JPH06220072A - Activation of prodrug by catalyst antibody - Google Patents

Abstract

PURPOSE:To produce the subject antibody useful for reduction of toxicity of a medicine, improvement of long-acting properties and improvement of various problems such as specificity to a focus by activating a prodrug using a catalyst antibody. CONSTITUTION:The objective antibody can be produced by specifically hydrolyzing a prodrug compound of formula II which is obtained by immunization with a compound of formula I as a hapten.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a catalytic antibody (Catalytic).
Antibodies) for activating prodrugs. Specifically, the present invention decomposes a compound (prodrug) chemically modified into a non-pharmacologically active derivative for the purpose of reducing toxicity of the drug or improving persistence, and the prodrug is converted into its original pharmacologically active form. It relates to an antibody that is converted into a drug. By using such an antibody in combination with a prodrug-form drug, various problems such as toxicity reduction of drug, improvement of persistence or specificity to lesion can be improved.

[0002]

2. Description of the Related Art Drugs having useful pharmacological activity but often accompanied by strong side effects / toxicity or unstable and difficult to administer are often used to improve their inconvenience. The drug is converted into a prodrug. Although the prodrug-form drug has no pharmacological activity by itself, it is decomposed by the change of pH in the body or the enzyme in the body after administration, and converted into the original drug to exert its original pharmacological action. Chemical modification to create such a prodrug is, for example, an anti-cancer drug with strong side effects, resists an enzyme in the body until the prodrug reaches the target lesion, and is decomposed only after reaching the lesion to produce an active drug. It is desirable that the modification be converted to. Therefore, in selecting a chemical modification for producing a prodrug, the presence or absence of an enzyme that performs degradation modification of the prodrug in vivo, and the reaction specificity of the enzyme when such an enzyme is present in vivo, are selected. , Substrate specificity or biodistribution should be considered. Even if the prodrug candidate compound obtained by a certain chemical modification is the most suitable compound for stabilizing the target drug or reducing toxicity, an enzyme that shows specificity to the compound and decomposes it If the compound is natural, that is, it is not present in the living body, the compound cannot be decomposed in the living body and therefore cannot be used as a prodrug. However, if an enzyme that exerts such a function is artificially produced and administered to a living body, a candidate compound that could not be used in the past can be used as a prodrug. Therefore, the present inventors have focused their attention on this point for the first time in the world, and have conducted intensive research in order to open up a new field for prodrug research. The present invention is a desired substrate (prodrug)
By developing a biocatalyst that specifically acts against the drug, it will greatly promote the conversion of drugs into prodrugs that could not be used due to problems such as toxicity and chemical modification of prodrugs specific to lesions. It makes a contribution to.

[0003]

As one of the embodiments of the present invention, the present invention provides an ester derivative prodrug of chloramphenicol represented by the following formula 9.

[Chemical 4] It has been confirmed by a biological test using Escherichia coli that the above ester derivative (9) of the present invention has no antibiotic activity. Therefore, this compound can certainly be used as a prodrug.

Furthermore, the present invention provides the above ester derivative.
An antibody capable of specifically hydrolyzing (9) to regenerate the original chloramphenicol (1) is provided. This antibody of the present invention specifically hydrolyzes the prodrug (9) of the present invention according to the following reaction formula to give chloramphenicol.
Generate (1).

[Chemical 5] Such an antibody is a catalytic antibody. Lerne
r, RA et al .: Science, 252 , 659 (19)
91)]. That is, the antibody of the present invention is prepared by immunizing a phosphoric acid ester derivative (4), which is a transition state analog of the hydrolysis reaction represented by the following formula, as a hapten.

[Chemical 6]

The basic structure of the antibody of the present invention has a peptide chain structure in which two homologous H chains and two L chains are linked by a disulfide bond and a noncovalent bond. As one aspect of the present invention, an antibody in which the H chain contains the amino acid sequence represented by SEQ ID NO: 1, an antibody in which the L chain contains the amino acid sequence represented by SEQ ID NO: 2, and the H chain comprises SEQ ID NO: 1 containing the above amino acid sequence and L
It also provides an antibody whose chain comprises the above amino acid sequence of SEQ ID NO: 2. In the present invention, the amino acid sequence of the first hypervariable region in the H chain is represented by the formula: Asn Tyr Ala Met Ser and the amino acid sequence of the second hypervariable region is represented by the formula: Ser Ser Gly Gly Ser Ile Tyr Tyr Leu Asp. Ser Val Ly
s Gly and the amino acid sequence of the third hypervariable region is represented by the formula: Val Ser His Tyr Asp Gly Ser Arg Asp Trp Tyr Phe As
The antibody represented by p Val and the amino acid sequence of the first hypervariable region in the L chain have the formula: Arg Ser Ser Gln Thr Ile Val His Ser Asn Gly Asp Th
r Tyr Leu Asp, the amino acid sequence of the second hypervariable region is represented by the formula: Lys Val Ser Asn Arg Phe, and the amino acid sequence of the third hypervariable region is represented by the formula: Phe Gln Gly Ser His Val Pro Pro Thr And the hypervariable regions thereof are H
It also provides an antibody having a chain and an L chain.

The phosphoric acid ester derivative (4) can be synthesized according to the synthetic route shown in the following reaction formula 1.

[Chemical 7] That is, an acetylated product in which only the primary hydroxyl group is protected by reacting chloramphenicol (1) in acetic anhydride
After (2), N-trifluoroacetyl-4-aminobenzylphosphoric acid and pyridine are reacted with N, N-dicyclohexylcarbodiimide as a condensing agent to form a condensate (3).
To get Deprotection of the acetyl group with 0.1N sodium hydroxide in acetone gave the desired phosphate ester
Get (4).

Then, as a preparation for binding a carrier to the above phosphoric acid ester derivative (4), glutaric anhydride is reacted with this in the presence of triethylamine in dimethylformamide to attach a side chain portion to form a terminal carboxylic acid. N-hydroxysuccinide is reacted to obtain an activated ester body (6) (Scheme 2).

[Chemical 8]

The activated ester form (6) thus obtained was treated with KLH ( k
ey hole limpet hemocyanin) and phosphate buffer (pH 7.
4) Condensate in to make an antigen. Immunization was performed on Balb / c mice using this KLH condensate. 10 days later 2
The second immunization was performed, and 10 days later, the third immunization was performed. One month later, the final immunization was performed, and three days later, the spleen was extracted. Common method [Milstein, C et al., Nature (Na
ture), 256 , 495 (1975)], and spleen cells and myeloma cells were fused to prepare hybridomas. The hybridoma producing the monoclonal antibody specific to the phosphoric acid ester (4) used as the hapten is BS.
Enzyme-labeled immunosorbent assay (ELI using A-condensate)
SA) method. Finally, 12 kinds of monoclonal antibodies specific for hapten binding were obtained.

As described above, chloramphenicol is used in the present specification in order to prove the utility and possibility of the antibody of the present invention. However, it will be understood by those skilled in the art that an antibody similar to that of the present invention can be prepared by using another useful drug. It will also be understood that the antibody of the present invention may be used as a starting material for producing a derivative that specifically catalyzes the reaction represented by the following formula.

[Chemical 9] [Wherein R means any residue]

Ester derivative used as prodrug
(9) is synthesized according to the synthetic route shown in the following reaction formula 3.

[Chemical 10]

That is, chloramphenicol (1) was reacted with tert-butyldimethylsilyl chloride in a dimethylformamide-dichloromethane mixed solvent to form a silyl compound (7) in which the primary hydroxyl group was protected with a silyl group, and then N-tri A condensate (8) was obtained by reacting with fluoroacetyl-4-aminophenylacetic acid. This was deprotected in a mixed solution of acetic acid: THF: water 3: 1: 1 to obtain the target ester derivative (9).

[0012]

ACTION AND EFFECT OF THE INVENTION The catalytic activity for the hydrolysis reaction of the ester derivative (9) was examined for the 12 kinds of prepared antibodies. When the amount of chloramphenicol produced by hydrolysis was traced by liquid chromatography (HPLC), catalytic activity was observed in 6 out of 15 antibodies. The kinetics of the catalytic reaction was determined by examining the reaction rate in more detail. The obtained value is Km = 64 × 10 ⁻⁶ M,
kcat = 0.133 min ⁻¹ (see Example 15).

Next, the activation of the prodrug by conversion of the ester derivative (9) to chloramphenicol (1) was tested for the resulting antibody by an Escherichia coli growth inhibition experiment. When a paper roll impregnated with a solution of the antibody and the ester derivative was placed on the medium and incubated with Escherichia coli, an inhibition circle due to growth inhibition was observed around the paper roll. As a comparative experiment, the same experiment was conducted only with the ester derivative (9) or the antibody, but no inhibition circle was observed. From the above, the antibody obtained by immunization with phosphate ester (4) as a hapten catalytically hydrolyzes the ester derivative (9) which is a prodrug of chloramphenicol, and further the ester derivative (9) It has been found that is selectively hydrolyzed only by this antibody and cannot be degraded by the hydrolase present in E. coli. This is the first time in the world that prodrug activation by antibodies has been proved. The new prodrug activation method proved by the present invention can be applied not only to chloramphenicol but also to other useful drugs in the future, and can be expected to make a great contribution in the field of medicine. Hereinafter, the present invention will be described in more detail with reference to examples.

[0014]

【Example】

Example 1 Synthesis of compound (2) Chloramphenicol compound (1) (500 mg, 1.55 mm
ol) was suspended in acetic anhydride (5 ml) and stirred at 100 ° C. for 1.5 hours. After concentration under reduced pressure, the residue was dissolved in dichloromethane and purified by silica gel column chromatography (elution solution: ethyl acetate-hexane (1: 1)). The fraction containing the target substance was concentrated under reduced pressure to obtain an oily substance. Yield: 335 mg
(59%).

Example 2 Synthesis of Compound (3) Compound (2) (200 mg, 548 μmol) was added to pyridine (5 ml).
Suspended in N-trifluoroacetyl-4-aminobenzylphosphoric acid (258 mg, 1.096 mmol) and N, N-dicyclohexylcarbodiimide (565 mg, 2.739 mmol).
Was added and the mixture was stirred at 40 ° C. overnight. After dichlorohexylurea was filtered off and concentrated under reduced pressure, the residue was purified by HPLC (ODS column, 10 × 250 mm, acetonitrile-0.1% trifluoroacetic acid aqueous solution (2: 3), flow rate 3 ml /
Min, detection 254 mm, holding time 180 minutes). Hold time 18
The peak at 0 minutes was collected and freeze-dried to obtain the desired product. Yield 57 mg (16%). Mass spectrum: 630 (M ⁺⁺ 1).

Example 3 Synthesis of compound (4) Compound (3) (10 mg, 15.9 μmol) was added to acetone (2.5 m).
l), cooled to -20 ° C, and cooled with ice to 0.1N-
Aqueous sodium hydroxide solution (2.5 ml) was added, and the mixture was stirred for 1 hr. The mixture was neutralized with 1N-hydrochloric acid and then concentrated under reduced pressure. The residue is H
Purified by PLC (ODS column, 10 x 250 m
m, acetone tolyl-0.1% trifluoroacetic acid aqueous solution,
Gradient 30% -60% acetonitrile (20 minutes),
Flow rate 3 ml / min, detection 254 mm, retention time 14.8 min). The peak with a retention time of 14.8 minutes was collected and freeze-dried to obtain the target compound (4). Yield: 8.4 mg (78%). Mass spectrum: 588 (M ⁺⁺ 1). NMR (δ, ppm): 3.18 (dd, J = 6.5, 14.7 Hz, 1
H), 3.22 (dd, J = 6.6, 14.7 Hz, 1 H), 3.4
9 (dd, J = 5.9,11.2Hz, 1H), 3.71 (dd, J =
7.1, 11.2Hz, 1H), 4.21 (m, 1H), 5.69 (d
d, J = 3.6, 19.7 Hz, 1H), 6.22 (s, 1H), 7.
26, 7.48 (ABX, J = 8.3, 2.1 Hz, 4 H), 7.
47, 8.11 (ABq, J = 8.7Hz, 4H). NMR is C
It was measured by D ₃ OD.

Example 4 Synthesis of Compound (5) Compound (4) (20 mg, 34.1 μmol) was dissolved in dimethylformamide (3 ml) to prepare glutaric anhydride (37.6 mg, 37.6 mg).
30 μmol) and triethylamine (116 μl, 830 μmo)
l) was added and the mixture was stirred at 80 ° C. for 2 hours. HPLC reaction solution
(ODS column, 10 × 250 mm, acetonitrile-0.1% trifluoroacetic acid aqueous solution (2: 3),
Flow rate 3 ml / min, detection 254 mm, retention time 15.1 min). The peak with a retention time of 15.1 minutes was collected and freeze-dried to obtain the desired product. Yield: 20.9 mg (87%). Mass spectrometry: 702
(M ⁺⁺ 1).

Example 5 Synthesis of compound (6) Compound (5) (9 mg, 12.8 μmol) was suspended in dried acetonitrile (2 ml), and N-hydroxysuccinide (1.
8 mg, 15.4 μmol) and N, N-dicyclohexylcarbodiimide (7.9 mg, 38.5 μmol) were added, and the mixture was stirred at room temperature for 1 hr. Dicyclohexylurea was removed by filtration, concentrated under reduced pressure, and purified by HPLC (ODS column,
10 × 250 mm, acetonitrile-0.1% trifluoroacetic acid aqueous solution (1: 1), flow rate 3 ml / min, detection 254 mm,
(Holding time 11.2 minutes). The peak with a retention time of 11.2 minutes was collected and freeze-dried to obtain the target compound (6). Yield: 2.4 mg (24%). Mass spectrum: 799 (M ⁺⁺ 1).

Example 6 of hapten and carrier protein
Condensation carrier protein, KLH (6.6 mg) was added to phosphate buffer (pH
7.4, 1.32 ml), the solution of compound (6) (3.3 mg) in dimethylformamide was added, and the mixture was gently stirred overnight at room temperature. After purification by gel filtration, the amount of protein was determined by the Bradford method (concentration protein 1.1 mg /
ml). Similarly, a condensate was prepared using BSA as a carrier protein, and the obtained condensate was used in the ELISA method for measuring the antibody titer of the antibody produced by the KLH condensate.

Example 7 Synthesis of Compound (7) Compound (1) (4 g, 12.38 mg) was dried to prepare a mixed solvent of dimethylformamide and dichloromethane (5 ml / 100 m).
l) and dissolved in t-butyldimethylsilyl chloride (2.
2 g, 14.85 mmol) and triethylamine (2.07 ml,
14.85 mmol) and dimethylaminopyridine (75.5 mg,
620 μmol) was added and the mixture was stirred at room temperature for 2.5 hours. The reaction solution was extracted with chloroform using a separating funnel, washed with water, and dried with magnesium sulfate. After concentration under reduced pressure, the residue was purified by silica gel column chromatography (elution solution: ethyl acetate-hexane (1: 4)). The target fraction was concentrated under reduced pressure to give a white solid. Yield: 4.3 g (80%). Mass spectrometry: 437
(M ⁺ ).

Example 8 Synthesis of compound (8) N-trifluoroacetyl-4-aminophenylacetic acid
(319.9 mg, 1.295 mmol) was dried and mixed solvent of dimethylformamide and dichloromethane (2 ml / 20 ml).
Dissolved in N, N-dicyclohexylcarbodiimide (1
67 mg, 809 μmol) was added and the mixture was stirred at room temperature for 1.5 hours. Dicyclohexylurea was removed by filtration, and the compound (7) (141.5 mg, 324 μmol) and triethylamine were added to the filtrate.
(135.8 μl, 971 μmol) and dimethylaminopyridine (7.9 mg, 64.8 μmol) were added, and the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, the residue was purified by silica gel column chromatography (elution solution: ethyl acetate-hexane (1: 2)).
The target fraction was concentrated under reduced pressure to give a white solid. Yield: 1
32.7 mg (62%). Mass spectrum: 665 (M ⁺ ).

Example 9 Synthesis of compound (9) 5 ml of a solution of compound (8) (482 mg, 723 μmol) in THF
After adding 0.5 ml of water and 15 ml of acetic acid to the mixture, warm bath (50
(° C) for 5 hours. Then, the excess solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (elution solution: ethyl acetate: hexane = 1: 1) to obtain the target ester derivative (9). Yield: 221.5mg
(56%). Mass spectrum: 551 (MH) ^- . NMR (δ, ppm): 3.46 (dd, J = 5.3, 11.4 Hz, 1
H), 3.60 (dd, J = 5.9, 11.4 Hz, 1H), 3.7
6 (d, J = 16.2Hz, 1H), 3.77 (d, J = 16.2H
z, 1H), 4.33 (m, 1H), 6.10 (d, J = 5.5Hz,
1H), 6.24 (s, 1H), 7.32, 7.60 (ABq, J
= 8.41Hz, 4H), 7.50, 8.16 (ABq, 8.74)
Hz, 4H). NMR was measured by CD ₃ OD.

Example 10 Preparation of monoclonal antibody: Immunogen (KLH condensate) 50 μg / 50 μl physiological saline was mixed with an equal amount of complete Freund's adjuvant, and the mixture was Balb / c mouse (4 weeks old, female). Was injected intraperitoneally. Ten days later, booster immunization was performed with a mixed solution of 50 μg / 50 μl of antigen and physiological saline and an equal amount of incomplete Freund's adjuvant, and further 10 days later, booster immunization was performed. Seven days later, blood was collected from the tail vein of the mouse, and the antibody titer was determined by enzyme-labeled immunosorbent assay (ELISA) using BSA-condensate (secondary antibody).
A titer of 1:10 ⁶ was obtained by measurement with a biotinylated anti-mouse IgG antibody, avidin, and biotinylated peroxidase. One month after the 2nd booster immunization, the antigen 100μ
g / 100 μl physiological saline was administered through the tail vein (final immunization).

Example 11 Preparation of hybridoma Three days after the final immunization, the spleen was excised from the mouse, and then 10 ⁸ spleen cells and 2.5 × 10 ⁷ myeloma cells were extracted.
(× 63 / Ag8.653) was fused with polyethylene glycol to obtain 10 ⁵ cells / well of feeder cells.
HAT selection medium containing (mouse thymocytes) (0.1 mM hypoxanthine, 0.4 μM aminopterin, 0.016 mM)
Thymidine, 10% fetal bovine serum RPMI medium), 8 96-well plates were used for selection (37 ° C. 1
0% carbon dioxide). Colonies appeared after 8-14 days. The supernatant was taken from the wells and screened by ELISA. As a result, 165 positive wells were found, out of which 45 wells were cloned.
A clone of the strain was obtained. As a result of an ELISA method using an anti-mouse IgG heavy chain antibody, all 36 clones produced IgG. As a result of the competitive inhibition experiment in which the hapten was added during the ELISA, 28 kinds of antibodies produced from 36 strains bound to the hapten.

Example 12 Cultivation of hybridoma and purification of monoclonal antibody Twelve hybridomas were cultured in a complete medium (10% fetal bovine serum, RPMI medium) for 7 days, respectively, and 1 × 10 ⁷
Cells / 500 μl of each cell suspension in physiological saline were administered to Balb / c mice (8
(Week-old females) by intraperitoneal injection, and then 2 weeks later,
5 ml each was collected. The centrifugation supernatant of each ascites fluid was precipitated with an equal amount of saturated ammonium sulfate solution, and then subjected to cation exchange chromatography with an S-Sepharose column and affinity chromatography with a protein G column to obtain 10 to 20 mg of purified antibody, respectively. .

Example 13 Measurement of catalytic activity 22.2 μ each of the 12 purified antibodies obtained according to Example 12
M solution (55.6 mM Tris buffer, pH 8.0) 90μ
leave l at room temperature. To this, 10 μl of a solution of the compound (9) in dimethylsulfoxide (25 mM) was added, and after rapid shaking, the absorption of chloramphenicol was determined by HPLC.
The increase in (278 nm) was followed for 4 hours each (ODS column, 4.6 x 250 mm, acetonitrile-0.1% aqueous trifluoroacetic acid solution (1: 1), flow rate 1 ml / min, retention time 4.5 min). As a result, 6 kinds of antibodies showing catalytic activity were identified, and the antibody having the strongest activity was named 6D9. The hybridoma producing the antibody 6D9 has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology (deposit date: November 27, 1992, accession number: Microbiology Research Institute No. 13308).

Example 14 Further Investigation of Antibody 6D9 The amino acid sequence of the 6D9 antibody obtained in Example 13 was determined. M from hybridoma producing antibody 6D9
The nucleotide sequence encoding the 6D9 antibody was sequenced by cloning the RNA and then making the cDNA. The obtained results are shown in FIGS. 1 and 2.
(Each sequence corresponds to SEQ ID NO: 3 and SEQ ID NO: 4). The underlined portion in the figure is the primer site used when the cDNA was subjected to the PCR method. Next, the amino acid sequence of the 6D9 antibody was determined by deducing the amino acid from this nucleotide sequence. The obtained results are shown in FIGS. 3 and 4 (the respective sequences correspond to SEQ ID NO: 1 and SEQ ID NO: 2). In the figure, CDR-1, CDR-2 and CDR-
3 indicates a first hypervariable region, a second hypervariable region, and a third hypervariable region, respectively. The nucleotide sequence shown in FIG. 3 is an amino acid sequence which occupies most of the Fd fragment of H chain.

Example 15 Determination of kinetic amount 90 μl of a 2.2 μM solution of antibody 6D9 (55.6 mM Tris buffer, pH 8.0) is kept at 30 ° C. in a constant temperature bath. Various concentrations of substrate (0.5 mM, 0.75 mM, 1 mM, 1.2
5 mM, 1.5 mM) dimethyl sulfoxide solution 10 μl
After rapid shaking, the increase in chloramphenicol absorption (278 nm) was monitored by HPLC for 80 minutes.
(ODS column, 4.6 x 250 mm, acetonitrile-
0.1% trifluoroacetic acid aqueous solution (1: 1), flow rate 1 ml /
Minutes, retention time 4.5 minutes). From the results obtained, the Km value for the substrate of antibody 6D9 was 64 × 10 ⁻⁶ M, and the kcat value was 0.1.
It was determined to be 33 min ^-1 .

Example 16 Bioassay LB agar medium incubated at 45 ° C. (0.7% agarose, p
H. 8.0) and Bacillus subtilis ( B. subtilis ISW1214) were mixed and overlaid on LB agar plates (1.5% agar pH 8.0). After the agar has set, place a paper disk (6 mm diameter) on it and add 10 μl of 20 μM antibody (6D9) and 10 μl of 100 mM substrate (ester derivative) or 10 μl of 20 μM.
M antibody (6D9) or 10 μl of 100 mM substrate (ester derivative) was soaked and incubated at 37 ° C. overnight. As a result, no growth inhibition circle was found around the paper disk impregnated with antibody (6D9) or substrate (ester derivative), but growth inhibition was observed when antibody and substrate (ester derivative) were impregnated at the same time. A circle appeared. A similar experiment Escherichia coli (E. Coli XLI-Blue), even with no line, confirmed the growth inhibition circle of only when impregnated both at the same time. The amount of chloramphenicol produced was calculated to be 0.7 mM from the diameter of the inhibition circle.

[0030]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 222 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Val Ser Ile Ser Ser Gly Gly Ser Ile Tyr Tyr Leu Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Phe Cys Ala Arg Val Ser His Tyr Asp Gly Ser Arg Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Thr Ser

SEQ ID NO: 2 Sequence length: 219 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile Val His Ser Asn Gly Asp Thr Tyr Leu Asp Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile V al Lys Ser Phe Asn Arg Asn Glu Cys

SEQ ID NO: 3 Sequence length: 666 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA sequence 10 20 30 40 50 60 CTCGAGTCTG GGGGAGGCTT AGTGAAGCCT GGAGGGTCCC TGAAACTCTC CTGTGCAGCC GAGCTCAGAC CCCCTCCGAA TCACTTCGGA CCTCCCAGGG ACTTTGAGAG GACACGTCGG 70 80 90 100 110 120 TCTGGATTCA CTTTCAGTAA CTATGCCATG TCTTGGGTTC GTCAGACTCC AGAGAAGAGG AGACCTAAGT GAAAGTCATT GATACGGTAC AGAACCCAAG CAGTCTGAGG TCTCTTCTCC 130 140 150 160 170 180 CTGGAGTGGG TCGTATCCAT TAGTAGTGGT GGTTCCATTT ACTATCTGGA CAGTGTGAAG GACCTCACCC AGCATAGGTA ATCATCACCA CCAAGGTAAA TGATAGACCT GTCACACTTC 190 200 210 220 230 240 GGCCGATTCA CCGTCTCCAG AGATAATGCC AGAAACATCC TGTACCTGCA AATGACCAGT CCGGCTAAGT GGCAGAGGTC TCTATTACGG TCTTTGTAGG ACATGGACGT TTACTGGTCA 250 260 270 CG 280 ACT CATCG 360 CATCG 350 ACT GCA GGGACCTCGG TCACCGTCTC CTCAGCCAAA GCGCTGACCA TGAAGCTACA GACCCCGCGT CCCTGGAGCC AGTGGCAGAG GAGTCGGTTT 370 380 390 400 410 420 ACGACACCCC CATCTGTCTA TCCACTGGCC CCTGGATCTG CTGCCCAAAC TAACTCCATG TGCTGTGGGG GTAGACAGAT AGGTGACCGG GGACCTAGAC GACGGGTTTG ATTGAGGTAC 430 440 450 460 470 480 GTGACCCTGG GATGCCTGGT CAAGGGCTAT TTCCCTGAGC CAGTGACAGT GACCTGGAAC CACTGGGACC CTACGGACCA GTTCCCGATA AAGGGACTCG GTCACTGTCA CTGGACCTTG 490 500 510 520 530 540 TCTGGATCCC TGTCCAGCGG TGTGCACACC TTCCCAGCTG TCCTGCAGTC TGACCTCTAC AGACCTAGGG ACAGGTCGCC ACACGTGTGG AAGGGTCGAC AGGACGTCAG ACTGGAGATG 550 560 570 580 590 600 ACTCTGAGCA GCTCAGTGAC TGTCCCCTCC AGCACCTGGC CCAGCGAGAC CGTCACCTGC TGAGACTCGT CGAGTCACTG ACAGGGGAGG TCGTGGACCG GGTCGCTCTG GCAGTGGACG 610 620 630 640 650 660 AACGTTGCCC ACCCGGCCAG CAGCACCAAG GTGGACAAGA AAATTGTGCC CAGGGATTGT TTGCAACGGG TGGGCCGGTC GTCGTGGTTC CACCTGTTCT TTTAACACGG GTCCCTAACA ACTAGT TGATCA

SEQ ID NO: 4 Sequence length: 666 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA sequence 10 20 30
40 50 60 GAGCTCGTGA TGACCCCAGAC TCCACTCTCC CTG
CCTGTCA GTCTTGGAGA TCAAGCCTCC CTCGAGCACT ACTGGGTTCTG AGGTGAGAGG GAC
GGACAGT CAGAACCTCT AGTTCGGAGG 70 80 90
100 110 120 ATCTCTTGCA GATCTAGTCA GACCATTGTA CAT
AGTAATG GAGAC ACGTA TTTAGATTGG TAGAGAACGT CTAGATCAGT CTGGTAACAT GTA
TCATTAC CTCTTGTGCAT AAATCTAACC 130 140 140 150
160 170 180 TTCCTGCAGA AACCAGGCCA GTCTCCAAAG CTC
CTGATCT ACAAAGTTTC CAACCGATTT AAGGACGTCT TTGGTCCGGT CAGAGGTTTC C GAG
GACTAGA TGTTTCAAAG GTTGGCTAAA 190 200 200 210
220 230 240 TCTGGGGTCC CAGACAGGTT CAGTGGCAGT GGA
TCAGGGA CAGATTTCAC ACTCAAGATC AGACCCCAGG GTCTGTCCAA GTCACCGTCA CCT
AGTCCCCT GTCTAAAGTG TGAGTTCTAG 250 260 270
280 290 300 AGCAGAGTGG AGGCTGAGGA TCTGGGAGTT TAT
TACTGCT TTCAAGGTTC ACATGTTCCT TCGTCTCACC TCCGAACTCCT AGACCTCAA ATA
ATGACGA AAGTTCCAAG TGTACAAGGA 310 320 320 330
340 350 350 CCGACGTTTCG GTGGAGGCAC CAAGTTTGGAA ATC
AAACGGG CTGATGCTGC ACCAACTGTA GGCTGCAAGC CACCTCCGTG GTTCAACCTT TAG
TTTGCCCC GACTACGACG TGGTTGGACAT 370 380 390
400 410 420 TCCATCTTCC CACCATCCAG TGAGCAGTTA ACA
TCTGGAG GTGCCTCAGT CGTGTGCTTC AGGTAGAAGG GTGGTAGGTC ACTCGTCAAT TGT
AGACCTC CACGGAGTCA GCACACGAAG 430 440 450
460 470 480 TTGAACAACT TCTACCCCAA AGACATCAAT GTC
AAGTGGA AGATTGATGG CAGTGAACGA AACTTGTTGA AGATGGGGGT TCTGTAGTTTA CAG
TTCACCT TCTAACTACC GTCACTTGCT 490 500 510
520 530 540 CAAAATGGCG TCCTGAACAG TTGGACTGAT CAG
GACAGCA AAGACAGCAC CTACAGCATG GTTTTACCGC AGGACTTGTC AACCTGACTA GTC
CTGTCGT TTCTGTCGTG GATGTCGTAC 550 560 570
580 590 600 AGCAGCACCC TCACGTTGAC CAAGGACGAG TAT
GAACGAC ATAACAGCTA TACCTGTGAG TCGTCGGTGGGG AGTGCAACTG GTTCCTGCTC ATA
CTTGCTG TATTGTCGAT ATGGACACTC 610 620 630
640 650 660 GCC ACTCACA AGACATCAAC TTCACCCCATT GTC
AAGAGCT TCAACAGGAA TGAGTGTTAA CGGTGAGTGT TCTGTAGTG AAGTGGGTAA CAG
TTCTCGA AGTTGTCCTT ACTCACAATT TTCTAGA AAGATCT

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence (SEQ ID NO: 3) encoding the amino acid sequence that constitutes the majority of the Fd fragment of the H chain of the 6D9 antibody.

FIG. 2 shows the nucleotide sequence (SEQ ID NO: 4) encoding the amino acid sequence that constitutes the majority of the L chain of the 6D9 antibody.

FIG. 3 shows the amino acid sequence (SEQ ID NO: 1) that constitutes the majority of the Fd fragment of the H chain of the 6D9 antibody.

FIG. 4 shows the amino acid sequence (SEQ ID NO: 2) that constitutes the majority of the L chain of the 6D9 antibody.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location C12N 9/00 ZNA 9359-4B 15/52 C12P 13/02 8931-4B 21/08 8214-4B / / C12N 15/06 (C12P 21/08 C12R 1:91)

Claims (17)

[Claims] 1. A compound represented by the following formula 4. [Chemical 1] 2. An antibody produced by antigen stimulation using the compound according to claim 1 as a hapten. 3. The antibody according to claim 2, which specifically hydrolyzes a prodrug compound represented by the following formula 9. [Chemical 2] 4. The amino acid sequence of the first hypervariable region in the H chain is represented by the formula: Asn Tyr Ala Met Ser, and the amino acid sequence of the second hypervariable region is represented by the formula: Ser Ser Gly Gly Ser Ile Tyr Tyr Leu Asp Ser. Val Ly
s Gly and the amino acid sequence of the third hypervariable region is represented by the formula: Val Ser His Tyr Asp Gly Ser Arg Asp Trp Tyr Phe As
The antibody according to claim 3, which is represented by p Val. 5. The amino acid sequence of the first hypervariable region in the L chain has the formula: Arg Ser Ser Gln Thr Ile Val His Ser Asn Gly Asp Th
r Tyr Leu Asp, the amino acid sequence of the second hypervariable region is represented by the formula: Lys Val Ser Asn Arg Phe, and the amino acid sequence of the third hypervariable region is represented by the formula: Phe Gln Gly Ser His Val Pro Pro Thr The antibody according to claim 3, which is represented by 6. The amino acid sequence of the first hypervariable region in the H chain is represented by the formula: Asn TyrAla Met Ser, and the amino acid sequence of the second hypervariable region is represented by the formula: Ser Ser Gly Gly Ser Ile Tyr.
Tyr Leu Asp Ser Val Lys Gly, the amino acid sequence of the third hypervariable region is represented by the formula: Val Ser His Tyr Asp Gly Ser
Arg Asp Trp Tyr Phe Asp Val and the amino acid sequence of the first hypervariable region in the L chain is represented by the formula: Arg Ser Ser
Gln Thr Ile Val His Ser Asn Gly Asp Thr Tyr Leu As
The amino acid sequence of the second hypervariable region is represented by the formula: Lys V
The antibody according to claim 3, which is represented by al Ser Asn Arg Phe and whose amino acid sequence of the third hypervariable region is represented by the formula: Phe Gln Gly Ser His Val Pro Pro Thr. 7. The antibody according to any one of claims 2 to 6, wherein the H chain contains the amino acid sequence represented by SEQ ID NO: 1. 8. The antibody according to any one of claims 2 to 6, wherein the L chain contains the amino acid sequence shown by SEQ ID NO: 2. 9. The method according to any one of claims 2 to 6, wherein the H chain contains the amino acid sequence represented by SEQ ID NO: 1 and the L chain contains the amino acid sequence represented by SEQ ID NO: 2. Antibody. 10. The antibody according to claim 7 or claim 9, wherein the H chain is encoded by the nucleotide sequence represented by SEQ ID NO: 3. 11. The antibody according to claim 8 or 9, wherein the L chain is encoded by the nucleotide sequence shown by SEQ ID NO: 4. 12. The amino acid sequence represented by SEQ ID NO: 1. 13. The amino acid sequence shown in SEQ ID NO: 2. 14. A nucleotide sequence represented by SEQ ID NO: 3. 15. A nucleotide sequence represented by SEQ ID NO: 4. 16. A prodrug compound represented by the following formula 9. [Chemical 3] 17. A method for regenerating chloramphenicol by hydrolyzing the prodrug compound according to claim 13 with the antibody according to any one of claims 2 to 8.