WO1999033968A1 - Process for producing antibody catalyst and method for utilizing the antibody catalyst thus obtained - Google Patents

Abstract

A process for producing an antibody catalyst capable of cleaving and/or decomposing a target protein or peptide. This process comprises constructing a monoclonal antibody by using as an antigen the target protein or peptide or a peptide fragment constituting the same, and then recovering from the thus constructed monoclonal antibody the antibody catalyst capable of cleaving and/or decomposing the target protein or peptide. To recover the antibody catalyst, it is preferable to separate the monoclonal antibody into its light chain and heavy chain. For example, the light chain of a monoclonal antibody constructed by using a peptide YP41-1 present on the constant region of an HIV surface protein gp41 has a capability of cleaving the peptide YP41-1 and the protein gp41.

Description

 ,

Method for producing antibody catalyst and method for using antibody catalyst obtained by the method

Technical field

 The present invention relates to a method for producing an antibody catalyst having an ability to cleave and / or degrade a target protein or peptide. The antibody catalyst obtained by the present invention, for example, cleaves the constant region of HIV (human immunodeficiency virus).

And or have the ability to decompose. In addition, the present invention

Antibody catalysts can also be expected to be used as other antiviral agents, anticancer agents, antithrombotic agents, etc. Furthermore, the present invention relates to a method for using the antibody catalyst obtained by the above production method.

Background art

 Conventionally, various reports have been made on antibodies having catalytic activity (antibody catalysts). For example, in 1989, S. Pau1 et al. Confirmed the presence of autoantibodies that decompose VIP (Vasoactive Intestinal Peptide) in patients with autoimmune diseases, and their catalytic activity was particularly low. It is reported to be on the chain. In 1992, G. Gabib • V et al. Confirmed that autoantibodies that degrade DNA were also present in patients with autoimmune diseases.

HIV, which causes AIDS (acquired immunodeficiency syndrome), is known to be severely mutated, but mutates, for example, the surface protein gp41 that passes through the membrane (envelope). There is an area (storage area) that is highly saved without being saved. Therefore, it is necessary to obtain an antibody catalyst that cleaves and / or degrades the protein or peptide constituting the conserved region. If this can be achieved, there is a possibility that the function of HIV can be suppressed by the antibody catalyst, and it can be expected to contribute to the treatment of AIDS.

 The present invention has been made in view of the above circumstances, and is intended to provide a protein or peptide which is desired to be cleaved and / or degraded, for example, a protein or peptide constituting a conserved region of HIV, or a core protein p24, and vice versa. An object of the present invention is to provide a method for producing an antibody catalyst capable of producing an antibody catalyst having the ability to cleave, Z or degrade proteins or peptides constituting a transcriptase or the like. Disclosure of the invention

 The present invention provides a method for producing an antibody catalyst which cleaves and / or degrades a protein or peptide of interest in order to achieve the above-mentioned object, comprising the protein or peptide of interest or the protein or peptide. An antibody characterized in that after preparing a monoclonal antibody using a partial peptide as an antigen, an antibody catalyst capable of cleaving and Z or degrading the target protein or peptide is collected from the prepared monoclonal antibody. A method for producing a catalyst is provided. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing an example of a time-dependent change of GP41-1 peptide when an anti-gp41 antibody light chain is reacted with GP41-1 peptide. FIG. 2 is a graph showing an example of the time-dependent change of the YP41-1 peptide when the anti-gp41 antibody light chain is reacted with the YP41_1 peptide. FIG. 3 is an SDS_PAGE photograph showing an example of degradation of gp41 protein when an anti-gp41 antibody light chain is reacted with gp41 protein. Figure 4 shows that the heavy chain of the anti-gp41 antibody reacted with YP41-1 peptide. FIG. 6 is a graph showing an example of a change with time of YP 4 1-1 peptide at the time. FIG. FIG. 5 is a graph showing an example of a time-dependent change of YP41-1 peptide when an anti-gp41 antibody heavy chain is reacted with YP41-1 peptide. FIG. 6 is a graph showing the relationship between the concentration of YP41-1 peptide and the initial velocity in the reaction of Experiment 10.

 FIG. 7 is a graph showing a Hanes-Woof1f plot in the reaction of Experiment 10.

 FIG. 8 is a photograph showing the result of the immunoplotting in Experiment 11.

 FIGS. 9A and 9B are photographs showing the results of SDS-PAGE in Experiment 12 in which FIGS.

 FIG. 10 is a graph showing the relationship between the peak area of YP41-1 peptide and the reaction time in the reaction of Experiment 13.

 FIG. 11 shows the amino acid sequence of the antibody light chain gene variable region used in the present invention.

 FIG. 12 is a photograph showing the results of SDS-PAGE in Experiment 16.

 FIG. 13 shows the amino acid sequence of the variable region of the anti-p24 monoclonal antibody light chain gene used in the present invention.

 FIG. 14 is a graph showing an example of a time-dependent change in RT peptide when a heavy or light chain of an anti-RT antibody is reacted with an RT peptide.

 FIG. 15 is a photograph showing the results of SDS-PAGE in Experiment 19.

 FIG. 16 is a photograph showing the results of SDS-PAGE in Experiment 19.

Figure 17 shows that anti-gp41 antibody light chain and anti-gp41 antibody heavy chain are mixed simultaneously. 4 is a graph showing an example of the temporal change of YP41-1 peptide reacted with YP41-1 peptide. BEST MODE FOR CARRYING OUT THE INVENTION

 In the present invention, first, a monoclonal antibody is prepared using a target protein or peptide or a partial peptide constituting the protein or peptide as an antigen.

 In this case, the antigen for preparing the monoclonal antibody is selected according to the protein or peptide to be cleaved and Z or degraded. For example, when the purpose is to cleave and Z or degrade proteins or peptides existing in the HIV conserved region, as an antigen for preparing a monoclonal antibody,

A peptide (GP41-1) having an amino acid sequence of the following formula (1), which is present in the constant region of the surface protein gP41 of HIV, may be used.

 R G P D R P E G I E E E GGE RD RD "-(1)

 In the present invention, the method for producing the monoclonal antibody is not limited. For example, the monoclonal antibody can be produced by any known method such as production using a hybridoma or production using a genetic engineering technique.

 Next, in the present invention, an antibody catalyst having the ability to cleave and / or degrade the target protein or peptide is collected from the prepared monoclonal antibody.

In this case, it is particularly preferable to collect the antibody catalyst having the ability to cleave and / or degrade the target protein or peptide by separating the monoclonal antibody into a light chain and a heavy chain. Then, whether or not the light and heavy chains have the above-mentioned ability is assayed by an appropriate method such as an immunoblotting method, an antigen-antibody reaction and a Z or antigen degradation reaction, and one of the light and heavy chains is tested. When only the light chain or heavy chain has the above ability, the light chain or the heavy chain is used as the antibody catalyst, and when both the light chain and the heavy chain have the above ability, the light chain alone or the heavy chain alone constitute the antibody catalyst, The antibody catalyst may be constituted by mixing the light chain and the heavy chain. Preferred methods for using the antibody catalyst include, for example, the following methods.

 (1) When cleaving and / or decomposing a target protein or peptide, one of an antibody heavy chain and an antibody light chain having the ability to cleave and / or decompose a target protein or peptide is used alone. A method for using an antibody catalyst, characterized in that:

 (2) When cleaving and / or degrading the target protein or peptide, simultaneously mix both the antibody heavy chain and the antibody light chain capable of cleaving and / or degrading the target protein or peptide. A method for using an antibody catalyst, characterized in that it is used.

 (3) The antibody heavy chain and Z or a partial region of the antibody light chain are used as the antibody heavy chain and antibody light chain having the ability to cleave and Z or degrade the target protein or peptide (1) or (2) Use of the antibody catalyst.

 (4) Use of antibody heavy chains and antibody light chains that have the ability to cleave and / or degrade the target protein or peptide produced by genetic manipulation. Use of the antibody catalyst of (1) to (3) Method. Example

 Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to the following examples.

 (Experiment 1) Preparation of anti-gp41 antibody

A monoclonal antibody (anti-gP41 antibody) was prepared as follows using the 19-mer GP41-1 peptide shown in the above formula (1) as an antigen. GP41-1 peptide was synthesized as a C-terminal Cys-added product by the F-moc method using an automatic peptide synthesizer (431A manufactured by Applied Biosystems, USA). Next, the obtained GP41-1-peptide was used as a binder with N- (ε-maleimidocaproxy) succinic acid imide as a binding agent, and as an immunogen for Jinkasa momosyanin and an enzyme immunoassay (ELIS II). ) Was conjugated to bovine serum albumin (BSA). BalbZc mice were immunized with the purified conjugate. The first and second immunizations were performed using a complete adjuvant of the oral int at 2 week intervals. A third immunization was performed using incomplete Freund's adjuvant two weeks after the second immunization. The last booth, Yuichi, was performed two weeks after the third immunization. Three days after the last booster, cell fusion of bal bZc mouse spleen cells and their myeloma cells (SP2Z0-Agl4) was performed, followed by HAT selection and screening. After two rounds of cloning, finally, hybridomas secreting the monoclonal antibodies were selected by competitive ELISA (19mer GP41-1 peptide bound to BSA was coated on an Imno plate). Its class and subclass were IgG2b: κ.

 (Experiment 2) Purification of anti-gP41 antibody using Affigel-protein A-MAPS-II kit

To obtain a higher purity anti-gP41 antibody, a BIO-RAD Affigel-Protein A-MAPS-II kit was used. Anti-gp41 antibody ascites was obtained by administering 1 × 10 ⁶ hybridomas per mouse. The binding buffer was obtained by dissolving 31.4 g of the binding buffer powder in 100 ml of distilled water (314 mg Zm 1). The elution buffer was obtained by dissolving 22 g of elution buffer powder in 10 Om 1 of distilled water (22 mg / m 1). As a neutralizing solution, a 2 M Tris-HC1 solution was used. (pH 8.7, 18.5 ° C). The sample was prepared by mixing ascites fluid and binding buffer in a ratio of 1: 1.5 and filtering through filter paper. The binding buffer, elution buffer and samples were degassed before purification. The column was washed with binding buffer and packed with Affi-Gel-Protein A with Pasteur Pit. The gel was washed with binding buffer until the baseline was settled, monitoring at 280 nm. The flow rate was adjusted to 0.2 ml Zmin, and the sample was supplied when the surface of the gel almost coincided with the level of the binding buffer. Next, washing was performed until the peak of the contaminants that had not been adsorbed on the gel was settled in the binding buffer. The flow rate was again adjusted to 0.2 ml Zmin, and after the gel surface almost coincided with the liquid level of the binding buffer, elution was carried out with an elution buffer, and fractionation was performed while looking at the monitor. At this time. Since the elution buffer contained antibodies, the eluate was immediately neutralized with a neutralizing solution. Gels, the 0.0 5% N a N ₃ stored at 4 in PBS. The neutralized eluate was dialyzed against PBS for 2 days, and the purity of the antibody was confirmed by SDS-PAGE (12% separation gel, 3% concentrated gel, Coomassie staining). The protein concentration was determined using the standard assay (BIO-RAD).

 (Experiment 3) Collection of anti-gp41 antibody light chain by open column method

7 mg 1 of anti-gp41 antibody solution purified by Affigel-Protein A—MAPS—II kit was concentrated to 2 ml by ultrafiltration, and 2 mM—EDTA, 0.5 MT ris-HC1 solution After one run for (pH 8.0), the solution was adjusted to 2.5 ml. The packing materials (Sephadex-G25 Fine and G-100) were swollen with 1 M propionic acid, packed into biocolumns (BIO-RAD), and the gel was stabilized with 1 M propionic acid. A column was made. Add 0.175 M—DTT to the sample to reduce disulfide bonds and cover with parafilm. ₀

After that, two holes were made with a Pasteur pit, N ₂ was added from one of the holes, the container was sealed again with parafilm, and the mixture was reacted at room temperature with stirring for 1 hour. Thereafter, 275 1 of 0.21 M monoacetic acid was added, and the resulting SH group was alkylated. Next, 20 zl of 0.1 M-DTT was added, and the mixture was stirred and reacted at room temperature for 15 minutes. To remove excess reagent, perform gel filtration on Sephadex-G25 Fine prep column equilibrated with 1 M propionic acid, and monitor at UV280 nm to collect the first peak. did. The first peak was concentrated by ultrafiltration to 2 ml and gel filtration was performed on a Sephadex-G100 column equilibrated with 1 M p-pionic acid to separate heavy and light chains. Fractions corresponding to the heavy and light chains were collected and dialyzed 4 to 5 times against PBS. Finally, the purity of the antibody fragment was confirmed by SDS-PAGE (12% separation gel, 3% concentrated gel, Coomassie staining), and the protein concentration was determined by DC Protein Standard Atssay (BIO-RAD).

 (Experiment 4) Collection of anti-gp41 antibody light and heavy chains by liquid chromatography

2.7 ml (5 mg) of the anti-gp41 antibody solution purified with the Affigel-Protein A—MAP S—II kit was concentrated to 0.5 ml by ultrafiltration and degassed. ris-HC0.15 M—NaCl buffer (pH 8.0) 5 ml was added, and the mixture was concentrated by ultrafiltration to 1 ml. _c . The above degassed buffer was added to 5 ml 1 In addition, the mixture was concentrated by ultrafiltration to 1 ml and made up to 2.7 ml with the above degassed buffer. To this was added 0.3 ml of 2 M / 3-mercaptoethanol, and the pH was adjusted to 8.0. Then, the vessel was sealed with N ₂ and reacted at 15 ° C. with stirring for 3 hours. Then, 3 ml of 0.6 M eodoacetamide as an alkylating agent was added to adjust the pH to 8.0, and the mixture was reacted at 15 ° C with stirring for 15 minutes. Reaction mixture ^

7.4 ml was subjected to particle removal using a 0.5 m filter, and concentrated to about 0.4 ml by ultrafiltration. Then, the reaction mixture was separated into a heavy chain and a light chain in two times by HPLC. The fractions corresponding to the separated heavy and light chains were diluted with 6 M guanidine hydrochloride (pH 6.5) and dialyzed eight times against PBS. Finally, the purity of the antibody fragment was confirmed by SDS-PAGE (12% separation gel; 3% concentrated gel, silver staining), and the protein concentration was determined by DC Protein Microassay (BIO-RAD).

 (Experiment 5) Reaction of anti-gp41 antibody light chain produced by open column method with GP41-1 peptide

 The anti-gp41 antibody light chain (0.8 M = 20 g / m1) collected by the open column method and the GP41-1 peptide (20 M = 50 μg / m1) The reaction was carried out in 50 mM PBS (pH 7.4), and the time course of the GP41-1 peptide was monitored by HPLC. The reaction temperature was 25 ° C, and sterilized test tubes were used for the reaction vessel. The results are shown in Figure 1. The symbols in Fig. 1 indicate the following.

 A: When the anti-gp41 antibody light chain was reacted with the GP41-1 peptide (Example of the present invention).

 B: When the same operation as in A was performed without adding the anti-gp41 antibody light chain (Comparative Example).

 (Experiment 6) Reaction of anti-gp41 antibody light chain prepared by liquid chromatography with YP41-1 peptide

A 21-mer YP4 1-1 peptide containing a GP41-1 peptide sequence containing the anti-gp41 antibody light chain (0.68 M = 17 g / m1) separated and purified by liquid chromatography (YPRGPDRPEGIEEE GGE RD RD) (4.l ^ M = 10 g / m 1) and 150 mMP BS (p ^ _Q

The reaction was carried out in H7.4), and the time course of the YP41-1 peptide was monitored by HPLC. The reaction temperature was 25 ° C, and sterilized test tubes were used for the reaction vessels. The result is shown in figure 2. The symbols in FIG. 2 indicate the following.

A: The case where the anti-gp41 antibody light chain was reacted with the YP41-11 peptide (Example of the present invention).

 B: When the same operation as in A was performed without adding the anti-gp41 antibody light chain (Comparative Example).

 C: When the same operation as in A was performed using an anti-methamphetamine antibody light chain (IgG2b: κ) purified in exactly the same manner as the anti-gp41 antibody light chain (comparative example).

 (Experiment 7) Reaction of gp41 protein with anti-gp41 antibody light chain prepared by liquid chromatography

 Separation by liquid chromatography '' Purified anti-gp41 antibody light chain (0.64 M) and gp41 protein (Intracell) 1.8 M with 15 mM PB (pH 6.5) 2 in 5 C. gps41 SDS-PAGE (silver stained) photo showing an example of protein degradation is shown in Figure 3. The lanes in FIG. 3 show the following.

L an e 1: molecular weight mer TJ—

 Lane2: the result of a reaction time of 0 hour when the anti-gp41 antibody light chain was reacted with the gp41 protein (Example of the present invention).

 Lane3: the result of the same reaction time of 14 hours (Example of the present invention).

Lane4: The result of a reaction time of 0 hour when the same operation was performed without adding the anti-gp41 antibody light chain (Comparative Example).

 Lane5: The result of the same reaction time of 14 hours (Comparative Example).

(Experiment 8) Reaction of anti-gp41 antibody heavy chain prepared by liquid chromatography with YP41-1 peptide The experiment was performed in the same manner as in Experiment 6, except that the anti-gρ41 antibody heavy chain was used instead of the anti-gρ41 antibody light chain. Fig. 4 shows the results. The symbols in Fig. 4 indicate the following.

 A: When the anti-gp41 antibody heavy chain was reacted with the YP411 peptide (Example of the present invention).

 B: When the same operation as in A was performed without adding the anti-gp41 antibody heavy chain (Comparative Example).

 C: When the anti-gp41 antibody itself was reacted with YP41-1 peptide (Comparative Example).

 As can be seen from FIGS. 1 and 2, when GP41-1 and YP41-1 peptides were reacted with the anti-gP41 antibody light chain, these peptides were reduced and eventually Thus, it was confirmed that the anti-gP41 antibody light chain had protease activity on GP411 and YP41-1 peptide. In addition, the mass spectrum confirmed that in the above reaction, the cleavage between the arginine and glycine of the YP41-1 peptide of the following formula (underlined portion) was performed. Further, as shown in FIG. 3, it was proved that the anti-gp41 antibody light chain completely degraded gp41 protein. In addition, as shown in FIG. 4, it was confirmed that the heavy chain of the anti-gp41 antibody also had the same protease activity after the induction period with respect to the YP41-1 peptide.

 R G P D R P E G I E E E G G E RD D-(1)

 (Experiment 9) Reaction of anti-gp41 antibody light chain prepared by liquid chromatography with YP41-1 peptide

Separation and purification of the anti-gp41 antibody light chain (0.8 M) separated by liquid chromatography and YP41-1 peptide (120 M) in 15 Mm phosphate buffer (pH 6. 5) Time-dependent change of YP4 1-1 peptide ₁ ^ was tracked by HPLC. The reaction temperature was 25 ° C, and sterilized reaction vessels and buffers were used. Fig. 5 shows the results. The symbols in Fig. 5 indicate the following.

 A: The case where the anti-gp41 antibody light chain was reacted with the YP41-1 peptide (Example of the present invention).

 B: Anti-methamphetamine antibody (MA-15) light chain reacted with YP41-1 peptide (Comparative example)

 C: Anti-gp41 antibody (complete antibody) reacted with YP41-1 peptide (Comparative Example).

D: When the same operation as A was performed without adding the anti-gp41 antibody light chain (Comparative Example).

 (Experiment 10) Kinetic study of the reaction between the anti-gp41 antibody light chain prepared by liquid chromatography and YP41-1 peptide

The anti-gp41 antibody light chain (0.64 M), which was separated and purified by liquid chromatography, was combined with various concentrations of YP41-1 peptide (0.64 to 30.7 Μ). The reaction was performed in a 5 Mm phosphate buffer (pH 6.5), and the kinetics of the reaction was examined. The reaction temperature was 25 ° C, and sterilized reaction vessels and buffers were used. The results are shown in FIGS. Figure 6 shows the relationship between YP41-1 peptide concentration and initial velocity, and Figure 7 shows the Hanes-Woolf plot. When obtaining the result from Michaelis one Menten constant (Km) and the catalytic rate constant (kcat), it was respectively 2 and ^{2 X 1 0- 7 M, 0} . 0 6 min- 1. That was the kcat / Km = 2. 8 X 1 0 5 M one ¹ min- ^1.

 (Experiment 1 1)

The reactivity of the anti-gp41 antibody light and heavy chains and anti-gp41 antibody collected in Experiment 4 with the AIDS virus protein was determined by Western blotting. ^ ₃ examined by The method used NovaPath IMMUNOBLOT A SSA kit (For Detection of Antibody to Human Immunodeficiency Virus Type-1, Nippon BIO-RAD, Tokyo, Japan) as a reagent and basically followed the manual. However, when adding a secondary antibody, the positive control contains goat anti human IgG, in gp4 1 geometry, its light chain, and heavy II as rabbit anti mouse Ig (G + M + A) (afinity purified grade (F ab ') ₂ : Zymed, USA) was used. FIG. 8 shows a photograph showing an example of the result of immunoblotting on the AIDS virus protein. The lanes in Fig. 8 show the following.

L an e 1: ositive control (comparative example)

 Lane 2: anti-gp41 antibody light chain (41S—2—L) (example of the present invention) Lane 3: anti-gp41 antibody heavy chain (41S—2-H) (example of the present invention) Lane 4: anti-gp41 antibody (41S—2 mAb) (Example of the present invention)

 (Experiment 1 2)

 Under the same conditions as in Experiment 9, except for the antigen concentration, recombinant gp41 (r-gp41), BSA (pserum albumin) and HSA (human serum albumin), and the anti-gp41 antibody light chain Was reacted. The results of SDS-PAGE at that time are shown in FIGS. 9a and 9b. [Intracel Corp. Catalog No.036001 HIV-1 gp41-IIIb, Cambridge, MA, USA] was used as r-gp41. Its amino acid sequence is as follows and consists of 19 residues. Some unnecessary parts have been deleted. In SDS—PAGE, it appears thick at the position of molecular weight 28.2 kDa (r-gp41, 0hr in FIG. 9a).

AVGIGS RQ LLSGI VQQQNNL L RA IE AQQHL LQL TVWG I KQ L QAR IL AVE RYI KDQQ LLGI WG CS GKL ICT TAV PWNA SWS NK SLEQI WNNMTWMEW DR EI NN YT SLIHSLIEES QNQQEKNEQE LLEL DKWA S LWNWFN I TNWL VNRVRQGY SPLS FQTH LPIP RG PDR PEGIEEE GG E RD RD RSIRL VN GS

 The lanes in FIG. 9 show the following.

Figure 9a (Example of the present invention)

 L an e 1 marker

 L an e 2 0 h r, r-g p 4 1 (control)

 L an e 3 0 h r, 4 1 S-2-L (control)

 L a n e 4 0 h r, r-g p 4 1 + 4 1 S-2-L

 L an e 5 6 h r, r-g p 4 1 + 4 1 S-2-L

 L an e 6 1 1 h r, r-g p 4 1 + 4 1 S-2-L

 L an e 7 16 h r, r-g p 4 1 + 4 1 S-2-L

 L an e 8 16 h r, r-g p 4 1 (control)

Fig. 9 b (Comparative example)

 L an e 1: Marker

 L an e 2: 0 h r, B S A + 4 1 S-2-L

 L an e 3: 3 3 h r, B S A + 4 1 S-2-L

 L an e 4: 0 h r, B S A

 L an e 5: 3 3 h r, B S A

 L an e 6: 0 h r, H S A + 4 1 S-2-L

 L an e 7: 33 h r, H S A + 4 1 S-2— L

 L an 8: 0 h r, H S A

 L an 9: 3 3 h r, H S A

 The time course of the degradation reaction of r-gp41 (4.5 M) by the anti-gp41 antibody light chain (41 S—2—L: 0.2 × M) is shown in FIG. 9A. Reaction progress

A new band appears at 25.2 kDa after 6 hours, and a little after 11 hours ". It became thin and almost disappeared after 16 hours. The band of 25.2 kDa is smaller than the original gp41 by 3 kDa, and the band of A rg— G 1 y This is in good agreement with the difference in the molecular weight (29997) when the gap was cleaved.Figure 9b shows a similar procedure to the above in which BSA and HSA (both 1. were replaced with the anti-gp41 (S—2—L: 0.64 / xM), and there was no change until 33 hours, indicating that the anti-gp41 antibody light chain was able to reach the antigen protein. It turns out that it is specifically degraded (Experiment 13)

 Most of the light chain of the anti-gp41 antibody collected in Experiment 4 was removed from the reaction system by immunoprecipitation using Kappa Lock (C ALBIOCHEM, Oncogene Research Products, MA, USA). An experiment was performed in which it was removed. The conditions were as follows.

Antibody light chain = 0.8 nU

Peptide = 100 to 120 zM in PBS at 25 ° C

 As a result, as shown in FIG. 10, when the antibody light chain was removed from the reaction system, the YP41-1 peptide degrading activity was significantly reduced. The symbols in FIG. 10 indicate the following.

 A: Y P 4 1-1 + light chain + Kappa Lock (Example of the present invention)

B: YP4 1—1 + light chain (comparative example)

C: Y P 4 1-1 (Comparative example)

 D: Y P 4 1-1 + Kappa Lock (Comparative example)

 (Experiment 1 4)

 The amino acid sequence of the antibody light chain gene variable region used in the present invention was examined. As a result, the amino acid sequence was as shown in FIG.

 (Experiment 1 5)

The amino acid sequence determined in Experiment 14 was expressed by genetic engineering. This ,

 At 1D, PET 21 — a (+) (Novagen) was used as a plasmid and expressed as a protein with a T7 tag at the N-terminus and six His tags at the C-terminus. . BamH1 and XhoI were provided as restriction enzyme sites for excision. As an expression system Escherichia coli, BL21 (DE3) deficient for protease was used. When induced by IPTG, the target protein was expressed as inclusion bodies. After sonication of Escherichia coli, purification was carried out using His'bind resin, and PBS was subjected to 4 days at 4 ° C for 2 days.

 0.8 g of the recombinant protein thus obtained was reacted with Ρ-peptidyl 120 under the same conditions as in Experiment 9. As a result, about 80% of YP41-1 was decomposed 190 hours after the start of the reaction. At this time, there was no change in the control 系 Ρ 4 1 — 1 only system. In the anti-gρ41 antibody light chain used as a positive control, YP41-1 peptide was almost completely degraded in about 100 hours. As a result, it was found that even the antibody light chain expressed by genetic engineering has a lower activity than the intact light chain, but has an antigen-degrading activity. (Experiment 16)

Using an antibody against P24, which is a core protein of AIDS virus (anti-p24 monoclonal antibody IgGi (k)), for the antibody light chain prepared by the method described in Experiments 1, 2 and 4 above, The reactivity with the antigen p24 was examined. The experimental conditions were almost the same as in Experiment 12. Anti-p24 monoclonal antibody light chain = 0.8 M, p24 = 5.6, 4.2, 2.1, 1.0 1, reaction temperature = 25 ° C. In addition, sterilized laboratory equipment and reagents were used, and the operation was performed in a clean bench. As p24, one prepared by recombinant was used. This p24 is a single band in SDS-PAGE and also specifically reacted with anti-p24 monoclonal antibody. _χ ^ I responded.

 The reaction between the p24 protein and the antibody light chain was examined by following the time course of the p24 band (27.1 kDa) by SDS-PAGE. About 22 hours after the start of the reaction, the band at p24 became lighter, and a new band appeared at 25.0 kDa (Fig. 12). From this, it was found that the enzyme activity was expressed even for the p24 which had not yet been determined, when the antibody was separated into light chains and allowed to react.

 (Experiment 17)

 The amino acid sequence of the variable region of the anti-P24 monoclonal antibody light chain gene used in the present invention was examined. As a result, the amino acid sequence was as shown in FIG.

 In the amino acid sequences shown in FIGS. 11 and 13, molecular modeling reveals that the catalytic triad (Asp, Ser, His) is present in both. The presence of this catalytic triad may be necessary for the antibody light chain to exhibit enzymatic activity.

 (Experiment 18)

 As described in Experiments 6 and 8, the heavy chain of the anti-RT antibody (antibody to the reverse transcriptase (RT) of AIDS virus IgG G i (k)) separated and purified by liquid chromatography Using the light chain, an RT peptide (partial peptide contained in reverse transcriptase: KLLRGTKALTEC) was reacted in PBS in the same manner as in Experiment 6 and Experiment 8. Peptide reduction was tracked by HPLC. Figure 14 shows the results. The symbols in Figure 14 indicate the following:

A: When the anti-RT antibody heavy chain is reacted with the RT peptide (Example of the present invention) B: When the same operation as A is performed without adding the anti-RT antibody heavy chain and light chain (Comparative Example) 丄 g

C: Reaction of the anti-RT antibody light chain with the RT peptide (Comparative Example) From the results in Fig. 14, the reaction of the heavy chain of the anti-RT antibody with the RT peptide allows the reaction of the RT peptide as an antigen Premature elimination occurred, confirming that the heavy chain of the antibody had beptidase activity.

 As a result of measuring the mass spectrum of one peak detected by HPLC (9 minutes: RT peptide was detected at 12.5 minutes by reference), it was determined that the divalent ions were 500.0.3 and monoisotopic mass = 500. 3 X 2-2 = 9 9 8.6. This showed a very good agreement with the theoretical value of 99.8.7, indicating that this peak was KL L R GTKAL. Therefore, it can be said that the antibody heavy chain cleaves the peptide bond between L and T of KLLRGTTCALTEC in the sequence of the RT peptide.

 (Experiment 19)

 Using the anti-RT antibody heavy chain (anti RT (H); 0.78 M) used in Experiment 18, RT (reverse transfer enzyme; Advanced Biotechnologies Incorporated); ABI, Columbia, Mayland, USA , 0.46 M) as an antigen. Degradation or reduction of RT was monitored by SDS-PAGE as in Experiment 7. The reaction conditions were the same as those in Experiment 7, except that the anti-RT antibody heavy chain and RT were used, and the reaction temperature was 25 ° C. The results are shown in FIG.

 The numbers just above SDS—PAGE in FIG. 15 indicate the reaction times. The molecular weight markers are also shown on the right.

 Figure 15 As can be seen, recombinant RT (r-RT; 72.4 kDa) was partially degraded in 9 hours of reaction time, and 6

A new band is created at 8 kDa. When the reaction was further continued, the r_RT band (72.4 kDa) almost disappeared in 23 hours. Also, 6 6

The band at 8 kDa was also considerably thinner at the reaction time of 40 hours. _It can be seen that _{1 g of the} compound is also undergoing decomposition and disappearing (Example of the present invention).

 On the other hand, in the reaction system containing only the RT without the anti-RT antibody heavy chain, no 66.8 kDa band appeared between the reaction times of 0 and 40 hours, and no decomposition was performed. (Comparative Example)

 In addition, even in the reaction system containing only the anti-RT antibody heavy chain without the addition of RT, no band other than the anti-RT antibody band was detected between the reaction times of 0 and 40 hours (Comparative Example).

 From the above results, it can be said that r-RT is degraded by the anti-RT antibody heavy chain and disappears.

 (Experiment 20)

 The reaction was carried out in the same manner as in Experiment 19, except that HSA and BSA (both 0.46 M) were used. Here, HSA and BSA were used to prepare a system mixed with the anti-RT antibody heavy chain used in Experiment 19, respectively, and a system containing only HSA and BSA without the addition of the anti-RT anti-weight chain, and reacted. Figure 16 shows the results.

 The numbers just above SDS—PAGE in the figure represent the reaction times. The molecular weight marker is shown on the right of the figure.

 Both HSA and BSA, with and without anti-RT antibody heavy chain, give exactly the same bands at reaction times of 0 and 40 hours, and the anti-RT antibody heavy chain degrades both proteins at all. (Comparative example).

 (Experiment 2 1)

 The anti-gp41 antibody light chain and heavy chain prepared in Experiments 6 and 8 were used alone or in combination to react with YP41-1. The results are shown in FIG. The symbols in the figure indicate the following.

A: When the anti-gp41 antibody light chain is reacted with the YP41-1 peptide _{2 Q}

(Comparative example)

 B: Reaction of anti-gp41 antibody heavy chain with YP41-1 peptide (Comparative Example)

 C: Anti-gp41 antibody light chain and anti-gp41 antibody heavy chain were simultaneously mixed and reacted with YP411-11 peptide (Example of the present invention)

 D: Reaction of anti-gp41 antibody with YP41-1 peptide (Comparative Example)

 E: A case where the same operation as A was carried out using only YP411-peptide without adding the anti-gp41 antibody light chain and the anti-gp41 antibody heavy chain (Comparative Example)

 From FIG. 17, it can be seen that when the light chain and the heavy chain are simultaneously added to the reaction system, the activity is higher than when the light chain or the heavy chain is reacted alone. Industrial applicability

 As described above, according to the present invention, an antibody catalyst having the ability to cleave and / or degrade a target protein or peptide can be obtained. That is, a protein or a peptide to be cleaved and / or degraded or a partial peptide constituting them is used as an antigen to prepare a monoclonal antibody, thereby obtaining an antibody catalyst having the above ability. Can be. Therefore, according to the present invention, by selecting an antigen according to the purpose and appropriately collecting an antibody catalyst from the obtained monoclonal antibody, an antiviral agent such as an anti-HIV agent, an anticancer agent, It is possible to obtain an antibody catalyst that can be used as a thrombotic agent or a general enzyme.

Claims

The scope of the claims 1. A method for producing an antibody catalyst which cleaves and degrades or degrades a target protein or peptide, wherein the target protein or peptide or a partial peptide constituting the protein or peptide is used as an antigen to prepare a monoclonal antibody A method for producing an antibody catalyst, comprising: preparing an antibody; and collecting an antibody catalyst capable of cleaving and / or degrading the target protein or peptide from the prepared monoclonal antibody. 2. The antibody catalyst according to claim 1, wherein the antibody catalyst capable of cleaving and / or degrading the target protein or peptide is collected by separating the prepared monoclonal antibody into a light chain and a heavy chain. Production method.  3. A GP41-1 peptide having an amino acid sequence of the following formula (1), which is present in the constant region of the surface protein gρ41 of ΗIV, is used as an antigen for preparing a monoclonal antibody. The method for producing an antibody catalyst according to the above.  R G P D R P E G I E E E GG E D RD-(1)  4. The method for producing an antibody catalyst according to claim 3, wherein the obtained monoclonal antibody is separated into a light chain and a heavy chain, and the light chain and / or heavy chain is collected as an antibody catalyst.  5. In cleaving and / or decomposing a target protein or peptide using the antibody catalyst obtained by the production method according to claim 2 or 4, cleaving and / or decomposing the target protein or peptide. A method of using an antibody catalyst, comprising using one of an antibody heavy chain and an antibody light chain having the ability alone. 6. When the target protein or peptide is cleaved and Z or degraded using the antibody catalyst obtained by the production method according to claim 2 or 4, A method for using an antibody catalyst, comprising simultaneously mixing both an antibody heavy chain and an antibody light chain having the ability to cleave and / or degrade a target protein or peptide.  7. The antibody heavy chain and Z or a partial region of the antibody light chain are used as the antibody heavy chain and antibody light chain having the ability to cleave and Z or degrade the target protein or peptide. Use of the described antibody catalyst.  8. The antibody catalyst according to any one of claims 5 to 7, wherein the antibody heavy chain and the antibody light chain having the ability to cleave and / or degrade the target protein or peptide are produced by genetic manipulation. How to use