09-30 2023-12-22 凯奥目生物科学株式会社 抗体的取得方法
WO2018147432A1 (fr) 2017-02-10 2018-08-16 株式会社カイオム・バイオサイエンス Procédé favorisant la diversification d'une région variable d'anticorps
EP3974521A1 (fr) 2017-02-10 2022-03-30 Chiome Bioscience Inc. Procédé favorisant la diversification d'une région variable d'anticorps
US11572402B2 (en) 2017-02-10 2023-02-07 Chiome Bioscience Inc. Method for promoting diversification of antibody variable region

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