JP2008228701A - Recombinant protein immunotetanus toxin - Google Patents

Abstract

The present invention provides a novel immunotoxin cell targeting method capable of temporarily suppressing the function of a target cell and means therefor.
A variable region polypeptide of an antibody that selectively recognizes human interleukin-2 receptor α chain (IL-2Rα) in order from the N-terminal side, green-concentrated toxin PII domain polypeptide, tetanus toxin L A recombinant protein comprising a chain polypeptide, a tag and a KDEL sequence in this order, and the recombinant protein contacted with a target neuron expressing a human interleukin-2 receptor α chain, the variable region polypeptide and The recombinant protein is incorporated into the target nerve cell through specific binding with human interleukin-2 receptor α chain, and VAMP− in the target nerve cell is activated by the action of the tetanus toxin L chain polypeptide. 2 or a method of transiently inhibiting neurotransmitter release in the target nerve cell.
[Selection] Figure 1

Description

The present invention relates to a recombinant protein immunotetanus toxin, a method using the recombinant protein, and a method for temporarily suppressing a specific function of the cell without destroying a target nerve cell.

Various brain functions are based on the mechanisms of information processing and regulation in complex neural circuits. In order to understand the neural mechanisms that mediate brain functions, it is essential to elucidate the behavioral physiological roles of specific neurons that make up the neural circuit. It is well known that certain neurological diseases are associated with abnormal activity in specific brain regions. The development of technology that suppresses the function of specific neuron types provides an important approach for elucidating the mechanisms of brain function and developing therapeutic methods for neurological diseases.

The inventor's research group has previously developed a gene modification technique (immunotoxin cell targeting method) that induces the removal of specific neurons based on the specificity of gene expression (Patent Document 1, Non-Patent Document 1). . In this technique, a transgenic mouse expressing a human interleukin-2 receptor α subunit (IL-2Rα) is produced using a promoter specific to the cell type, and a recombinant immunotoxin (human IL) is expressed in this mouse. -2Rα monoclonal antibody variable site and Pseudomonas aeruginosa toxin fusion protein) to induce removal of the desired cell type. This method was applied to research to selectively remove specific neurons constituting the cranial nerve circuit and clarify their behavioral physiological roles (Non-patent Documents 2 and 3).
JP-A-8-322427 Kobayashi et al., Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 1132-1136, 1995 Sano et al., J. Neurosci., Vol.23, pp. 9078-9088, 2003 Yasoshima et al., J. Neurosci., Vol. 25, pp. 7743-7753, 2005

  However, in conventional immunotoxin cell targeting methods, the target cell is completely destroyed or damaged, and then the neural circuit mechanism is elucidated by restoring the function involved in the target cell as necessary. Was impossible. Therefore, development of an immunotoxin cell targeting method that is effective for such elucidation and that can temporarily suppress the function of the target cell is desired. Accordingly, an object of the present invention is to provide means for solving this problem.

That is, the present invention relates to the following aspects.
[1] A recombinant protein (immunotetanus toxin) comprising a variable region polypeptide of an antibody that selectively recognizes human interleukin-2 receptor α chain (IL-2Rα) and a tetanus toxin L chain polypeptide.
[2] A nucleic acid molecule encoding the recombinant protein of the present invention.
[3] An expression vector containing the nucleic acid molecule of the present invention.
[4] A method for producing the recombinant protein of the present invention.
[5] A method for cleaving VAMP-2 in a target nerve cell by the action of a tetanus toxin L chain polypeptide of the recombinant protein of the present invention.
[6] A method for transiently inhibiting neurotransmitter release in target neurons by the action of the recombinant protein tetanus toxin L chain polypeptide of the present invention.
[7] A method for temporarily suppressing a specific function of a target nerve cell without inducing damage to the target nerve cell by the method of the present invention.
[8] A method of inducing a transient behavioral disorder in an animal by the method of the present invention.

The recombinant protein of the present invention, immunotetanus toxin, acts on a specific cell type that expresses human IL-2Rα and transiently suppresses the function of the cell without inducing damage to the target cell type. be able to.

Hereinafter, embodiments for carrying out the present invention will be described in detail.

  The recombinant protein of the present invention selectively (specifically) a human interleukin-2 receptor α chain (IL-2Rα) that is specifically expressed in a target cell (eg, displayed on the cell surface). Immunotetanus toxin (hereinafter also abbreviated as “ITet”), which is a derivative of “immunotoxin”, comprising as an element a variable region polypeptide of an antibody that recognizes tetanus toxin and a light chain polypeptide of tetanus toxin (tetanus toxin) It is.

Tetanus toxin L chain is a protease that cleaves vesicle-associated membrane protein-2 (VAMP-2), which plays an essential role in neurotransmitter release. The amino acid sequence of the tetanus toxin L chain is described in, for example, Eisel et al., EMBO J., Vol. 12, pp. 3365-3372, 1993 (GenBank accession number L19522).

  Here, the variable region polypeptide may be derived from any antibody that selectively recognizes IL-2Rα, and preferred examples thereof include an anti-Tac antibody (Uchiyama, T) which is a monoclonal antibody against human IL-2Rα. , et al., J Immunol (1981) 126: 1393-1397). More specifically, a single chain Fv fragment in which the variable regions of the anti-Tac antibody H chain and L chain are linked via a peptide linker can be exemplified (Chaudhary, VK, et al., Nature (1989). 339: 394-397; Batra, JK, et al., J Biol Chem (1990) 265: 15198-15202).

Furthermore, it is preferable to have a polypeptide involved in transport from the endosome to the cytoplasm, for example, a green-condensed toxin PII domain polypeptide, between the variable region polypeptide and the tetanus toxin L chain polypeptide. Therefore, as a preferred example of the recombinant protein of the present invention, in order from the N-terminal side, an antibody variable region polypeptide that selectively recognizes human IL-2Rα, green-concentrated toxin PII domain polypeptide, tetanus toxin L chain Mention may be made of recombinant proteins comprising a polypeptide, a tag tag sequence and a KDEL sequence.
The amino acid sequence of the toxin PII domain polypeptide and the base sequence encoding it are described in Gray et al., Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2645-2649 (1984).

  Here, examples of the label tag sequence include, for example, polyhistidine sequences, MBP fusion proteins, thioredoxin fusion proteins, and FLAG fusion proteins (FLAG sequences) known to those skilled in the art. When these labeling tags are included, the recombinant protein of the present invention can be easily purified using various affinity chromatography. The KDEL sequence is a sequence that specifically binds to the KDEL receptor present in the Golgi membrane.

  The expression vector of the present invention comprises a nucleic acid molecule (DNA sequence) encoding each of the above elements (polypeptides). For example, any method known to those skilled in the art as described in the above-mentioned cited documents. Can be prepared. In the expression vector, the nucleic acid molecule encoding the polypeptide is bound under the expression control of an expression regulatory sequence. The structure of an expression vector (pEX-ITet), which is a preferred specific example of the present invention, is shown in FIG.

  “Under expression control” means that a DNA encoding a predetermined amino acid sequence has the ability to express a protein having the amino acid sequence under predetermined conditions. When DNA encoding a predetermined amino acid sequence is bound under the expression control of an expression regulatory sequence, the DNA expresses a predetermined protein under predetermined conditions. Here, “expression control sequence” means a nucleic acid sequence that regulates the expression of other nucleic acid sequences, and also controls and regulates the transcription and preferably translation of other nucleic acid sequences. Expression control sequences include appropriate promoters, enhancers, transcription terminators, initiation codons (ie, ATG) in the gene encoding the protein, splicing signals for introns, polyadenylation sites, and stop codons.

  “Promoter” means a minimal sequence necessary for transcription. Promoters also include promoter elements that control the expression of genes in a cell-type-specific, tissue-specific manner, or by an external signal or regulator in a promoter-dependent manner. The promoter element is linked to either the 5 ′ region or the 3 ′ region of the expressed DNA. Promoters include both constitutive and inducible promoters. For example, when producing an expression vector in bacteria, promoters such as T7 RNA polymerase promoter, bacteriophage γ pL, lac, ptrp, ptac (ptrp-lac hybrid promoter) can be used as inducible promoters.

  With such an expression vector, for example, according to the method described in the above cited reference, a suitable host known to those skilled in the art such as E. coli is transformed, and the resulting transformant is cultured and its cell suspension The recombinant protein of the present invention can be purified from the liquid.

  “Transformation” means that by introducing new (foreign) DNA into a cell, the cell undergoes a permanent genetic change. If the cell is a mammalian cell, permanent genetic changes are achieved by introducing DNA into the cell's genome.

A “transformant” is a cell into which the cell, or an ancestor of the cell, has been introduced with a DNA (including a vector) encoding the recombinant protein of the present invention by recombinant DNA technology. It means that. Transformation of host cells with recombinant DNA can be performed by methods well known to those skilled in the art. When the host is a prokaryotic cell (for example, E. coli), competent cells having the ability to take up DNA can be prepared by treating with the calcium chloride method by procedures well known to those skilled in the art. Transformation can also be performed by alternative methods such as electroporation.

  The recombinant protein of the present invention can be obtained by expressing a nucleic acid encoding the protein in a prokaryotic cell-derived transformant. Examples of prokaryotic cells include microorganisms (for example, bacteria) transformed with a recombinant vector for expressing bacteriophage, plasmid DNA, and cosmid DNA containing the nucleic acid sequence encoding the protein of the present invention. Technical scope of the present invention Is not limited thereto. The recombinant protein of the present invention can be expressed by culturing Escherichia coli on a large scale in an in vitro test system.

  Regarding the technique of isolation or purification after the recombinant protein of the present invention is expressed, any means known to those skilled in the art, for example, various chromatography such as ion exchange, affinity and gel filtration, ammonium sulfate precipitation, and It can be carried out by an immunological separation method using an antigen or antibody (monoclonal antibody, polyclonal antibody).

When the recombinant protein of the present invention contacts a target neuron expressing the human interleukin-2 receptor α chain, the variable region polypeptide and the human interleukin-2 receptor α chain are mediated through specific binding. The recombinant protein is taken into the target nerve cell, and VAMP-2 (vesicle-associated membrane protein-2) is cleaved by the action of the tetanus toxin L chain polypeptide, resulting in the release of the neurotransmitter. Is inhibited (FIG. 2). Thereby, for example, specific functions of the cell such as nerve transmission can be suppressed. This suppression is transient and does not induce cell death of target neurons.

  In order to bring the recombinant protein of the present invention into contact with a target nerve cell expressing the human interleukin-2 receptor α chain, for example, an animal can apply the recombinant protein to a target cell or tissue by an appropriate method known to those skilled in the art. To administer. If the target nerve cell (tissue) is susceptible to (close to) blood flow, simple injection or other systemic administration is used. However, when nerve cells in the brain are target cells, the recombinant protein is preferably administered (injected) directly into an appropriate compartment (eg, striatum) in the cerebral ventricle. The recombinant protein of the present invention is preferably administered in the form of an appropriate preparation. Such a preparation suitably contains a solvent other than the recombinant protein of the present invention and various auxiliary components, taking into consideration the administration method and the properties of the protein itself, etc., and a liquid or the like using a method known in the art. It can be prepared in any form. For example, suitable preparation methods are described in Remington's Remington's Pharmaceutical Science latest edition, Mac Publishing Company, Easton, PA (Mack Publishing Company, Easton, PA).

The recombinant protein of the present invention is used in an appropriate amount so as to selectively and effectively transiently inhibit the release of neurotransmitters in the target nerve cell or tissue.

Examples of animals include transgenic animals in which human interleukin-2 receptor α chain is expressed under control of the expression of the dopamine D2 receptor gene promoter in target neurons such as striatal neurons (eg, mice and rats). Mammals including laboratory rodents, except for humans). An example of such a transgenic animal is a transgenic mouse produced by the present inventors (Non-patent Document 2).

  Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples. These examples show a part of the present invention, and the technical scope of the present invention is not limited by these examples. Unless otherwise specified, the experimental conditions in each operation were in accordance with standard methods in the technical field.

Example 1: Purification and activity of ITet A) Production and purification of ITet: pEX-ITet, which is an expression vector of the present invention, was obtained by using a genetic engineering technique well known to those skilled in the art (Chaudhary, VK, et al., Nature (1989) 339: 394-397; Batra, JK, et al., J Biol Chem (1990) 265: 15198-15202) described in anti-Tac (Fv) -PE40 (derived from anti-Tac antibody) Based on an expression vector encoding a single chain Fv fragment and a truncated protein of Pseudomonas exotoxin truncated PE40), a part of the sequence encoding the PE40 (catalytic site) It was prepared by replacing with a sequence encoding the tetanus toxin L chain polypeptide prepared based on the description.

The pEX-ITet thus prepared was transformed into E. coli, induced with IPTG, and a cell suspension was prepared. The soluble fraction was purified by combining ion exchange chromatography, affinity chromatography, and gel filtration chromatography until it showed a single band on SDS electrophoresis (FIG. 3A). In the figure, the peak fractions obtained from (1) cell suspension, (2) DEAE ion exchange chromatography, (3) FLAG affinity chromatography, and (4) Superdex G-200 gel filtration chromatography are subjected to SDS electrophoresis. Shows the result of analysis.

B) Binding activity to IL-2Rα: Purified human IL-2Rα (Nikaido et al. Nature Vol.311, pp.631-635 (1984)) was coated in 96 wells and incubated with various concentrations of purified ITet. . Furthermore, it was incubated with an antibody against the PII domain (Prior TI, FitzGerald DJ, Pastan I .: Cell 64, 1017-1023 (1991)) and an HRP-conjugated secondary antibody (Jackson laboratory). The bound protein was detected by chromogenic method and the absorbance at 450 nm was measured. As a result, it was revealed by the binding test by ELISA that purified ITet has binding activity to human IL-2Rα (FIG. 3B).

C) In vitro protease activity: Various amounts of ITet were added to synaptosomes, reacted for 3 hours, and a certain amount of protein was separated by SDS electrophoresis. The protein was transferred to a nitrocellulose membrane, and Western blotting was performed using an antibody against VAMP-2 (Wako Pure Chemical Industries) or an antibody against Syntaxin-1A (Wako Pure Chemical Industries). Bands were quantified and ratios to ITet-free samples were calculated and plotted (n = 6, * p <0.05, ** p <0.01). As a result, it was confirmed that ITet has a protease activity that cleaves VAMP-2 (FIG. 3C).

Example 2: Transient behavioral disorder caused by ITet injection ITet was injected into one side striatum (4 locations) of wild type and transgenic mice (described in Non-Patent Document 2) using a stereotaxic apparatus. Rotational motion was measured every other day to examine the effect on brain tissue. According to the results shown in FIG. 4, the ITet injection induced a rotational movement opposite to the treatment in the transgenic mice, and this rotational movement became prominent 4 days after the injection (n = 5-6 , * p <0.05). After that, the rotational movement was attenuated by 6 days after the injection and became the same level as the wild type. This result indicates that ITet acts on neurons expressing human IL-2Rα, and this cell function may be temporarily suppressed. As a result of Nistle staining and in situ hybridization, no damage to striatal neurons due to ITet injection was observed. In addition, the effects of mesencephalic dopamine cells entering the striatum on nerve endings and cell bodies were not observed. Therefore, it became clear that administration of ITet did not induce cell damage.

Example 3: Transient change of striatum VAMP-2 level by ITet injection ITet was injected into one side striatum (4 places) of wild type and transgenic mice with a brain stereotaxic apparatus. Before treatment (day 0), 4 days after treatment (day 4), and 10 days (day 10), the striatum on the untreated side (-) and treated side (+) is removed to prepare a tissue suspension. Then, Western blotting using an anti-VAMP-2 antibody or an anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) antibody (Chemicon) was performed. For each striatum, the ratio of the intensity of the VAMP-2 band to the intensity of the GAPDH band was determined. The relative values of the treated side relative to the ITet untreated side were calculated and compared for the two genotypes (n = 5-6, * p <0.05). According to the results (FIG. 5), a significant decrease in the VAMP-2 protein level was observed in the transgenic mouse 4 days after the injection, and the striatal VAMP-2 level in the transgenic mouse mouse was 10 days after the injection. It recovered to the same level as the mold. This result supported the possibility that ITet cleaves VAMP-2 and transiently suppresses specific cell functions even in vivo.

  In addition to the conventional immunotoxin cell targeting method, the present invention is an extremely effective technique for elucidating a neural circuit mechanism that mediates behavioral control. In the conventional immunotoxin cell targeting method, specific cells were removed, but in the immunotoxin cell targeting method of the present invention, the activity can be temporarily suppressed using the same mouse strain without destroying the cells. The present invention can be applied to various experimental animals including monkeys as well as rodents by devising gene transfer methods such as viral vector systems, and is a promising technology in the study of brain function and behavior. Is expected to be. Furthermore, it is known that excessive activity of specific nerve cells is related to the pathology in certain neurological diseases, and application of the present invention may lead to treatment of diseases.

  The contents described in the documents cited in the present specification constitute the disclosure of the present specification as a part of the present specification.

The structure of the expression vector (pEX-ITet) of the present invention is schematically shown. It has a sequence encoding the recombinant protein (ITet) of the present invention downstream of the promoter of T7 RNA polymerase. ITet consists of a human IL-2Rα monoclonal antibody variable site, Pseudomonas aeruginosa toxin PII domain, tetanus toxin L chain, FLAG sequence, and KDEL sequence. The mechanism of action of recombinant protein (ITet) is shown. ITet binds to neurons that express human IL-2Rα and is taken up via endocytosis. Processing is performed in the small bubbles, and the tetanus toxin L chain moves to the cytoplasm. Tetanus toxin light chain cleaves VAMP-2 and inhibits neurotransmitter release. A: A photograph of electrophoresis showing the results of analyzing the peak fractions of various chromatography by SDS electrophoresis. B: Result of binding test by ELISA showing that purified ITet has binding activity to human IL-2Rα. C: Western blot results showing that ITet has protease activity to cleave VAMP-2. Figure 3 shows transient behavioral impairment in mice injected with ITet. Figure 6 shows transient changes in striatal VAMP-2 levels in mice injected with ITet.

Claims (20)

A recombinant protein comprising a variable region polypeptide of an antibody that selectively recognizes human interleukin-2 receptor α chain (IL-2Rα) and a tetanus toxin L chain polypeptide. The recombinant protein according to claim 1, wherein the variable region polypeptide is a single-chain Fv fragment formed by connecting the variable regions of the anti-Tac antibody H chain and L chain via a peptide linker. The recombinant protein according to claim 1 or 2, wherein the recombinant protein has a green-condensed toxin PII domain polypeptide between the variable region polypeptide and the tetanus toxin light chain polypeptide. In order from the N-terminal side, a variable region polypeptide of an antibody that selectively recognizes human interleukin-2 receptor α chain (IL-2Rα), Phytotoxic toxin PII domain polypeptide, tetanus toxin L chain polypeptide, The recombinant protein according to any one of claims 1 to 3, comprising a labeling tag and a KDEL sequence. The recombinant protein according to claim 4, wherein the labeling tag is a FLAG sequence. A nucleic acid molecule encoding the recombinant protein according to any one of claims 1 to 5. An expression vector comprising the nucleic acid molecule according to claim 6. The expression vector according to claim 6, wherein the nucleic acid molecule according to claim 6 is bound under the expression control of an expression regulatory sequence. The expression vector according to claim 8, wherein the expression regulatory sequence is a T7 RNA polymerase promoter. The expression vector according to claim 9, which is pEX-ITet. The host is transformed with the expression vector according to any one of claims 7 to 9, the obtained transformant is cultured, and the cell suspension is used to produce the transformant according to any one of claims 1 to 4. A method for producing the recombinant protein comprising purifying the recombinant protein. The production method according to claim 11, wherein the host is Escherichia coli. The recombinant protein according to any one of claims 1 to 5 is contacted with a target neuron expressing a human interleukin-2 receptor α chain, and the variable region polypeptide and human interleukin-2 receptor α are contacted. A method of cleaving VAMP-2 in a target nerve cell by the action of the tetanus toxin L chain polypeptide by incorporating the recombinant protein into the target nerve cell through specific binding to a chain. Furthermore, the method of transiently inhibiting the release | release of the neurotransmitter in this target nerve cell by the method of Claim 13. A method for temporarily suppressing a specific function of a target nerve cell without inducing damage to the target nerve cell by the method according to claim 13. A recombinant protein according to any one of claims 1 to 5 is administered into an animal body, thereby bringing the recombinant protein into contact with a target nerve cell expressing a human interleukin-2 receptor α chain. Item 16. The method according to any one of Items 13 to 15. The method according to claim 16, wherein the animal is a transgenic animal in which the human interleukin-2 receptor α chain is expressed under the expression control of the dopamine D2 receptor gene promoter in the target nerve cell. The method according to claim 16 or 17, wherein the target nerve cell is a striatal neuron. The method according to any one of claims 16 to 18, wherein the recombinant protein according to any one of claims 1 to 5 is administered into an animal body by injecting the unilateral striatum of the animal. A method for inducing a transient behavioral disorder in an animal by the method according to any one of claims 16-19.