WO2018025826A1 - Anticorps fusionné avec une protéine de marquage - Google Patents

Anticorps fusionné avec une protéine de marquage Download PDF

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WO2018025826A1
WO2018025826A1 PCT/JP2017/027791 JP2017027791W WO2018025826A1 WO 2018025826 A1 WO2018025826 A1 WO 2018025826A1 JP 2017027791 W JP2017027791 W JP 2017027791W WO 2018025826 A1 WO2018025826 A1 WO 2018025826A1
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antibody
gene
leucine zipper
sequence
chain
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PCT/JP2017/027791
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秀雄 中野
昭博 森
晃代 加藤
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国立大学法人名古屋大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to an antibody fused with a labeled protein. Specifically, the present invention relates to a labeled protein fusion Fab antibody and its use.
  • This application claims priority based on Japanese Patent Application No. 2016-152447 filed on August 3, 2016, the entire contents of which are incorporated by reference.
  • Antibody-enzyme fusions are used for ELISA, Western blotting, cell staining, in vivo substance imaging, cancer treatment, and the like (Non-Patent Documents 1 to 5).
  • enzyme modification of an antibody has been mainly carried out by chemical modification, but has the disadvantages that “the amount of enzyme to be modified cannot be quantitatively controlled” and “experiment of chemical modification is required”.
  • an example in which an antibody in which an enzyme is genetically fused is prepared has been reported (Non-patent Documents 6 and 7), but most are related to scFv antibodies (single-chain Fv antibodies).
  • the interaction between the V region of the L chain and the H chain is weak, and it is often impossible to form a stable antigen-binding site, resulting in a decrease in antigen affinity or a sufficient enzymatic activity.
  • an antibody fused with a labeled protein such as an enzyme can be used not only for various detection methods but also for medical applications such as diagnosis and treatment.
  • a labeled protein fusion antibody having a sufficient function suitable for use in various applications has not been developed. Therefore, an object of the present invention is to provide a novel antibody that is excellent in the original characteristics of an antibody (that is, recognition of an antigen) and that the fused labeled protein exhibits a sufficient function.
  • Non-Patent Document 6 which is one of the few reports, alkaline phosphatase (PhoA) is genetically fused to an anti-human tumor necrosis factor ⁇ (TNF) Fab antibody and produced in a secretory expression system of Escherichia coli.
  • Non-patent Document 9 there is an example in which a divided nanoluciferase gene is fused to an anti-HER2 Fab antibody and expressed in Escherichia coli.
  • the common point of these reports is that they use only one kind of antibody gene that is most manageable in each research environment.
  • Fab antibodies have been studied under conditions that can be stably produced in E. coli, and their versatility has not been shown.
  • secretory expression of Fab antibodies is very difficult, but Fab antibody expression technology with excellent versatility and practicality has not been realized.
  • Non-Patent Document 6 since Non-Patent Document 6 has been reported, there has been no report on a technique for fusing Fab antibodies and enzymes.
  • Fab antibodies have light chain and heavy chain interactions in their constant regions (CL and CH1), which makes them more stable than scFv and can be used for a short time when newly prepared from B cells.
  • Non-patent Document 8 Fab formation efficiency differs depending on the type of antibody, and a desired activity may not be obtained.
  • LZ leucine zipper
  • Hc and Lc of Fab antibody it is possible to efficiently fold in E. coli cell-free protein synthesis system and E. coli intracellular expression system.
  • An antibody molecule is developed and named Zipbody (Patent Document 1 and Non-Patent Document 10).
  • the present inventors considered the effectiveness of Zipbody and genetically fused a labeled protein (for example, an enzyme) to the C-terminus of Zipbody. It was thought that the presence of LZ could produce a Fab antibody-labeled protein fusion with a stable structure. Based on this idea, a detailed experiment was planned using the knowledge and experience accumulated so far, and a detailed study was conducted. As a result, it was found that the above-mentioned strategy is extremely effective, and a fusion antibody that can achieve both recognition of the antigen and expression of the function of the labeled protein at a high level can be obtained (see Examples described later).
  • a labeled protein for example, an enzyme
  • the present inventors succeeded in providing labeled protein fusion antibodies that are extremely effective for various detection methods.
  • One thing that should be noted is that the effects were demonstrated in both mouse-derived antibodies and rabbit-derived antibodies, and in a plurality of labeled proteins (specifically, GFP and luciferase), and high versatility was confirmed. is there. Also, during the study, important and interesting findings related to practicality were obtained. Furthermore, when another enzyme (alkaline phosphatase) was fused, the desired effect was obtained, confirming the high versatility.
  • the following invention is mainly based on the above results and considerations.
  • [4] The labeled protein-fused Fab antibody according to [3], wherein the fluorescent protein is GFP and the luminescent enzyme is luciferase.
  • a peptide tag consisting of an amino acid sequence of SK, SKX, SKXX, AKXX or KKXX (where X represents any amino acid residue) is linked to the N-terminus of the H chain and L chain, respectively.
  • [6] The labeled protein-fused Fab antibody according to any one of [1] to [5], which is refolded.
  • [7] The labeled protein-fused Fab antibody according to any one of [1] to [5], which is synthesized in the cytoplasm of E. coli.
  • a detection reagent comprising the labeled protein-fused Fab antibody according to any one of [1] to [7].
  • a detection kit comprising the detection reagent according to [8].
  • [10] The following steps: (A) co-expressing antibody H chain gene encoding VH region and CH1 region and antibody L chain gene encoding VL region and CL region, or (B) antibody H chain encoding VH region and CH1 region A gene and an antibody L chain gene encoding a VL region and a CL region, respectively, and then mixing the expression product, Including Of the pair of peptides constituting the leucine zipper, the first tag sequence encoding one of them is at the 3 ′ end of the antibody H chain gene, and the second tag sequence encoding the other is at the 3 ′ end of the antibody L chain gene.
  • a labeled protein gene is linked to the antibody H chain gene and / or the antibody L chain gene via the first or second tag sequence, Preparation method of labeled protein fusion Fab antibody.
  • the expression system is an expression system using E.
  • a protease recognition sequence is interposed between the sequence encoding the peptide tag and the antibody H chain gene, and between the sequence encoding the peptide tag and the antibody L chain gene, [13] or [14 ]
  • the preparation method as described in. [16] The preparation method according to any one of [13] to [15], wherein the expression system using E. coli is an expression system using a T7 promoter or an expression system using a low-temperature expression promoter. [17] The preparation method according to any one of [13] to [15], wherein the expression system using E. coli is a cell-free protein synthesis system using E. coli-derived components.
  • [18] The preparation method according to any one of [10] to [17], wherein the antibody H chain gene and the antibody L chain gene are prepared by the following steps (i) to (viii): (i) providing mRNA derived from a single B cell; (ii) preparing cDNA by reverse transcription PCR using the mRNA as a template; (iii) PCR is performed using a primer set consisting of multiple primers containing the same third tag sequence at the 5 ′ end and capable of amplifying the antibody H chain gene encoding the VH region and CH1 region, and using the cDNA as a template.
  • Step to do (iv) PCR is performed using a primer set consisting of multiple primers containing the same fourth tag sequence at the 5 'end and capable of amplifying the antibody L chain gene encoding the VL region and CL region, and using the cDNA as a template Step to do; (v) performing PCR using a single primer containing the third tag sequence and using the amplification product of step (iii) as a template; (vi) performing PCR using a single primer containing the fourth tag sequence and using the amplification product of step (iv) as a template; (vii) adding the first tag sequence to the antibody heavy chain gene that is the amplification product of step (v); (viii) A step of adding the second tag sequence to the antibody L chain gene that is the amplification product of step (vi).
  • a promoter A first cloning site for a gene encoding one of the antibody chains constituting the Fab antibody; A first leucine zipper sequence encoding one of a pair of leucine zipper peptides; A second cloning site for a gene encoding the other antibody chain constituting the Fab antibody; A second leucine zipper sequence encoding the other of the pair of leucine zipper peptides, and A sequence encoding a labeled protein is arranged downstream of the first leucine zipper sequence and / or the second leucine zipper sequence.
  • Vector for preparing labeled protein fusion Fab antibody [21]
  • the promoter is a promoter that functions in E.
  • coli The vector according to [20], wherein a ribosome binding site is disposed between the promoter and the first cloning site. [22] Immediately before the first cloning site and immediately before the second cloning site, A sequence encoding a peptide tag consisting of an amino acid sequence of SK, SKX, SKXX, AKXX or KKXX (X represents an arbitrary amino acid residue) arranged immediately after the initiation codon is arranged. The vector according to [20] or [21].
  • a promoter A first antibody gene encoding one of the antibody chains constituting the Fab antibody; A first leucine zipper sequence encoding one of a pair of leucine zipper peptides; A second antibody gene encoding the other antibody chain constituting the Fab antibody; A second leucine zipper sequence encoding the other of the pair of leucine zipper peptides, and A sequence encoding a labeled protein is arranged downstream of the first leucine zipper sequence and / or the second leucine zipper sequence.
  • Vector for preparing labeled protein fusion Fab antibody [24]
  • the promoter is a promoter that functions in E.
  • coli The vector according to [23], wherein a ribosome binding site is arranged between the promoter and the first antibody gene. [25] Immediately before the first antibody gene and immediately before the second antibody gene, A sequence encoding a peptide tag consisting of an amino acid sequence of SK, SKX, SKXX, AKXX or KKXX (X represents an arbitrary amino acid residue) arranged immediately after the initiation codon is arranged. The vector according to [23] or [24].
  • a promoter A first cloning site for a gene encoding one of the antibody chains constituting the Fab antibody; A first vector having a first leucine zipper sequence encoding one of a pair of leucine zipper peptides; A promoter, A second cloning site for a gene encoding one of the antibody chains constituting the Fab antibody; A second vector having a second leucine zipper sequence encoding the other of the pair of leucine zipper peptides, A sequence encoding a labeled protein is arranged downstream of the first leucine zipper sequence and / or the second leucine zipper sequence.
  • a vector set for preparing a labeled protein-fused Fab antibody A vector set for preparing a labeled protein-fused Fab antibody.
  • a promoter A first antibody gene encoding one of the antibody chains constituting the Fab antibody; A first vector having a first leucine zipper sequence encoding one of a pair of leucine zipper peptides; A promoter, A second antibody gene encoding one of the antibody chains constituting the Fab antibody; A second vector having a second leucine zipper sequence encoding the other of the pair of leucine zipper peptides, A sequence encoding a labeled protein is arranged downstream of the first leucine zipper sequence and / or the second leucine zipper sequence.
  • a vector set for preparing a labeled protein-fused Fab antibody A vector set for preparing a labeled protein-fused Fab antibody.
  • Zipbody, Zipbody-Luc and Zipbody-GFP structures A leucine zipper (LZ) was fused to the C-terminus of Hc and Lc to facilitate the association of Hc and Lc. Luc and GFP were genetically fused to the C-terminus of Zipbody Hc.
  • LZ leucine zipper
  • a secondary antibody that recognizes the primary antibody is used, and an antigen is detected by a signal from the secondary antibody.
  • ELISA using a Fab-fluorescent protein fusion fluorescence from the fluorescent protein is detected, and a secondary antibody is not required. Plasmid structure for expressing Zipbody-Luc and Zipbody-GFP.
  • Plasmids were constructed to express mouse-derived anti-E. Coli O157 Fab antibody m6Fab, mouse-derived anti-E. Coli O157 Zipbody, and rabbit-derived anti-L. Monocytogenes Zipbody.
  • Luc gene is m6Fab Hc (pET22 m6Fab Hc-Luc), m6Fab LZ Hc (pET22 m6Fab LZ Hc-Luc), m6Fab LZ Lc (pET22 m6Fab LZ Lc-Luc), m6Fab Lc Hc (both m6Fab LZ W-Luc) and r4Fab LZ Hc (pET22 r4Fab LZ Hc-Luc).
  • the GFP gene was fused to m6Fab LZ Hc (pET22 m6Fab LZ Hc-GFP) and r4Fab LZ Hc (pET22 r4Fab LZ Hc-GFP).
  • the sequence of pET22 m6Fab LZ Hc-Luc is SEQ ID NO.1
  • the sequence of pET22 r4Fab LZ Hc-Luc is SEQ ID NO.2
  • the sequence of pET22 m6Fab LZ Hc-GFP is SEQ ID NO.3
  • pET22 r4Fab LZ Hc-GFP Is shown in SEQ ID NO: 4. Selection of host for expression of Zipbody-fluorescent protein fusion.
  • the affinity of each fusion protein for E.coli O157 was evaluated. Average values of three experiments are shown. The bar in the graph represents standard error (S.E.). Luminescent ELISA using anti-E. Coli O157 Zipbody-Luc. An immunoassay was performed using the Luc molecule of the fusion protein. Average values of three experiments are shown. The bar in the graph represents standard error (S.E.). ELISA using anti-L. Monocytogenes Zipbody-Luc or anti-L. Monocytogenes Zipbody-GFP. The affinity of each fusion protein for L. monocytogenes was evaluated. Average values of three experiments are shown. The bar in the graph represents standard error (S.E.). Luminescent ELISA using anti-L.
  • Monocytogenes Zipbody-Luc An immunoassay was performed using the Luc molecule of the fusion protein. Average values of three experiments are shown. The bar in the graph represents standard error (S.E.). Zipbody-GFP fluorescence. The GFP activity of each soluble fusion protein was evaluated. Average values of three experiments are shown. The bar in the graph represents standard error (S.E.). Fluorescence ELISA using Zipbody-GFP. An immunoassay using the GFP molecule of the fusion protein was performed. (A) Anti-E. Coli O157, (B) Anti-L.Lmonocytogenes. Average values of three experiments are shown. The bar in the graph represents standard error (S.E.). A list of primers.
  • the first aspect of the present invention relates to a labeled protein fusion Fab antibody (hereinafter, sometimes referred to as “the antibody of the present invention” for convenience of explanation).
  • the labeled protein-fused Fab antibody is a Fab antibody fused with a labeled protein, and has a function as an antibody that specifically recognizes a specific antigen, and also has a function of the labeled protein (in the case of an enzyme, a specific protein). Enzyme activity of catalyzing the reaction).
  • the Fab antibody is a fragment antibody composed of an H chain having a VH region and a CH1 region and an L chain having a VL region and a CL region, and does not contain an Fc region.
  • the H chain constituting the Fab antibody may be referred to as Hc and the gene encoding it may be referred to as the Hc gene, respectively, according to common practice.
  • the L chain constituting the Fab antibody may be referred to as Lc, and the gene encoding it may be referred to as Lc gene.
  • the antibody of the present invention contains a leucine zipper as part of its structure.
  • Leucine zippers are used to improve the rate of Fab antibody formation.
  • the binding force unique to the leucine zipper assists the association of Hc and Lc, and the Fab formation efficiency is improved.
  • stabilization of the structure of the Fab antibody can be expected by using a leucine zipper. Stabilization of the structure leads to improvement of antigen recognition ability or specificity.
  • Leucine zipper is a characteristic structure found as a secondary structure motif of proteins (Science. 1988 Jun 24; 240 (4860): 1759-64.).
  • the leucine zipper has a basic skeleton in which 4 to 5 leucine residues are arranged every 7 in an amino acid sequence that tends to have an ⁇ -helix structure. By this skeleton, leucine residues are arranged in almost one row in the ⁇ -helix axis direction, and are hydrophobically linked to the leucine residue sequence in another leucine zipper structure.
  • a pair of peptides referred to as leucine zipper peptide A and leucine zipper peptide B) containing such a characteristic motif is used.
  • Leucine zipper peptide A and leucine zipper peptide B have high affinity and form leucine zippers.
  • Leucine zipper peptide A contains leucine every 7 residues so that a leucine zipper can be formed.
  • leucine zipper peptide B contains leucine every 7 residues. These leucines are arranged so as to correspond to leucine in leucine zipper peptide A (those constituting leucine zipper motif).
  • the length of leucine zipper peptide A and leucine zipper peptide B is not particularly limited, but if it is too short, the desired effect, i.e., the binding force due to the leucine zipper structure, cannot be fully exerted, and if it is too long, the length of Hc and Lc may be reduced due to steric hindrance, etc. May affect association and antibody binding (antigen recognition). Therefore, the length of leucine zipper peptide A and leucine zipper peptide B is, for example, 25 to 50 residues, preferably 28 to 35 residues.
  • the lengths of leucine zipper peptide A and leucine zipper peptide B are basically the same, but may be different in length as long as a leucine zipper structure can be formed.
  • leucine zippers that exert binding force by electrostatic interaction between positively charged amino acids and negatively charged amino acids in addition to hydrophobic bonds between leucine and leucine are adopted. To do. Such leucine zippers (referred to as “charged leucine zippers” for convenience of description) exhibit high binding forces.
  • leucine zipper peptide A and leucine zipper peptide B have the following structural features (a) or (b).
  • (a) Leucine zipper peptide A contains a positively charged amino acid (basic amino acid).
  • Leucine zipper peptide B contains a negatively charged amino acid (acidic amino acid) at a position corresponding to the positively charged amino acid of leucine zipper peptide A.
  • Leucine zipper peptide A contains an acidic amino acid.
  • Leucine zipper peptide B contains a basic amino acid at a position corresponding to the acidic amino acid of leucine zipper peptide A.
  • Examples of basic amino acids are lysine (K), arginine (R), and histidine (H). Among these, K or R may be selected for reasons of charge strength.
  • examples of acidic amino acids are aspartic acid (D) and glutamic acid (E).
  • D aspartic acid
  • E glutamic acid
  • Increasing the number of charged amino acids in the leucine zipper peptide increases the electrostatic interaction, which can be expected to improve the binding power of the leucine zipper. Therefore, the proportion of charged amino acids in the leucine zipper peptide is, for example, 15% to 50%, preferably 20% to 40%.
  • LZA and LZB can be used as a pair of leucine zipper peptides rich in charged amino acids.
  • LZA and LZB The sequences of LZA and LZB are shown below.
  • LZA Leucine zipper peptide that retains negative charge
  • AQLEKELQALEKENAQLEWELQALEKELAQK SEQ ID NO: 5
  • glutamic acid E
  • E glutamic acid
  • LZB Leucine zipper peptide that retains positive charge
  • AQLKKKLQALKKKNAQLKWKLQALKKKLAQK SEQ ID NO: 6
  • lysine (K) is arranged at the 4th, 6th, 11th, 13th, 18th, 20th, 25th, and 27th positions.
  • the antibody of the present invention one of a pair of peptides constituting a leucine zipper (leucine zipper peptide A) is added in a state linked to the C terminus of the H chain (Hc), that is, CH1, and the other (leucine zipper peptide B) is added in a state of being linked to the C terminus of the L chain (Lc), that is, CL.
  • the antibody of the present invention is a Fab antibody in which a leucine zipper is added to the C-terminal, that is, the constant region side.
  • the antibody exhibits specific binding properties depending on the variable region, with the leucine zipper added.
  • the leucine zipper peptide is linked to the H chain and L chain directly or via a linker sequence.
  • a peptide linker is used as the linker.
  • a peptide linker is a linker consisting of a peptide in which amino acids are linked in a straight chain.
  • a typical example of a peptide linker is a linker composed of glycine and serine (GGS linker or GS linker).
  • a GGS linker consists of a sequence in which GGS is repeated one to several times. The number of repetitions is not particularly limited, but is preferably 2 to 6 times, more preferably 2 to 4 times.
  • the GS linker is a sequence in which GGGGS (SEQ ID NO: 7) is repeated one to several times.
  • GGS linker and glycine and serine which are amino acids constituting the GS linker, have a small size per se and are difficult to form higher-order structures in the linker. Therefore, it is unlikely to become an obstacle during the association of Hc and Lc and the formation of the leucine zipper structure.
  • a labeled protein is linked to the H chain, L chain, or both via a leucine zipper peptide. Therefore, the antibody of the present invention can take the following three forms.
  • the form (1) is particularly preferred because high expression efficiency and high antigen affinity were observed when the labeled protein was linked only to the H chain (see Examples below).
  • the labeled protein linked to the H chain and the labeled protein linked to the L chain may not be the same.
  • the labeled protein is not particularly limited. That is, various labeled proteins can be employed as a component of the antibody of the present invention.
  • a labeled protein is a protein whose label is its own luminescence or fluorescence, or an enzyme reaction product.
  • Antibodies fused with labeled proteins are (1) detection and measurement of specific antigens (qualitative or quantitative), (2) labeling, staining or visualization of cells, tissues or organs / organs (eg in vivo imaging), Used for treatment.
  • labeled proteins examples include GFP (green fluorescent protein), EGFP, GFP2, ECFP, EBFP, YFP, mBanana, mOrange, DsRed2, mStrawberry, mCherry, mRasberry, mPlum, Kaede, and other fluorescent proteins, luciferase (Luc), nano Luminescent enzymes such as lanthanum, peroxidase (for example, HRP), alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, and hydrolase.
  • Proteins such as luciferase and GFP are widely used for monitoring gene translation control (Ghim CM, Lee SK, Takayama S, Mitchell RJ. 2010. The art of reporter proteins in science: past, present and future applications. Bmb Reports 43 (7): 451-460.). These proteins can be genetically fused to the protein to be monitored and their behavior detected as a luminescent signal. Luciferase is a general term for enzymes that generate luminescence by oxidizing a substrate (Hastings JW. 1996. Chemistries and colors of bioluminescent reactions: A review. Gene 173 (1): 5-11.).
  • Luciferase from L. cruciata was first reported in 1989 as an enzyme that catalyzes the oxidation of luciferin in the presence of ATP and oxygen molecules (MasudasT, Tatsumi H, Nakano E. 1989.
  • 257 Tyr is known to affect the emission wavelength, and it is yellow-green in the wild type (WT), but Y257F, Y257A, Y257E, Y257R show yellow, orange, red, and yellow, respectively (Wang Y , Akiyama H, Terakado K, Nakatsu T. 2013. Impact of Site-Directed Mutant Luciferase on Quantitative Green and Orange / Red Emission Intensities in Firefly Bioluminescence. Scientific Reports 3.).
  • GFP is a protein that emits green fluorescence. It has been discovered from Aequorea aequorea, etc., and is frequently used for protein expression monitoring (Zimmer M. 2002. Green fluorescent protein (GFP): Applications, structure, and related photophysical behavior. Chemical Reviews 102 (3): 759-781.). Moreover, it is known that misfolding is likely to occur when GFP is expressed in E. coli (Tsien RY. 1998. The green fluorescent protein. Annual Review of Biochemistry 67: 509-544.).
  • Pedelacq and others have developed super folder GFP with high folding efficiency by introducing cycle-3 'mutation (F99S, M153T, V163A) and enhanced GFP mutateon (F64L, S65T) into GFP derived from Pyrobaculum aerophilum (Pedelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. 2006. Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24 (1): 79-88.).
  • a peptide tag consisting of an amino acid sequence of SK, SKX, SKXX, AKXX or KKXX (where X represents any amino acid residue) is linked to the N-terminus of the H chain and L chain, respectively. is doing.
  • the antibody of the present invention is prepared as a protein (tag added protein) in which a specific peptide tag is linked to each antibody chain.
  • the peptide tag used in the present invention is composed of an amino acid sequence of SK, SKX, SKXX (SEQ ID NO: 8), AKXX (SEQ ID NO: 9) or KKXX (SEQ ID NO: 10).
  • X represents any amino acid residue.
  • SK for example, I (Ile), K (Lys), S (Ser), A (Ala) or the like is used.
  • K (Lys), I (Ile) or the like is used.
  • X following AK is preferably I (Ile).
  • AKX is preferably I (Ile) or K (Lys).
  • KK is preferably K (Lys).
  • KKX is preferably K (Lys).
  • SK is a peptide (Ser-Lys) in which serine (Ser) and lysine (Lys) are linked in this order from the N-terminal side to the C-terminal side.
  • SKX is a peptide in which one amino acid residue is added to SK
  • SKXX (SEQ ID NO: 8) is a peptide in which two amino acid residues are added to SK.
  • a specific example of the peptide represented by SKX is SKI (Ser-Lys-Ile).
  • SKXX (SEQ ID NO: 8) is preferably SKIX (SEQ ID NO: 11), that is, contains SKI.
  • SKIX SKIX
  • SKIK SKIK
  • SKII Ser-Lys-Ile-Ile
  • SEQ ID NO: 13 SKII
  • SKKK Ser-Lys-Lys -Lys
  • AKXX is a peptide in which two amino acid residues are added to a peptide (Ala-Lys) in which alanine (Ala) and lysine (Lys) are linked in this order from the N-terminal side to the C-terminal side. It is.
  • Specific examples of the peptide represented by AKXX (SEQ ID NO: 9) are AKIK (Ala-Lys-Ile-Lys) (SEQ ID NO: 15) and AKII (Ala-Lys-Ile-Ile) (SEQ ID NO: 16).
  • KKXX (SEQ ID NO: 10) is a peptide in which two amino acid residues are added to a peptide (LysL-Lys) in which lysine (Lys) and lysine (Lys) are linked in this order from the N-terminal side to the C-terminal side. It is.
  • a specific example of the peptide represented by KKXX (SEQ ID NO: 10) is KKKK (Lys-Lys-Lys -Lys) (SEQ ID NO: 17).
  • the peptide tag used in the present invention is composed of the amino acid sequence of SK, SKX, SKXX (SEQ ID NO: 8), AKXX (SEQ ID NO: 9) or KKXX (SEQ ID NO: 10) as described above, and typically 2-4 Consists of amino acid residues.
  • other amino acid residues may be added to the N-terminal side and / or C-terminal side as long as the function (improving the expression level of the target protein) is not affected.
  • the total length is 5 to 13 amino acid residues, preferably 5 to 10 amino acid residues, more preferably 5 to 7 amino acid residues.
  • a plurality of the above peptide tags (SK, SKX, SKXX (SEQ ID NO: 8), AKXX (SEQ ID NO: 9) or KKXX (SEQ ID NO: 10)) may be used.
  • SK, SKX, SKXX (SEQ ID NO: 8), AKXX (SEQ ID NO: 9) or KKXX (SEQ ID NO: 10) may be used.
  • 2 to 5 peptide tags are linked in tandem.
  • the above peptide tag and other tags for example, His tag, HA tag, FLAG tag, etc.
  • the second aspect of the present invention provides a preparation method of the labeled protein fusion Fab antibody.
  • the preparation method of the present invention comprises antibody H chain gene encoding VH region (heavy chain variable region) and CH1 region (heavy chain constant region 1), VL region (light chain variable region) and CL region (light chain constant region). And expressing an antibody L chain gene encoding).
  • the antibody H chain gene and the antibody L chain gene are coexpressed (step (A), hereinafter referred to as “coexpression step”). That is, the antibody H chain gene and the antibody L chain gene are expressed in the same expression system.
  • the antibody H chain gene and the antibody L chain gene are each expressed.
  • the expression product is mixed after expression, and the antibody H chain and the antibody L chain are associated.
  • a characteristic antibody H chain gene and antibody L chain gene are subjected to expression. That is, as an antibody gene used in the expression step, an Hc gene to which a base sequence (first tag sequence) encoding one (leucine zipper peptide A) of a pair of peptides constituting leucine zipper is added, and the other (leucine) An Lc gene to which a base sequence (second tag sequence) encoding zipper peptide B) has been added is used.
  • the Hc gene and / or Lc gene, or both encodes a labeled protein via a base sequence encoding a leucine zipper peptide (first tag sequence in the case of Hc gene, second tag sequence in the case of Lc gene) Keep the genes linked.
  • a leucine zipper peptide first tag sequence in the case of Hc gene, second tag sequence in the case of Lc gene
  • Such a characteristic Hc gene expression construct and Lc gene expression construct are expressed, and a leucine zipper is added, and a Fab antibody in which a labeled protein is fused to the H chain, the L chain, or both is obtained.
  • the addition position of the first tag sequence is the 3 ′ end of the Hc gene.
  • the addition position of the second tag sequence is the same, and is added to the 3 ′ end of the Lc gene.
  • the first tag sequence and the second tag sequence are linked to the antibody gene directly or via a linker sequence (sequence encoding the linker). In the latter case, a Fab antibody in which leucine zippers are linked by a linker is obtained.
  • the gene encoding the tag protein is a base sequence encoding the leucine zipper peptide (first tag sequence in the case of the Hc gene, second tag in the case of the Lc gene), either directly or via a linker sequence (sequence encoding the linker). Array).
  • the antibody gene (Hc gene, Lc gene) can be directly or via a sequence encoding the peptide tag.
  • An expression construct comprising a structure linked to is used.
  • various sequences can be employed. For example, if the peptide tag is SK, tctaaa or tcg aag sequence, etc.
  • the peptide tag is SKI, tct ⁇ ⁇ aaa ata or tcg aag atc sequence, etc., if the peptide tag is SKIK
  • the peptide tag is SKKK, such as the sequence of tct aaa ata aaa (SEQ ID NO: 18) or tcg aag atc aag (SEQ ID NO: 19), the sequence of tct aaaaaaaa (SEQ ID NO: 20), the peptide tag is SKII
  • tct aaa att att SEQ ID NO: 21
  • the peptide tag is AKIK
  • gca aaa att aaa SEQ ID NO: 22
  • the peptide tag is AKII
  • the sequence of gca ⁇ aa att att SEQ ID NO: 23) and the sequence of aaaaaaaaa
  • protease recognition sequence is an amino acid sequence that is recognized by a specific protease and necessary for cleavage of the protein by the protease.
  • a sortase recognition sequence, an HRV3C recognition sequence, or a TEV protease recognition sequence can be used.
  • a functional sequence such as a sequence encoding a protease recognition sequence, a sequence not having a specific function may be interposed between the sequence encoding the peptide tag and the antibody gene.
  • Hc gene and Lc gene can be prepared by any method.
  • Our research group has developed a method for obtaining Fab antibodies called the SICREX method (Single-Cell RT-PCR Linked In Vitro Expression).
  • the SICREX method makes it possible to obtain a desired antibody from antibody-producing cells in a short time.
  • the Hc gene and the Lc gene are prepared easily and in a short time, and the series of operations is accelerated. In a typical operation of the SICREX method (Biotechnol Prog.
  • B cells are isolated from spleen and peripheral blood of rats and rabbits) or human peripheral blood. Dilute the solution containing the isolated B cells so that the cell count is 1 cell / well. Alternatively, B cells are isolated using a micromanipulator or the like.
  • cDNA is synthesized from mRNA in B cells using reverse transcription PCR (RT-PCR). Subsequently, the Hc gene and the Lc gene are amplified separately by two-step PCR.
  • first primer set a plurality of cDNA-specific primers (first primer set) having the same tag sequence added to the 5 ′ end are used.
  • the second stage PCR is performed using the amplification product of the first stage PCR as a template.
  • a single primer with the same tag sequence added to the 5 'end as the tag sequence used for the first primer set is used, and the amplification product of the first stage PCR is specifically and efficiently used. Amplify to.
  • the single primer used for the second stage PCR is complementary to the 5 ′ end part and the 3 ′ end part of the amplification product obtained by the first stage PCR. Therefore, specific amplification with a single primer becomes possible.
  • Step of preparing mRNA derived from a single B cell ii) Step of preparing cDNA by reverse transcription PCR using said mRNA as a template
  • Step of preparing cDNA by reverse transcription PCR using said mRNA as a template iii) Tag sequence identical to the 5 ′ end (third tag)
  • step Iv performing PCR using a primer set that can amplify an antibody H chain gene (Hc gene) encoding a VH region and a CH1 region, and using the cDNA as a template.
  • a primer set consisting of a plurality of primers containing the same tag sequence (4th tag sequence) at the end and capable of amplifying an antibody L chain gene (Lc gene) encoding VL region and CL region, and using the cDNA as a template (V) performing PCR using a single primer including the third tag sequence and using the amplification product of step (iii) as a template (vi) performing a single step including the fourth tag sequence Using the primers, the amplification product of step (iv) A step of performing PCR using as a template (vii) a step of adding a first tag sequence (sequence encoding leucine zipper peptide A) to the antibody H chain gene (Hc gene) which is an amplification product of step (v) (viii) A step of adding a second tag sequence (sequence encoding leucine zipper peptide B) to the antibody L chain gene (Lc gene) which is an amplification product of step (vi)
  • steps (vii) and (viii) are particularly characteristic of the present invention.
  • the tag sequence in steps (vii) and (viii) can be added by overlap PCR in the same manner as the addition of elements necessary for expression in the cell-free protein synthesis system in the SICREX method.
  • a promoter is used as “an element necessary for expression in a cell-free protein synthesis system”.
  • a promoter and a terminator are used in combination. More preferably, a promoter, a terminator and a ribosome binding site are used in combination.
  • T7 promoter, T3 promoter, SP6 promoter and the like can be used.
  • the terminator for example, a T7 terminator can be used.
  • an improved technique of SICREX method that uses two-step PCR in one operation (one-step PCR) by simultaneously using the primers used for the first-step PCR and the second-step PCR at a predetermined quantitative ratio.
  • the antibody gene is amplified by one-step PCR instead of the conventional two-step PCR.
  • the following steps (a) and (b) are performed in place of the steps (iii) to (vi).
  • a primer set comprising a plurality of primers containing the same tag sequence (fifth tag sequence) at the 5 ′ end and capable of amplifying an antibody H chain gene (Hc gene) encoding a VH region and a CH1 region;
  • B performing PCR using a single primer containing the fifth tag sequence used at a higher concentration than the primer set and using the cDNA as a template
  • a primer set that can amplify an antibody L chain gene (Lc gene) encoding a VL region and a CL region, and a primer set that is used at a higher concentration than the primer set.
  • the amount ratio of the primer set to the single primer is, for example, 1: 2 to 1:50, preferably 1: 5 to 1:20, more preferably 1: 8 to 1:15.
  • the antibody cDNA is specifically amplified by a two-step PCR method (nested PCR method) using an outer primer and an inner primer. Also good.
  • steps (I) to (IV) are performed.
  • IV Amplifying the antibody L chain gene by a nested PCR method using the cDNA as a template.
  • the Hc gene (the first tag sequence is added.
  • the tag protein gene is also linked.
  • the Lc gene (the first tag sequence is added.
  • the co-expression step of the labeled protein gene is also performed in an expression system using a host cell or a cell-free protein synthesis system.
  • an expression vector holding the Hc gene so that it can be expressed and an expression vector holding the Lc gene so that it can be expressed are prepared, and an appropriate host is transformed with these expression vectors.
  • the host is transformed with an expression vector capable of co-expressing the Hc gene and the Lc gene.
  • the obtained transformant is cultured under conditions that allow expression of the antibody gene from the expression vector, and then the labeled protein-fused Fab antibody, which is an aggregate of expression products, is recovered from the transformant or culture medium .
  • an appropriate host is transformed with an expression vector that holds the Hc gene so that the Lc gene can be expressed.
  • An appropriate host is transformed with an expression vector retained so as to allow expression.
  • the expression product is recovered from the transformant or the culture solution. Thereafter, the expression products are mixed and associated to obtain a labeled protein fusion Fab antibody.
  • E. coli bacterial cells
  • yeast cells for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris
  • filamentous fungi cells for example, Aspergillus oryzae, Aspergillus niger
  • mammalian cells for example, CHO cells, Sp2 / 0 cells, NS0 cells
  • E. coli is employed as the host.
  • E. coli is suitable for the efficient and large-scale preparation of the target protein.
  • T7 promoter Various expression systems using T7 promoter, lac promoter, tac promoter, trp promoter, T3 promoter, SP6 promoter, low-temperature expression promoter (cold shock gene cspA promoter) and the like can be used.
  • the T7 promoter and the low-temperature expression promoter are particularly preferred promoters because they have the advantages of being easily induced and capable of strong expression control. Therefore, it is preferable to use an expression system using a T7 promoter or a low temperature expression promoter.
  • E. coli expression system the expression system using E. coli as a host
  • E. coli expression system the cell-free protein synthesis system using E. coli-derived components (described later) are comprehensively used. Expression system ".
  • the expression vector may be selected in consideration of the relationship with the host.
  • Each operation and conditions such as transformation, culture, and recovery may be in accordance with conventional methods. Alternatively, each operation may be performed according to past reports.
  • the cell-free protein synthesis system does not use living cells, but uses ribosomes derived from living cells (or obtained by genetic engineering techniques), transcription / translation factors, etc. It means to synthesize in vitro.
  • a cell-free protein synthesis system a cell extract obtained by purifying a cell disruption solution as needed is generally used.
  • Cell extracts generally contain ribosomes necessary for protein synthesis, various factors such as initiation factors, and various enzymes such as tRNA.
  • other substances necessary for protein synthesis such as various amino acids, energy sources such as ATP and GTP, and creatine phosphate are added to the cell extract.
  • a ribosome, various factors, and / or various enzymes prepared separately may be supplemented as necessary during protein synthesis.
  • the cell-free protein synthesis system has the following advantages. First, since there is no need to maintain live cells, operability is good and the degree of freedom of the system is high. Therefore, it is possible to design a synthetic system with various modifications and modifications according to the properties of the target protein. Next, in the synthesis of cell systems, it is basically impossible to synthesize proteins that are toxic to the cells used, but in the cell-free system, even such toxic proteins can be produced. In addition, high throughput can be easily achieved because many types of proteins can be synthesized simultaneously and rapidly. It also has the advantage that the produced protein can be easily separated and purified, which is advantageous for high throughput. In addition, it also has the advantage that non-natural proteins can be synthesized by incorporating non-natural amino acids.
  • a cell-free protein synthesis system If a cell-free protein synthesis system is adopted, a series of operations of the preparation method of the present invention can be performed according to the SICREX method, and a labeled protein-fused Fab antibody can be prepared efficiently and rapidly.
  • E. coli S30 extract system prokaryotic cell system
  • wheat germ extract system eukaryotic cell system
  • rabbit reticulocyte lysate system eukaryotic cell system
  • the E. coli 30S fraction is prepared through steps of E. coli collection, cell disruption, and purification.
  • the preparation of the 30S fraction of E. coli and the cell-free transcription / translation coupling reaction were performed by the method of Pratt et al. (Pratt, J. M .: Chapter 7, in “Transcription and Translation: A practical approach”, ed. By B. D. Hames & S. J. Higgins, pp. 179-209, IRL Press, New York (1984)) and Ellman et al. (Ellman, llJ. Et al .: Methods Enzymol., 202, 301-336 (1991)) This can be done with reference.
  • the wheat germ extract system has the advantage of efficiently synthesizing high-quality eukaryotic proteins, and is often used to synthesize eukaryotic proteins that are difficult to synthesize using the E. coli S30 extract system. Is done. Recently, it has been reported that a highly efficient and stable synthetic system is constructed by preparing an extract from germs from which seed endosperm components have been washed away (Madin, K. et al .: Proc. Natl. Acad. Sci. USA, 97: 559-564, 2000). After that, technical developments such as mRNA untranslated sequence with high translation promoting ability, protein synthesis method for multi-item function analysis using PCR, construction of dedicated high expression vector, etc. were carried out (Sawasaki, T. et al .: Proc Natl. Acad. Sci. USA, 99: 14652-14657, 2002), is expected to be applied in various fields.
  • the wheat germ extract can be obtained by grinding and centrifuging wheat germ and then separating the supernatant by gel filtration.
  • Anderson et al. the method of Anderson et al. (Anderson, C. W. et al .: Methods Enzymol., 101, 638-644 (1983)) can be referred to. Improved methods have also been reported, such as the method of Kawarazaki et al. (Kawarasaki, Y. et al .: Biotechnol. Prog., 16, 517-521 (2000)) and the method of Madin et al. (Madin, K. et al. : Proc. Natl. Acad. Sci. USA, 97: -559-564, 2000).
  • Rabbit reticulocyte lysate system is suitable for globulin production.
  • Rabbit reticulocyte lysate is made anemic by injecting phenylhydrazine intravenously into rabbits for several days, blood is collected after a predetermined period (for example, day 8), and then subjected to ultracentrifugation from the hemolyzed solution. can get.
  • the preparation of rabbit reticulocyte lysate can be performed with reference to the method of Jackson and Hunt (Jackson, R. J. and Hunt, T .: Methods Enzymol., 96, 50-74 (1983)). .
  • the cell-free protein synthesis system that can be used in the practice of the present invention is not limited to the above-described ones, for example, bacterial extracts other than E. coli and plant extracts other than wheat, insect-derived extracts, animal cell-derived extracts, Alternatively, a system constructed based on genome information may be used. Preferably, an E. coli S30 extract system (prokaryotic cell system) or a system reconstituted based on the E. coli genome as described above is used. These systems are also commercially available as kits and can be used easily.
  • the expressed or synthesized labeled protein-fused Fab antibody can be recovered by conventional methods (centrifugation, filtration, affinity chromatography, etc.). If a collection tag sequence (for example, histidine tag) is incorporated into the Hc gene and the Lc gene, the tag sequence can be used for easy and simple collection.
  • a collection tag sequence for example, histidine tag
  • Refolding operation may be performed after expression.
  • Refolding is an operation of rewinding to an active natural structure, and is roughly divided into a dilution method and a dialysis method.
  • the expression product is solubilized with a denaturing agent such as guanidine hydrochloride (in combination with a reducing agent such as DDT or ⁇ -mercaptoethanol to form a disulfide bond), and then diluted in a refolding buffer.
  • a denaturing agent such as guanidine hydrochloride (in combination with a reducing agent such as DDT or ⁇ -mercaptoethanol to form a disulfide bond)
  • dialysis method after treatment with a denaturant, dialysis is performed using a dialysis solution in which the concentration of the denaturant is reduced stepwise to remove the denaturant.
  • the peptide tag is linked to the N-terminus of the H chain and L chain with the protease recognition sequence interposed.
  • the peptide tag is cleaved by prosthesis treatment ( It is possible to obtain a labeled protein-fused Fab antibody that has been separated.
  • Vector for preparing labeled protein-fused Fab antibody The present invention also provides a vector for preparing labeled protein-fused Fab antibody.
  • a promoter, a first cloning site for a gene encoding one antibody chain constituting a Fab antibody, and a first leucine zipper sequence encoding one of a pair of leucine zipper peptides A second cloning site for a gene encoding the other antibody chain constituting the Fab antibody, and a second leucine zipper sequence encoding the other of the pair of leucine zipper peptides, and the first leucine zipper A sequence encoding a labeled protein is arranged downstream of the sequence and / or the second leucine zipper sequence.
  • the Hc gene and the Lc gene are inserted into the first cloning site and the second cloning site (the antibody gene that has not been inserted into the first cloning site is inserted into the second cloning site).
  • a vector expressing the desired labeled protein-fused Fab antibody is completed. Therefore, it can be said that it is a versatile vector.
  • a promoter a first antibody gene encoding one antibody chain constituting a Fab antibody, a first leucine zipper sequence encoding one of a pair of leucine zipper peptides, and the Fab
  • a second antibody gene encoding the other antibody chain constituting the antibody and a second leucine zipper sequence encoding the other of the pair of leucine zipper peptides, and the first leucine zipper sequence and / or the first leucine zipper sequence
  • a sequence encoding a labeled protein is arranged downstream of the 2 leucine zipper sequence.
  • a pair of antibody genes (Hc gene and Lc gene) corresponding to the Fab antibody are incorporated.
  • the antibody gene is obtained by inserting a pair of antibody genes into the vector of the first embodiment. Can do.
  • the vector of the present invention can be constructed, for example, as a vector using E. coli as a host or a vector using yeast as a host.
  • Escherichia coli When Escherichia coli is used as a host, a promoter that functions in Escherichia coli, such as T7 promoter, lac promoter, tac promoter, trp promoter, T3 promoter, SP6 promoter, cold expression promoter ( Cold shock gene cspA promoter) and the like.
  • a ribosome binding site is usually incorporated downstream of the promoter (between the promoter and the first cloning site in the first embodiment and between the promoter and the first antibody gene in the second embodiment).
  • the ribosome binding site contains a sequence (SD sequence) to which the liposome binds.
  • SD sequence is a sequence rich in adenine and guanine, and consists of, for example, the AGGAGG sequence.
  • the expression vector is in the form of a plasmid.
  • the vector of the present invention When constructing the vector of the present invention as a vector using yeast as a host, it typically takes the form of a plasmid.
  • a shuttle vector having an origin of replication capable of replicating in E. coli may be used.
  • promoters examples include GAL1, GAL10, AOX1, pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1, or pPYK1.
  • Nutritional complement genes eg, URA3 gene, HIS3 gene, LYS2 gene, LEU2 gene, etc.
  • URA3 gene, HIS3 gene, LYS2 gene, LEU2 gene, etc. can also be incorporated into the vector.
  • the vector of the present invention may contain, in addition to the above-described elements, elements necessary for propagation in host E. coli, elements necessary or useful for expression of antibody genes, elements useful for detection and identification, and the like.
  • elements that can be incorporated into the vector of the present invention include replication origin, terminator (eg, T7 terminator), drug resistance gene (ampicillin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, streptomycin resistance gene, etc.) It is.
  • a sequence encoding a peptide tag may be incorporated in the vector so that the antibody is expressed as a protein (tag added protein) in which a peptide tag is linked to each antibody chain.
  • the amino acid sequence of SK, SKX, SKXX, AKXX, or KKXX arranged immediately before the first cloning site and immediately before the second cloning site and immediately after the start codon.
  • a sequence encoding a peptide tag consisting of (wherein X represents any amino acid residue) is arranged.
  • similar sequences are arranged immediately before the first antibody gene and immediately before the second antibody gene.
  • the sequence encoding the peptide tag and the first and second cloning sites (in the first embodiment), or the sequences encoding the first and second antibody genes (in the second embodiment) are directly or other sequences. It is connected via.
  • Hc and Lc are expressed by one vector, but Hc and Lc can also be expressed by separate vectors. Further aspects of the invention (third and fourth aspects) provide a set of vectors used for such expression. In addition, about the matter which is not demonstrated especially, the corresponding description of a 1st aspect or a 2nd aspect is used.
  • a promoter, a first cloning site for a gene encoding one antibody chain constituting a Fab antibody, and a first leucine zipper encoding one of a pair of leucine zipper peptides are provided.
  • a sequence encoding a labeled protein is arranged downstream of the first leucine zipper sequence and / or the second leucine zipper sequence.
  • a first vector having a promoter, a first antibody gene encoding one antibody chain constituting a Fab antibody, and a first leucine zipper sequence encoding one of a pair of leucine zipper peptides A vector set comprising a second vector having a promoter, a second antibody gene encoding one of the antibody chains constituting the Fab antibody, and a second leucine zipper sequence encoding the other of the pair of leucine zipper peptides
  • a sequence encoding a labeled protein is disposed downstream of the first leucine zipper sequence and / or the second leucine zipper sequence.
  • a sequence encoding a peptide tag can be incorporated as in the first and second aspects.
  • a further aspect of the present invention relates to use of the labeled protein fusion Fab antibody.
  • the labeled protein-fused Fab antibody of the present invention has a function as an antibody, that is, an affinity for a specific antigen, and also has a labeling ability due to fusion of the labeled protein, and a specific target (antigen) It is useful as a reagent for specific detection or measurement. For example, it can be applied to various immunological methods (for example, ELISA method), labeling, staining or visualization (for example, in vivo imaging) of cells, tissues or organs / organs.
  • a labeled protein-fused Fab antibody derived from a specific subject for example, a patient suffering from a specific disease
  • diagnosis evaluation of disease state and therapeutic effect
  • a protein capable of exerting a therapeutic effect such as an enzyme exhibiting anticancer activity
  • the labeled protein-fused Fab antibody can be used as a therapeutic antibody.
  • the present invention further provides a detection kit containing the reagent of the present invention.
  • detection and measurement using the reagent of the present invention can be performed more easily.
  • the kit of the present invention contains the reagent of the present invention as a main component. Other reagents, reaction solutions, containers / equipment necessary for detection and measurement may be included in the kit of the present invention. Usually, an instruction manual is attached to the kit of the present invention.
  • LZ leucine zipper
  • E. coli DH5 ⁇ was used as a host strain for gene manipulation.
  • E. coli SHuffle (registered trademark) T7 express (New England biolabs, fhuA2 lacZ :: T7 gene1 [lon] ompT ahpC gal ⁇ att :: pNEB3-r1-cDsbC (Spec R , lacI q ) ⁇ trxB sulA11 R (mcr-73 :: miniTn10--Tet S ) 2 [dcm] R (zgb-210 :: Tn10 --Tet S ) endA1 ⁇ gor ⁇ (mcrC-mrr) 114 :: IS10), E.
  • coli BL21 DE3) pLysS (Promega, F - , ompT, hsdS (r B -, m B -)., dcm, gal, ⁇ (DE3), pLysS, Cm r), E coli Rosetta-gami TM 2 (DE3) pLysS ( Merck Millipore, ⁇ (ara-leu) 7697 ⁇ lacX74 ⁇ phoA PvuII phoR araD139 ahpC galE galK rpsL (DE3) F ′ [lac + lacIq pro] gor522 :: Tn10 trxB pLysSRARE2 (CamR, StrR, TetR)) was used. E. coli was cultured at 37 ° C. or 16 ° C. using LB medium. Ampicillin was appropriately added as a selection marker so as to be 100 ⁇ g / mL.
  • Plasmids were extracted from the generated colonies, and DNA sequence analysis was performed using BigDye Terminator v3.1 cycle sequencing kit and ABI PRISM 3100 genetic analyzer. The prepared plasmid was designated as pCold orange Luc.
  • the prepared plasmid was designated as pCold green Luc.
  • Plasmid pET22m6Fab LZ Hc-Luc for expressing the protein m6Fab LZ Hc-Luc in which Luc was fused to the C-terminus of Hc of m6Fab LZ was prepared (FIG. 3).
  • primers L. cruciata Luc F and L. cruciata Luc R (FIG. 13) were used for PCR [2 minutes at 94 ° C.
  • Each DNA amplification product was treated with Dpn I (Takara), then purified with FastGene Gel / PCR Extraction Kit (Nippon Genetics Co., Ltd.), and Luc gene was converted to Hc C of m6Fab LZ with Gibson Assembly (New England BioLabs). Fused to the ends.
  • Each fused DNA was introduced into E. coli DH5 ⁇ , the generated colonies were cultured overnight in LB medium, and the plasmid was extracted with FastGene Plasmid Mini Kit] (Nippon Genetics). The sequence of each plasmid was confirmed by a DNA sequence analysis service (Fusmac Co., Ltd.). The operation after amplification of the DNA fragment was carried out in the same manner in the subsequent plasmid construction experiments (2-4) to (2-9).
  • the cells were suspended in 2 mL of PBS, and the cells were crushed with zirconia / silica beads 0.1 mm (Ieda Trading Co., Ltd.) and a bead-type cell crusher Micro Smash TM MS 100R. Centrifuged at 14,000 x g for 10 minutes at 4 ° C. The obtained supernatant was used as a soluble fraction. The precipitate was suspended in 2 mL of PBS to obtain an insoluble fraction.
  • the polyacrylamide gel after electrophoresis was transferred to a nitrocellulose membrane using iBlot (registered trademark) Dry Blotting System (Thermo Fisher Scientific Inc.) and iBlot (registered trademark) Gel Transfer Stacks Nitrocellulose, Mini (Thermo Fisher Scientific Inc.) .
  • iBlot registered trademark
  • Dry Blotting System Thermo Fisher Scientific Inc.
  • iBlot registered trademark
  • Gel Transfer Stacks Nitrocellulose, Mini Thermo Fisher Scientific Inc.
  • the plate was washed with PBS-T for 5 minutes.
  • 2,000-fold diluted anti-hemagglutinin, monoclonal antibody, peroxidase binding (Wako Pure Chemical Industries) or 5,000-fold diluted Anti-Flag, chicken-Poly, HRP (GeneTex, Inc.) were added and shaken for 30 minutes.
  • Emission-ELISA Zipbody-Luc was used as a primary antibody, and its luminescence was used as an index, and ELISA was performed without using a secondary antibody.
  • 96-well nunc white immunoplate maxi soap (Thermo scientific, Inc.) was coated with antigen, blocked, washed wells, and each sample was added. After further washing the wells, 20 ⁇ L of an equal volume of PBS and ONE-Glo TM Luciferase Assay System was added. Luminescence was measured per second with a microplate reader.
  • Fluorescence-ELISA Zipbody-GFP was used as a primary antibody, and its luminescence was used as an index, and ELISA was performed without using a secondary antibody.
  • 96-well nunc black immunoplate maxisorp (Thermo Fisher Scientific Inc.) was coated with antigen, blocked, washed wells, and each sample was added. After further washing the wells, 20 ⁇ L of PBS was added. The fluorescence per 20 ⁇ s at 530 nm with respect to the excitation light at 488 nm was measured with a microplate reader.
  • the insoluble fraction was dissolved in 100 mM Tris HCl (pH 7.5) containing 1 mL of 6 M GuHCl and 10 mM mercaptoethanol, and incubated overnight at 4 ° C. Subsequently, the mixture was transferred to a dialysis tube and dialyzed for 12 hours using 100 mM Tris HCl (pH 7.5) containing 6M GuHCl as a dialysis buffer. Thereafter, every 12 hours, the dialysis buffer was replaced with 100 mM Tris HCl (pH 7.5) containing GuHCl at concentrations of 3, 2, 1, 0.5, and 0 M (pH 7.5) only (1M dialysis contains 375 ⁇ M ⁇ ⁇ GSSG). The supernatant was collected at 14,000 ⁇ g for 5 minutes, and the retention of activity was confirmed by fluorescence ELISA.
  • the refolded Zipbodyzyme was purified using Ni-sepharose 6 Fast Flow (GE Health care) according to the manual. Subsequently, the antibody titer of m6Fab LZ Hc-GFP after purification was examined by BLitz using an Aminopropylsilane sensor.
  • the program is as follows: Initial Baseline 30 seconds, Loading 120 seconds, Baseline 30 seconds, Association 120 seconds, Dissociation 120 seconds.
  • E. coli BL21 (DE3) pLysS In particular, high expression was observed in both fractions in E. coli SHuffle (registered trademark) T7 express. On the other hand, expression was not confirmed from E. coli Rosetta-gami TM 2 (DE3).
  • E. coli SHuffle (registered trademark) T7 express was used, a signal 18.4 times as high as that of the clone into which pET22b, which was a negative control, was introduced.
  • E. coli BL21 (DE3) pLysS and E. coli Rosetta-gami TM 2 (DE3) were used, the activity was only as high as that of the clone into which the negative control pET22b was introduced. From this result, E. coli SHuffle (registered trademark) T7 express was used for the subsequent experiments.
  • E. coli SHuffle (registered trademark) T7 express E. coli BL21 (DE3) pLysS, E. coli Rosetta-gami TM 2 (DE3) pLysS. The productivity was compared (Fig. 4).
  • E. coli SHuffle (registered trademark) T7 express is characterized in that the chaperone DsbC that optimizes the disulfide bond originally present in the periplasm is highly expressed in the cytoplasm and the cytoplasm is in a reduced state.
  • BL21 (DE3) pLysS holds a plasmid pLysS encoding a T7 lysozyme gene that inhibits transcription of T7 RNA polymerase, and is a strain capable of strict expression control by IPTG.
  • Rosetta-gami TM 2 (DE3) pLysS can enhance disulfide bond formation due to the cytoplasm being oxidized by two types of reductase mutations.
  • E. coli has a tRNA corresponding to a codon having a small abundance and is a host capable of expressing a protein regardless of species differences.
  • E. coli SHuffle (registered trademark) T7 express had the highest expression level and the antigen affinity of the soluble fraction was high.
  • E. coli Rosetta-gami TM 2 (DE3) was also oxidative in the cytoplasm and suitable for disulfide bond formation, but both the expression level and ELISA signal were low. From this result, it is considered that DsbC expressed in the cytoplasm of E. coli SHuffle (registered trademark) T7 express is important for correct folding of m6Fab LZ.
  • E. coli SHuffle (registered trademark) T7 express has been reported by Robinson et al.
  • m6Fab LZ Hc-Luc in which Luc was genetically fused to the Hc of Zipbody m6Fab ⁇ ⁇ LZ, was expressed in the cytoplasm of E. coli SHuffle (registered trademark) T7 express.
  • E. coli SHuffle registered trademark
  • m6Fab Hc-Luc in which Luc was fused to Hc of m6Fab without LZ was also compared.
  • the expression of each fusion protein was confirmed by Western blotting and retained almost the same Luc activity and antigen affinity (FIGS. 5 to 7).
  • m6Fab LZ Hc-Luc showed a signal about 15 times higher than m6Fab Hc-Luc (FIG. 8). From this result, it was found that a complex in which an enzyme is fused to a Zipbody having LZ can be detected by highly sensitive antigen detection using the Luc moiety. The main factor is that the fusion protein is stabilized by the presence of LZ.
  • m6Fab LZ Lc-Luc fused to Lc and m6Fab LZ W-Luc fused to both Hc and Lc were examined in order to optimize the fusion site of Luc to Zipbody.
  • m6Fab LZ Lc-Luc and m6Fab LZ W-Luc were not detected in any of the Lc bands fused to Luc, and m6Fab LZ W-Luc had a band corresponding to the Hc-Luc fusion. Only a small amount was detected (FIG. 5). From this, it was found that the expression level was decreased by fusion of Luc to Lc.
  • Fab antibodies have been reported to produce fusion proteins with alkaline phosphatase (Weiss E, Orfanoudakis G. 1994. Application of an alkaline-phosphatase fusion protein system suitable for efficient screening and production of fab-enzyme conjugates in Escherichia coli. Journal of Biotechnology 33 (1): 43-53 .; Carrier A, Ducancel F, Settiawan NB, Cattolico L, Maillere B, Lreonetti M, Drevet P, Menez A, Boulain Jcomant conjugates for diagnosis of human iggs-application to anti-hbsag detection.
  • R4Fab ⁇ LZ Hc-Luc in which Luc was added to the Hc of r4Fab LZ, a rabbit-derived anti-L. Monocytogenes antibody, was expressed in order to investigate whether a fusion protein with Luc could be produced using a Zipbody other than m6Fab LZ. According to prior studies by the inventors, it has been known that r4Fab LZ requires purification in order to perform ELISA. Also in this study, a significant signal could not be detected by ELISA before the soluble fraction before purification, but a significant signal could be detected by luminescence ELISA using the same fraction (FIGS. 9 and 10).
  • a luminescence ELISA can be performed even with a Zipbdy-Luc fusion produced from a rabbit-derived Zibpdoy. Moreover, it means that the sensitivity of the luminescence ELISA is higher than that of a general ELISA using a secondary antibody. Because of the successful production of Zipbody-Luc that retains activity from both mouse-derived antibody m6Fab LZ and rabbit-derived antibody r4Fab LZ, Luc can be fused universally to Zipbody derived from various species. The possibility is suggested.
  • m6Fab ⁇ LZ ⁇ Hc-GFP and r4Fab LZ Hc-GFP were prepared by fusing GFP with mouse and rabbit-derived Zipbody Hc.
  • fluorescent ELISA was performed using the soluble fraction, a significant signal was obtained with m6Fab LZ Hc-GFP (FIG. 11).
  • no significant signal was obtained with r4Fab LZ Hc-GFP.
  • Western blotting analysis shows the expression of r4Fab LZ Hc-GFP in Hc-LZ-GFP and Lc-LZ (Fig. 5). Consideration is necessary.
  • GFP-ELISA Unlike Luc, HRP, and alkaline phosphatase, GFP-ELISA has the merit that it can be detected without the need for a substrate, and an assay at a low running cost is expected. Since GFP and Zipbody were successfully genetically fused not only with Luc, it is considered that Zipbody can be genetically fused with various enzymes and expressed in E. coli.
  • insoluble m6Fab LZ Hc-GFP expressed at 37 ° C was refolded to successfully prepare a fusion that retained both antibody and GFP activity.
  • the antibody titer of this molecule was equivalent to that without GFP fusion.
  • fusion proteins can be produced in the future by combining Zipbody derived from various biological species (human, goat, rat, etc.) and various types of enzymes (horseradish peroxidase, alkaline phosphatase, etc.). It is done.
  • Plasmid 1 the amino acid Ser-Lys-Ile immediately after the start codon of heavy chain and light chain of pET22 m6Fab LZ (plasmid 1) in which the gene of m6Fab LZ was introduced into the NdeI site of the expression vector pET22b PET22 SKIKm6Fab LZ (plasmid 2) into which the DNA sequence 5′-TCTAAAATAAAA-3 ′ (SEQ ID NO: 18) encoding -Lys (SKIK) was inserted was prepared.
  • AP alkaline phosphatase
  • Plasmid 3 includes a heavy chain-LZA-AP-His tag (amino acid sequence shown in SEQ ID NO: 35) and a light chain-LZB-FLAG tag (amino acid sequence shown in SEQ ID NO: 36) between the T7 promoter and T7 terminator. These genes are located in series so that both polypeptide chains can be expressed.
  • the sequence of the gene coding region of pET22b SKIK m6FabLZ-AP (plasmid 3) is shown in SEQ ID NO: 37.
  • Ni-NTA affinity chromatography (Wako Pure Chemicals, binding buffer: 50 mM TrisHCl (pH 8.0), 500 mM NaCl, 20 mM Imidazole, washing solution: 50 mM TrisHCl (pH 8.0), 500 mM NaCl, 50 mM Imidazole, eluent: 50
  • the collected fraction was applied to mM TrisHCl (pH 8.0), 500 mM NaCl, 500 mM Imidazole), and the collected fraction was concentrated by ultrafiltration Amicon centrifugal filter (10 KDa cut-off) to obtain 1.2 liter of a solution having a protein concentration of 0.13 mg / mL.
  • the AP activity measurement method is as follows. A substrate solution containing paranitrophenyl phosphate (pNPP) (0.1 M Tris-HCl (pH 8.0), 5 mM pNPP) was dispensed in 500 ⁇ L aliquots, 5 ⁇ L of enzyme solution was added thereto, and incubated at 37 ° C. for 30 minutes. . After 50 ⁇ L of 2 M NaOH was added to stop the reaction, absorbance at 410 nm was measured. An enzyme solution containing 1% BSA was used as a negative control.
  • pNPP paranitrophenyl phosphate
  • AP-ELISA is the same method as the light-emitting ELISA except that a transparent Maxisorp plate is used and color development is used as an index. Specifically, after washing the Zipbodyzyme solution, 350 ⁇ L of a substrate solution containing pNPP was added and reacted at 37 ° C. for 30 minutes. After 35 ⁇ L of 2M NaOH was added to stop the reaction, the absorbance at 410 nm was measured.
  • FIGS. 16B and 16C The results of normal ELISA and AP-ELISA are shown in FIGS. 16B and 16C, respectively.
  • a specific ELISA signal for the antigen E. coli O157 was detected at a sample concentration of 320 ⁇ ng / mL or more and in AP-ELISA at a concentration of 32000 ng / mL.
  • the antibody of the present invention has a stable structure and exhibits high affinity for an antigen. Moreover, the fused labeled protein (for example, an enzyme) exhibits a sufficient function.
  • the antibody of the present invention having such excellent characteristics includes various detection methods represented by ELISA, staining of cells or tissues, use for in vivo imaging, and further use / application for diagnosis and treatment of diseases. There is expected.
  • SEQ ID NO: 1 Description of artificial sequence: pET22 m6Fab LZ Hc-Luc SEQ ID NO: 2 Description of artificial sequence: pET22 r4Fab LZ Hc-Luc SEQ ID NO: 3 Description of artificial sequence: pET22 m6Fab LZ Hc-GFP SEQ ID NO: 4: Description of artificial sequence: pET22 r4Fab LZ Hc-GFP Sequence number 5: Description of artificial sequence: LZA Sequence number 6: Description of artificial sequence: LZB SEQ ID NO: 7: description of artificial sequence: linker SEQ ID NO: 8-24: description of artificial sequence: tag sequence SEQ ID NO: 25-34: description of artificial sequence: primer SEQ ID NO: 35: description of artificial sequence: heavy chain-LZA-AP -His tag SEQ ID NO: 36: Description of artificial sequence: light chain-LZB-FLAG tag SEQ ID NO: 37: Description of artificial sequence: gene coding region of pET22b SKIK m6FabLZ-AP

Abstract

Le problème à la base de la présente invention est de fournir un nouvel anticorps qui présente d'excellentes propriétés inhérentes aux anticorps et qui permet à une protéine de marquage fusionnée avec ceux-ci de fonctionner suffisamment. L'invention concerne un anticorps Fab fusionné avec une protéine de marquage, l'une d'une paire de peptides qui constituent une glissière à leucine étant ajoutée à l'extrémité C de la chaîne H et l'autre de ladite paire étant ajoutée à l'extrémité C de la chaîne L et la protéine de marquage étant fixée à la chaîne H et/ou à la chaîne L par l'intermédiaire du/des peptide(s).
PCT/JP2017/027791 2016-08-03 2017-08-01 Anticorps fusionné avec une protéine de marquage WO2018025826A1 (fr)

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Citations (4)

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US20090035821A1 (en) * 2005-08-30 2009-02-05 Novo Nordisk A/S Expression of Proteins in E.Coli
JP2010519894A (ja) * 2007-03-01 2010-06-10 ノボ・ノルデイスク・エー／エス 大腸菌におけるタンパク質の発現
JP2016002009A (ja) * 2014-06-13 2016-01-12 国立大学法人名古屋大学 タグ付抗体
WO2016204198A1 (fr) * 2015-06-16 2016-12-22 国立大学法人名古屋大学 Procédé d'expression de protéine

Patent Citations (4)

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US20090035821A1 (en) * 2005-08-30 2009-02-05 Novo Nordisk A/S Expression of Proteins in E.Coli
JP2010519894A (ja) * 2007-03-01 2010-06-10 ノボ・ノルデイスク・エー／エス 大腸菌におけるタンパク質の発現
JP2016002009A (ja) * 2014-06-13 2016-01-12 国立大学法人名古屋大学 タグ付抗体
WO2016204198A1 (fr) * 2015-06-16 2016-12-22 国立大学法人名古屋大学 Procédé d'expression de protéine

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HIDEO NAKANO ET AL.,: "Zipbody: Leucine Zipper o Mochiita Fab Kotai to Sono Riyo", DAI 5 KAI NEO BIO BUNSHI KENKYUKAI YOSHISHU, 10 June 2016 (2016-06-10), Retrieved from the Internet <URL:https://www.sci.kagoshima-u.ac.jp/yito/Neobio/NBKagoshima_201606.pdf> [retrieved on 20170831] *
OJIMA-KATO TERUYO. ET AL.: "`Zipbody' leucine zipper-fused Fab in E.coli in vitro and in vivo expression systems", PROTEIN ENGINEERING, DESIGN AND SELECTION, vol. 29, 21 February 2016 (2016-02-21), pages 149 - 157, XP055604740 *

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