JP2826435B2 - Activation of prodrugs by catalytic antibodies - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a catalytic antibody (Catalytic).
Antibody) to activate prodrugs. Specifically, the present invention decomposes a compound (prodrug) chemically modified into a non-pharmacologically active derivative for the purpose of reducing the toxicity or improving the sustainability of a drug, and converting the prodrug to the original pharmacologically active form. It relates to an antibody that is converted into a drug. By using such an antibody in combination with a drug that has been converted into a prodrug, it becomes possible to reduce various problems such as reduction in toxicity of the drug, improvement in persistence, and specificity to a lesion.

[0002]

2. Description of the Related Art Pharmaceuticals having useful pharmacological activity but accompanied by strong side effects and toxicity or unstable and difficult to administer are often required to improve their inconvenience. The prodrugs of pharmaceuticals are converted. Prodrugs have no pharmacological activity by themselves, but after administration, they are degraded by changes in pH in the body or by enzymes in the living body, converted into the original drug, and exert their original pharmacological action. The chemical modification for producing such a prodrug is, for example, an anticancer drug having a strong side effect, which resists enzymes in the living body until the prodrug reaches a target lesion, and is degraded only after reaching the lesion to become 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 degrades and modifies the prodrug in a living body, and when such an enzyme is present in a living body, the reaction specificity of the enzyme , Substrate specificity or biodistribution should be considered. Prodrug candidate compounds obtained by certain types of chemical modification, even if they are the most suitable compounds for stabilizing the target drug or reducing toxicity, show specificity for the compound and enzymes that degrade it If is not naturally occurring, i.e., not present in vivo, the compound cannot be used as a prodrug since it cannot be degraded in vivo. However, if an enzyme exhibiting such a function is artificially produced and administered into a living body, a candidate compound which could not be used conventionally can be used as a prodrug. Therefore, the present inventors have paid attention to this point for the first time in the world, and have conducted intensive research to open up a new field of prodrug research. The present invention provides the desired substrate (prodrug)
The development of biocatalysts that specifically act on steroids greatly promotes the conversion of drugs into drugs that could not be used due to problems such as toxicity, and the chemical modification of lesion-specific prodrugs, and greatly contributes to the medical field. It is a contribution of.

[0003]

The present invention provides, as one of its aspects, a prodrug of an ester derivative of chloramphenicol represented by the following formula 9.

Embedded image It has been confirmed by a biological test using Escherichia coli that the ester derivative (9) of the present invention does not have antibiotic activity. Therefore, the present compound can certainly be used as a prodrug.

Further, the present invention relates to the above ester derivative
(9) An antibody capable of specifically hydrolyzing (9) to regenerate the original chloramphenicol (1). The antibody of the present invention specifically hydrolyzes the above-mentioned prodrug (9) of the present invention according to the following reaction formula to give chloramphenicol.
(1) is generated.

Embedded image 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 produced by immunizing a phosphate derivative (4), which is a transition state analog of a hydrolysis reaction represented by the following formula, as a hapten.

Embedded image

[0005] The basic structure of the antibody of the present invention has a peptide chain structure in which two homologous H chains and two homologous L chains are linked by disulfide bonds and non-covalent bonds. In 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 an H chain comprising the amino acid sequence represented by SEQ ID NO: 2 Containing the above amino acid sequence and L
It also provides an antibody whose chain contains 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 Tyr Leu Asp Ser Val Ly
s Gly and the amino acid sequence of the third hypervariable region is of 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 their hypervariable regions are designated H
The present invention also provides an antibody having a chain and an L chain.

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

Embedded image That is, an acetylated product in which only the primary hydroxyl group is protected by reacting chloramphenicol (1) in acetic anhydride.
After (2), a condensate (3) is obtained by reacting N, N-dicyclohexylcarbodiimide in N-trifluoroacetyl-4-aminobenzylphosphoric acid and pyridine as a condensing agent.
Get. The acetyl group is deprotected with 0.1N sodium hydroxide in acetone, and the desired phosphoric ester is obtained.
Obtain (4).

Next, as preparation for binding the carrier to the above-mentioned phosphoric ester (4), glutaric anhydride is reacted with the carrier in the presence of triethylamine in dimethylformamide to attach a side chain portion, and the carboxylic acid at the terminal is reacted with carboxylic acid. The activated ester (6) is obtained by reacting N-hydroxysuccinide (reaction formula 2).

Embedded image

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

As described above, in this specification, chloramphenicol is used 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 the present invention can be produced using another useful drug. It will also be understood that the antibodies of the present invention may be used as a starting material for producing derivatives that specifically catalyze the reaction represented by the following formula.

Embedded image [Wherein, R represents any residue]

Ester derivatives used as prodrugs
(9) is synthesized according to the synthesis route shown in the following reaction formula 3.

Embedded image

That is, chloramphenicol (1) is reacted with t-butyldimethylsilyl chloride in a mixed solvent of dimethylformamide-dichloromethane to obtain a silyl compound (7) in which a primary hydroxyl group is protected with a silyl group, and then N-triol is added. It was reacted with fluoroacetyl-4-aminophenylacetic acid to obtain a condensate (8). This was deprotected in a 3: 1: 1 mixture of acetic acid: THF: water to give the desired ester derivative (9).

[0012]

Effects and Effects of the Invention The catalytic activity for the hydrolysis reaction of the ester derivative (9) was examined for the 12 kinds of produced antibodies. When the amount of chloramphenicol produced by the hydrolysis was monitored by liquid chromatography (HPLC), catalytic activity was observed in 6 of 15 antibodies. The reaction rate was examined in more detail, and the kinetic amount of the catalytic reaction was determined. The obtained value is Km = 64 × 10 ⁻⁶ M,
kcat = 0.133 min ^-1 (see Example 15).

Next, the resulting antibody was tested for the activation of the prodrug by conversion of the ester derivative (9) to chloramphenicol (1) by a growth inhibition experiment of Escherichia coli. When the paper containing the solution of the antibody and the ester derivative was soaked on the medium and cultured with Escherichia coli, an inhibition circle due to growth inhibition was observed around the paper. When a similar experiment was performed using only the ester derivative (9) or the antibody as a comparative experiment, no inhibition circle was observed. From the above, the antibody obtained by immunization with the phosphate ester (4) as a hapten can catalytically hydrolyze the ester derivative (9), which is a prodrug of chloramphenicol, and furthermore, the ester derivative (9) Was selectively hydrolyzed only by the present antibody and could not be degraded by the hydrolase present in E. coli. Here, activation of a prodrug by an antibody has been proved for the first time in the world. The novel 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 is expected to make a great contribution in the pharmaceutical field. Hereinafter, the present invention will be described in more detail by way of 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 (eluent: ethyl acetate-hexane (1: 1)). The fraction containing the desired product was concentrated under reduced pressure to obtain an oil. Yield: 335 mg
(59%).

Example 2 Synthesis of compound (3) Compound (2) (200 mg, 548 μmol) was added to pyridine (5 ml).
And N-trifluoroacetyl-4-aminobenzylphosphoric acid (258 mg, 1.096 mmol) and N, N-dicyclohexylcarbodiimide (565 mg, 2.739 mmol)
And stirred at 40 ° C. overnight. After dicyclohexylurea was removed by filtration, concentration was performed under reduced pressure, and 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, retention time 180 minutes). Retention time 18
The peak at 0 min was collected and freeze-dried to obtain the desired product. Yield 57 mg (16%). Mass spec: 630 (M ⁺⁺ 1).

EXAMPLE 3 Synthesis of Compound (4) Compound (3) (10 mg, 15.9 μmol) was added to acetone (2.5 mM).
l), cooled to -20 ° C, and cooled to 0.1 N- under ice-cooling.
An aqueous sodium hydroxide solution (2.5 ml) was added, and the mixture was stirred for 1 hour. After neutralization with 1N-hydrochloric acid, the mixture was concentrated under reduced pressure. Residue is H
Purified by PLC (ODS column, 10x250m
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 minutes). The peak having a retention time of 14.8 minutes was collected and freeze-dried to obtain the desired compound (4). Yield: 8.4 mg (78%). Mass spec: 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, 1H), 3.4
9 (dd, J = 5.9, 11.2 Hz, 1H), 3.71 (dd, J =
7.1, 11.2 Hz, 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, 4H), 7.
47, 8.11 (ABq, J = 8.7 Hz, 4H). NMR is C
Measured at D ₃ OD.

Example 4 Synthesis of Compound (5) Compound (4) (20 mg, 34.1 μmol) was dissolved in dimethylformamide (3 ml), and glutaric anhydride (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 the 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 minutes). The peak having 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-hydroxysuccinimide (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 hour. After dicyclohexylurea was removed by filtration, the mixture was 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,
Retention time 11.2 minutes). The peak having a retention time of 11.2 minutes was collected and freeze-dried to obtain the desired compound (6). Yield: 2.4 mg (24%). Mass spec: 799 (M ⁺⁺ 1).

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

Example 7 Synthesis of Compound (7) Compound (1) (4 g, 12.38 mg) was dried in a mixed solvent of dimethylformamide and dichloromethane (5 ml / 100 m2).
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) and stirred at room temperature for 2.5 hours. The reaction solution was extracted with chloroform using a separating funnel, washed with water and dried over magnesium sulfate. After concentration under reduced pressure, the residue was purified by silica gel column chromatography (eluent: ethyl acetate-hexane (1: 4)). The fraction of the desired product was concentrated under reduced pressure to obtain a white solid. Yield: 4.3 g (80%). Mass spec: 437
(M ⁺ ).

Example 8 Synthesis of compound (8) N-trifluoroacetyl-4-aminophenylacetic acid
(319.9 mg, 1.295 mmol) dried solvent mixture of dimethylformamide and dichloromethane (2 ml / 20 ml)
In N, N-dicyclohexylcarbodiimide (1
(67 mg, 809 μmol) and stirred at room temperature for 1.5 hours. The dicyclohexyl urea 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 (eluent: ethyl acetate-hexane (1: 2)).
The fraction of the desired product was concentrated under reduced pressure to obtain a white solid. Yield: 1
32.7 mg (62%). Mass spec: 665 (M ⁺ ).

Example 9 Synthesis of compound (9) 5 ml of a THF solution of compound (8) (482 mg, 723 μmol)
After adding 0.5 ml of water and 15 ml of acetic acid to the
C) for 5 hours. Thereafter, the excess solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate: hexane = 1: 1) to obtain the desired ester derivative (9). Yield: 221.5 mg
(56%). Mass spec: 551 (M−H) ⁻ . 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 = 16.2 Hz, 1 H), 3.77 (d, J = 16.2 H
z, 1H), 4.33 (m, 1H), 6.10 (d, J = 5.5 Hz,
1H), 6.24 (s, 1H), 7.32, 7.60 (ABq, J
= 8.41 Hz, 4H), 7.50, 8.16 (ABq, 8.74
Hz, 4H). NMR was measured by CD ₃ OD.

Example 10 Preparation of Monoclonal Antibody: Immunization antigen (KLH condensate) 50 μg / 50 μl physiological saline was mixed with an equal volume of complete Freund's adjuvant, and the resulting mixture was used as a Balb / c mouse (four weeks old, female). Was injected intraperitoneally. Ten days later, booster immunization was performed with a mixture of 50 μg / 50 μl of physiological saline and an equal volume 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-linked immunosorbent assay (ELISA) using a BSA-condensate (secondary antibody).
It was measured with a biotinylated anti-mouse IgG antibody, avidin, biotinylated peroxidase) to obtain a value of 1:10 ⁶ antibody titer. One month after the second boost, 100 μg of antigen
g / 100 μl physiological saline was administered via the tail vein (final immunization).

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

Example 12 Culture of Hybridoma and Purification of Monoclonal Antibody Twelve hybridomas were cultured in complete medium (10% fetal bovine serum, RPMI medium) for 7 days each, and 1 × 10 ⁷
Balb / c mice (8 weeks) received 500 μl of pristane two weeks ago.
Week-old female), injected intraperitoneally, and 2 weeks later the ascites was
Each 5 ml was collected. The centrifugal supernatant of each ascites was precipitated with ammonium sulfate in an equal volume of a saturated aqueous solution of ammonium sulfate, and then subjected to cation exchange chromatography using an S-Sepharose column and affinity chromatography using a protein G column to obtain 10 to 20 mg of purified antibody, respectively. .

Example 13 Determination of catalytic activity 22.2 μm of each of the 12 purified antibodies obtained in Example 12
M solution (55.6 mM Tris buffer solution, pH 8.0) 90 μ
Leave at room temperature. 10 μl of a dimethyl sulfoxide solution (25 mM) of the substrate and compound (9) was added thereto, and the mixture was quickly shaken. Then, absorption of chloramphenicol by HPLC was performed.
The increase in (278 nm) was followed for 4 hours (ODS column, 4.6 × 250 mm, acetonitrile-0.1% trifluoroacetic acid aqueous solution (1: 1), flow rate 1 ml / min, retention time 4.5 minutes). As a result, six types of antibodies exhibiting catalytic activity were specified, and the antibody having the strongest activity was named 6D9. The hybridoma producing antibody 6D9 has been deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology (Deposit date: November 27, 1992, accession number: Microtechnical Laboratory 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 generating cDNA from it. The obtained results are shown in FIGS.
(Each sequence corresponds to SEQ ID NO: 3 and SEQ ID NO: 4). The underlined portions in the figure are the primer sites used when the cDNA was subjected to the PCR method. Next, the amino acid sequence of the 6D9 antibody was determined by estimating the amino acid from this nucleotide sequence. The obtained results are shown in FIG. 3 and FIG. 4 (the respective sequences correspond to SEQ ID NO: 1 and SEQ ID NO: 2). In the figure, CDR-1, CDR-2 and CDR-
Reference numeral 3 denotes a first hypervariable region, a second hypervariable region, and a third hypervariable region, respectively. The nucleotide sequence described in FIG. 3 is an amino acid sequence that occupies most of the Fd fragment of the 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 thermostat. This is followed by various concentrations of the substrate (0.5 mM, 0.75 mM, 1 mM, 1.2 mM).
10 mM dimethyl sulfoxide solution (5 mM, 1.5 mM)
Was added, and the mixture was shaken quickly, and 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 /
Min, 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 (0.7% agarose, p.
H8.0) and mixed with Bacillus subtilis (B. Subtilis ISW1214), was overlaid on LB agar plates (1.5% agar pH 8.0). After the agar has set, place a paper disc (6 mm diameter) on which 10 μl of 20 μM antibody (6D9) and 10 μl of 100 mM substrate (ester derivative) or 10 μl of 20 μl
M antibody (6D9) or 10 μl of 100 mM substrate (ester derivative) was infiltrated and cultured at 37 ° C. overnight. As a result, a growth inhibition circle could not be formed around the paper disk impregnated with only the antibody (6D9) or the substrate (ester derivative), but the growth inhibition was not achieved when the antibody and the substrate (ester derivative) were simultaneously impregnated. A circle has 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 Gly Gly Gly Gly Gly Gly G 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 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 250 260 270 280 290 300 CTGAGGTCTG AGGACACGGC CATGTATTTC TGTGCAAGAG TCTCCCATTA CGACGGTAGT GACTCCAGAC TCCTGTGCCG GTACATAAAG ACACGTTCTC AGAGGGTAAT GCTGCCATCA 310 320 330 340 350 360 CGCGACTGGT ACTTCGATGT CTGGGGC 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: number of nucleic acid chains: double-stranded Topology: linear Sequence type: cDNA to mRNA sequence 10 20 30
40 50 60 GAGCTCGTGA TGACCCAGAC TCCACTCTCC CTG
CCTGTCA GTCTTGGAGA TCAAGCCTCC CTCGAGCACT ACTGGGTCTG AGGTGGAGAGG GAC
GGACAGT CAGAACCCTCT AGTTCGGAGG 70 80 90
100 110 120 ATCTCTTGCA GATCTAGTCA GACCATTGTA CAT
AGTAATG GAGACACGTA TTTAGATTGG TAGAGAACGT CTAGATCAGT CTGGTAACAT GTA
TCATTAC CCTTGTGCAT AAATCTAACC 130 140 150
160 170 180 TTCCTGCGAGA AACCAGGCCA GTTCCCAAAG CTC
CTGATCT ACAAAGTTTTC CAACCGATTT AAGGACGTCT TTGGTCCGGT CAGAGGTTTC GAG
GACTTAGA TGTTTCAAAAG GTTGGCTAAA 190 200 210
220 230 240 TCTGGGGTCC CAGACAGGTT CAGTGGCAGT GGA
TCAGGGA CAGATTTCAC ACTCAAGATC AGACCCCAGGG GTCTGTCCAA GTTCACCGTCA CCT
AGTCCCT GTTCAAAGTG TGAGTCTAG 250 260 270
280 290 300 AGCAGAGTGG AGGCTGAGGGA TCTGGGAGGTTT TAT
TACTGCT TTCAAGGTTC ACATGTTCCT TCGTCTCACC TCCGACTCCT AGACCCTCAA ATA
ATGACGA AAGTTTCCAAG TGTACAAGGA 310 320 330
340 350 360 CCGACGTTCG GTGGAGGCAC CAAGTTTGGAA ATC
AAACGGG CTGATGCTGC ACCAACTGTA GGCTGCAAGC CACCTCCGTGG GTTCAACCTT TAG
TTTGCCCC GACTACGACG TGGTTGACAT 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 TCTACCCCAAGACACATCAAT GTC
AAGTGGA AGATTGATGG CAGTGAACGA AACTTGTTGA AGATGGGGTT TCTGTAGTTTA CAG
TTCACCT TCTAACTACC GTCACTTGCT 490 500 510
520 530 540 CAAAATGGCG TCCTGAACAG TTGGACTGAT CAG
GACAGCA AAGACAGCAC CTACAGCATG GTTTTACGC AGGACTTGTC AACCTGACTA GTC
CTGTCGT TTCTGTCGTG GATGTCCGTAC 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 GCCACTCACA AGACATCAAC TTCACCCATT GTC
AAGAGCT TCAACAGGAA TGAGTGTTAA CGGTGAGTGT TCTGTAGTTTG AAGTGGGTAA CAG
TTCTCGA AGTTGTCCTT ACTCACAATT TTCTAGA AAGATCT

[Brief description of the drawings]

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

FIG. 3 shows the amino acid sequence (SEQ ID NO: 1) constituting 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) constituting the majority of the L chain of the 6D9 antibody.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12P 21/08 C12P 21/08 // C12N 15/02 C12N 15/00 A 15/09 C (C12P 21/08 C12R 1:91 )

Claims (17)

(57) [Claims] 1. A compound represented by the following formula 4. Embedded image 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. Embedded image 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 Tyr Leu Asp Ser Val Ly
s Gly and the amino acid sequence of the third hypervariable region is of 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 light 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
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, wherein the antibody is represented by al 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. 7. The antibody according to claim 2, wherein the H chain contains the amino acid sequence represented by SEQ ID NO: 1. 8. The antibody according to claim 2, wherein the L chain comprises the amino acid sequence represented by SEQ ID NO: 2. 9. The method according to claim 2, wherein the H chain has the amino acid sequence represented by SEQ ID NO: 1, and the L chain has the amino acid sequence represented by SEQ ID NO: 2. Antibodies. 10. The antibody according to claim 7, wherein the H chain is encoded by the nucleotide sequence represented by SEQ ID NO: 3. 11. The antibody according to claim 8, wherein the L chain is encoded by the nucleotide sequence represented by SEQ ID NO: 4. 12. An amino acid sequence represented by SEQ ID NO: 1. 13. An amino acid sequence represented by 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 compound represented by the following formula 9: Embedded image 17. The compound according to claim 16, wherein the compound is
A method for producing chloramphenicol by hydrolysis using the antibody according to any one of claims 1 to 8.