CN116284356A

CN116284356A - Neutralizing antibody P5-1H1 for broad spectrum neutralization of SARS-CoV-2 and application thereof

Info

Publication number: CN116284356A
Application number: CN202310046051.7A
Authority: CN
Inventors: 张林琦; 张政; 张绮; 鞠斌; 陈鹏; 周兵
Original assignee: Tsinghua University; Third Peoples Hospital of Shenzhen
Current assignee: Tsinghua University; Third Peoples Hospital of Shenzhen
Priority date: 2023-01-30
Filing date: 2023-01-30
Publication date: 2023-06-23

Abstract

The invention discloses a neutralization antibody P5-1H1 for broad spectrum neutralization of SARS-CoV-2 and application thereof. The invention provides an IgG antibody, named as P5-1H1 antibody, which consists of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region in the heavy chain are set forth in SEQ ID NO:1, 26 th to 33 th, 51 th to 57 th and 96 th to 106 th are shown; CDR1, CDR2 and CDR3 in the light chain variable region in the light chain are set forth in SEQ ID NO:4, 27 th to 32 th, 50 th to 52 th and 89 th to 96 th. The P5-1H1 antibody provided by the invention has the effect of broad-spectrum neutralization of SARS-CoV-2, and has strong neutralization capability on wild novel coronaviruses and natural variant strains. The invention has great application value for the prevention and control of novel coronaviruses and can generate profound social significance.

Description

Neutralizing antibody P5-1H1 for broad spectrum neutralization of SARS-CoV-2 and application thereof

Technical Field

The invention belongs to the field of biotechnology, and relates to a neutralization antibody P5-1H1 for broad-spectrum neutralization of SARS-CoV-2 and application thereof.

Background

The new coronavirus infection is caused by the new coronavirus infection, the main symptoms are low fever, hypodynamia and dry cough, few patients also have symptoms of upper respiratory tract and digestive tract such as nasal obstruction, nasal discharge, diarrhea and the like, and severe patients can develop acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulation dysfunction and multiple organ failure. Novel coronaviruses (SARS-CoV-2) belong to the genus beta coronavirus.

SARS-CoV-2 produces many mutations during its transmission process, enhancing its transmission and immune escape, and currently the main popular natural mutants include Alpha strain found in UK, beta strain found in south Africa, gamma strain found in Brazil, etc. Vaccine immunity against the novel coronavirus is also affected to a certain extent, neutralizing activity of the inactivated vaccine immune serum against the south African mutant is reduced by half, neutralizing activity of the mRNA vaccine immune serum against the south African mutant is reduced by about 10 times, and neutralizing activity of a plurality of monoclonal antibodies against the mutant is weakened or lost.

The monoclonal antibody can be industrially produced in large quantity, has high affinity and high specificity for combining with antigen, and greatly reduces adverse reaction in clinical application. While antibody molecules may be engineered to increase their antiviral efficacy. Antibodies, with their specificity and flexibility of use, are very promising approaches in the treatment of infectious diseases. There is a need to address the challenges presented by the natural mutant strains of novel coronaviruses, and the development of broad-spectrum humanized monoclonal antibodies against novel coronaviruses would provide more effective means for the prevention and treatment of infection by the natural mutant strains of novel coronaviruses.

Disclosure of Invention

The invention aims to provide a neutralizing antibody P5-1H1 for broad spectrum neutralization of SARS-CoV-2 and application thereof.

The invention provides an IgG antibody, named as P5-1H1 antibody, which consists of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region in the heavy chain are set forth in SEQ ID NO:1, 26 th to 33 th, 51 th to 57 th and 96 th to 106 th are shown; CDR1, CDR2 and CDR3 in the light chain variable region in the light chain are set forth in SEQ ID NO:4, 27 th to 32 th, 50 th to 52 th and 89 th to 96 th.

Specifically, the heavy chain variable region is shown in SEQ ID NO: 1.

Specifically, the light chain variable region is shown in SEQ ID NO: 4.

Specifically, the heavy chain is as follows (a) or (b): (a) SEQ ID NO:2 from position 20 to 466; (b) SEQ ID NO:2, and a protein represented by formula 2.

Specifically, the light chain is as follows (c) or (d): (c) SEQ ID NO:5 from position 20 to position 232; (d) SEQ ID NO: 5.

Genes encoding the IgG antibodies are also within the scope of the invention.

Specifically, the gene encoding the heavy chain is (1) or (2) as follows:

(1) SEQ ID NO:3 from nucleotide 949 to 2292;

(2) SEQ ID NO:3 from nucleotide 892 to 2292.

Specifically, the gene encoding the light chain is as follows (3) or (4):

(3) SEQ ID NO:6 from nucleotide 1095 to nucleotide 1736;

(4) SEQ ID NO:6 from nucleotide 1038 to nucleotide 1736.

The invention also provides the application of any of the IgG antibodies in preparing medicaments for inhibiting novel coronaviruses.

The invention also provides a medicine for inhibiting novel coronavirus, and the active ingredient of the medicine is any one of the IgG antibodies.

The invention also protects the application of any of the IgG antibodies in preparation of medicaments for neutralizing novel coronaviruses.

The invention also provides a medicine for neutralizing novel coronavirus, and the active ingredient of the medicine is any one of the IgG antibodies.

The invention also provides the application of any of the IgG antibodies in preparing medicaments for preventing and/or treating the new crown infection.

The invention also provides a medicine for preventing and/or treating the new crown infection, and the active ingredient of the medicine is any IgG antibody.

Any of the above described novel coronavirus infections are novel coronavirus infections.

Any of the above novel coronaviruses is a wild-type novel coronavirus or a natural variant of a wild-type novel coronavirus.

Any of the above novel coronaviruses is a novel coronavirus D614G mutant.

Any of the above novel coronaviruses is a novel coronavirus wild strain, a novel coronavirus Alpha strain, a novel coronavirus Beta strain, a novel coronavirus Gamma strain, a novel coronavirus Delta plus strain, a novel coronavirus Mu strain or a novel coronavirus Omiko strain.

Specifically, the novel coronavirus omnikon strain is a novel coronavirus omnikon BA.1 strain, a novel coronavirus omnikon BA.2.12.1 strain, a novel coronavirus omnikon BA.2.75 strain, a novel coronavirus omnikon BA.3 strain or a novel coronavirus omnikon BA.4/5 strain.

The invention uses spike protein of novel coronavirus as bait, and screens memory B cells generated by antibody from peripheral blood mononuclear cells of infected persons to obtain monoclonal antibody which can be specifically combined with spike protein and is named as P5-1H1 antibody. The P5-1H1 antibody provided by the invention has the effect of broad-spectrum neutralization of SARS-CoV-2, and has strong neutralization capability on wild novel coronaviruses and natural variant strains. The invention has great application value for the prevention and control of novel coronaviruses and can generate profound social significance.

Drawings

FIG. 1 shows the neutralizing activity of the P5-1H1 antibody against novel coronavirus pseudoviruses.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged. 293F cells and 293T cells are commercially available human embryonic kidney epithelial cells. The pMD18-T vector is a commercially available plasmid vector. pcDNA3.1 (+) vector: invitrogen, cat# V790-20.

hACE2-hela cells (i.e., "HeLa cell lines stably expressing the ACE2 molecules" in the literature) are described in the following literature: wang, r., zhang, q., ge, j, ren, w, zhang, r, lan, j, ju, b, su, b, yu, f, chen, p, liao, h, feng, y, li, X, shi, X, zhang, z, zhang, f, din, q, zhang, t, wang, X, zhang, l.analysis of SARS-CoV-2variant mutations reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species.Immunity 54,1611-1621.e1615, doi:10.1016/j.immini.2021.06.003 (2021).

Example 1 discovery and preparation of P5-1H1 antibodies

1. Discovery of P5-1H1 antibodies

Separating memory B cells from peripheral blood mononuclear cells of SARS-CoV-2 convalescence patient, and amplifying the antibody genes of memory B cells to obtain antibody sequence. Through a large number of alignments, analyses, preparations and effect verifications, the inventors of the present invention discovered 1 new SARS-CoV-2IgG antibody with excellent activity, which was designated as P5-1H1 antibody.

The amino acid sequence of the heavy chain variable region of the P5-1H1 antibody is shown in SEQ ID NO:1 (CDR 1, CDR2 and CDR3 are located at positions 26-33, 51-57 and 96-106 in this order; CDR1, CDR2 and CDR3 are GITVSSNY, IYSGGST, ARDLQVYGMDV in this order). The amino acid sequence of the full-length heavy chain of the P5-1H1 antibody is shown in SEQ ID NO:2 is shown in the figure; SEQ ID NO:2, amino acid residues 1-19 constitute the signal peptide (leading the protein to be secreted outside the cell), amino acid residues 20-136 constitute the heavy chain variable region, and amino acid residues 137-466 constitute the heavy chain constant region.

The amino acid sequence of the light chain variable region of the P5-1H1 antibody is shown in SEQ ID NO:4 (CDR 1, CDR2 and CDR3 are located at positions 27-32, 50-52 and 89-96 in this order; CDR1, CDR2 and CDR3 are QGISNF, AAS, QHLNDYPL in this order). The amino acid sequence of the full-length light chain of the P5-1H1 antibody is shown in SEQ ID NO:5 is shown in the figure; SEQ ID NO:5, amino acid residues 1-19 constitute the signal peptide (the guide protein is secreted outside the cell), amino acid residues 20-125 constitute the light chain variable region, and amino acid residues 126-232 constitute the light chain constant region.

2. Preparation of P5-1H1 antibodies

Construction of recombinant plasmid

The heavy chain DNA molecule is a double-chain DNA molecule, such as SEQ ID NO: 3. SEQ ID NO:3, the promoter is composed of nucleotides 1 to 891, the full-length heavy chain is encoded by nucleotides 892 to 2292, and the terminator is composed of nucleotides 2293 to 2438. The heavy chain DNA molecule is inserted into the pMD18-T vector to obtain a heavy chain expression vector.

The light chain DNA molecule is a double-chain DNA molecule, such as SEQ ID NO: shown at 6. SEQ ID NO:6, nucleotides 1 to 1037 constitute the promoter, nucleotides 1038 to 1736 encode the full length light chain, and nucleotides 1737 to 1884 constitute the terminator. The light chain DNA molecule is inserted into the pMD18-T vector to obtain a light chain expression vector.

Construction of recombinant cells

And co-transfecting 293F cells with the heavy chain expression vector and the light chain expression vector to obtain recombinant cells.

Preparation of (III) antibodies

1. Culturing the recombinant cells obtained in the step (II) in a DMEM medium containing 2% fetal bovine serum for 72 hours, centrifuging at 4 ℃ and 4000rpm for 30 minutes, and collecting supernatant.

2. Affinity chromatography

Chromatographic column specification for affinity chromatography: 3cm in length and 1cm in inner diameter;

column packing for affinity chromatography: protein A tags (Thermo, catalog number 10006D);

the operation steps are as follows: (1) loading 300mL of the supernatant obtained in the step 1 onto an affinity chromatography column, and incubating at 4 ℃ for 16 hours; (2) washing the column with 60ml of binding buffer; (3) the target protein was eluted with 30mL of elution buffer and the post-column solution was collected.

Binding buffer: 112.6g of glycine and 175.2g of sodium chloride are taken, dissolved in water and fixed to a volume of 1L by water, and the pH is adjusted to 8.0 by sodium hydroxide.

Elution buffer: 7.5g of glycine was taken, dissolved in water and brought to a volume of 500ml with water and adjusted to pH 3.0 with hydrochloric acid.

3. The post-column solution obtained in the step 2 was concentrated by an ultrafiltration concentration tube and the system was replaced with PBS buffer (pH 7.2, 10 mM) to obtain a P5-1H1 antibody solution (antibody concentration: about 1 mg/ml).

Example 2 neutralization test

1. Preparation of novel coronavirus pseudoviruses

The plasmids expressing the novel coronavirus membrane proteins and the skeleton plasmid pNL 4-3R-E-luciferases are transfected into 293T cells, and after incubation, the novel coronavirus pseudovirus with infectivity but no replication capacity can be obtained, and the infectivity of the novel coronavirus pseudovirus is similar to that of the novel coronavirus live virus. The backbone plasmid pNL 4-3R-E-luciferases, i.e.the backbone plasmid pNL4-3R-E containing luciferases (i.e. vector with the Luciferase gene containing backbone pNL4-3R-E in the literature) is described in the following literature: wang Q, liu L, ren W, getlie a, wang H, liang Q, shi X, montefiori DC, zhou T, zhang l.cell rep.2019.

The encoding gene of the novel coronavirus membrane protein is inserted between BamHI and EcoRI cleavage sites of pcDNA3.1 (+) vector to obtain a plasmid for expressing the novel coronavirus membrane protein. The plasmids expressing the novel coronavirus membrane protein and the skeleton plasmid pNL 4-3R-E-luciferases are co-transfected into 293T cells, the cells are subjected to standing incubation at 37 ℃ (adopting DMEM culture medium containing 10% bovine serum), and cell culture supernatant is collected after transfection for 60 hours, thus obtaining the virus liquid containing novel coronavirus pseudoviruses. Virus solutions of pseudoviruses of 12 novel coronavirus strains were prepared respectively. The 12 novel coronavirus strains refer to novel coronavirus D614G mutant strain, novel coronavirus Alpha strain, novel coronavirus Beta strain, novel coronavirus Gamma strain, novel coronavirus Delta plus strain, novel coronavirus Mu strain, novel coronavirus Omikon BA.1 strain, novel coronavirus Omikon BA.2.12.1 strain, novel coronavirus Omikon BA.2.75 strain, novel coronavirus Omikon BA.3 strain and novel coronavirus Omikon BA.4/5 strain.

The membrane protein of the novel coronavirus wild strain (WT) is shown in SEQ ID NO: shown at 7. The encoding genes of the membrane proteins of the novel coronavirus wild strain are described in GenBank: the 21563-25384 bits in the whole genome in MN908947.3 (18-MAR-2020).

The membrane protein of the novel coronavirus D614G mutant strain is mutated relative to the membrane protein of the wild strain as follows: D614G. The encoding gene of the membrane protein of the novel coronavirus D614G mutant strain is shown as SEQ ID NO: shown at 8.

The membrane protein of the novel coronavirus Alpha strain (Pango linear b.1.1.7) was mutated as follows relative to the membrane protein of the wild strain: 69-70del (HV), 144del (Y), N501Y, A570D, D614G, P681H, T716I, S982A, D1118H. The encoding gene of the membrane protein of the novel coronavirus Alpha strain is described in GISAID: EPI_ISL_601443.

The membrane protein of the novel coronavirus Beta strain (Pango linear B.1.351) was mutated as follows relative to the membrane protein of the wild strain: L18F, D80A, D G, 242-244del (LLA), S305T, K417N, E484K, N501Y, D G, A701V. The encoding gene of the membrane protein of the novel coronavirus Beta strain is described in GISAID: EPI_ISL_700450.

The membrane protein of the novel coronavirus Gamma strain (Pango linear P.1) has the following mutation relative to the membrane protein of the wild strain: L18F, T20N, P S, D138Y, R S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F. The encoding gene of the membrane protein of the novel coronavirus Gamma strain is described in GISAID: EPI_ISL_792681.

The membrane protein of the novel coronavirus Delta strain (Pango linear B.1.617.2) was mutated as follows relative to the membrane protein of the wild strain: T19R, G D, 156-157del (EF), R158G, A222V, L452R, T478K, D614G, P681R, D950N. The encoding gene of the membrane protein of the novel coronavirus Delta strain is described in GISAID: EPI_ISL_1534938.

The membrane protein of the novel coronavirus Delta plus strain (Pango linear AY. X) has increased the following mutations relative to the membrane protein of the Delta strain: K417N. The encoding gene of the membrane protein of the novel coronavirus Delta plus strain is described in GISAID: EPI_ISL_3019629.

The membrane protein of the novel coronavirus Mu strain (Pango linear b.1.621) was mutated as follows relative to the membrane protein of the wild strain: T95I, Y144T, Y145S, ins146N, R346K, E484K, N501Y, D G, P681H, D950N. The gene encoding the membrane protein of the novel coronavirus Mu strain is described in GISAID: EPI_ISL_3987640.

The membrane protein of the novel coronavirus amikau ba.1 strain (Pango linear ba.1) was mutated as follows relative to the membrane protein of the wild strain: a67V, Δ69-70 (HV), T95I, G142D, Δ143-145 (VYY), Δ211 (N), L212I, ins EPE, G339D, S371, 373P, S375F, K417N, N K, G446, 339 477N, T, K, E, 484A, Q, 4982, 496S, Q498, 4837, R, N614, 655, 679, R, N681, 764, R, N, 856, 5294, R, N, 5297 969, 5297, 981F. The encoding gene of the membrane protein of the novel coronavirus Omikovia BA.1 strain is recorded in GISAID: EPI_ISL_6752027.

The membrane protein of the novel coronavirus amikau strain ba.2.12.1 (Pango linear ba.2.12.1) was mutated as follows relative to the membrane protein of the wild strain: T19I, 24-26del (LPP) a27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N79440 452Q, S477N, T478A, Q498R, N493A, Q498 4837Y, Y614Y, Y655Y, Y679Y, Y681Y, Y764Y, Y796Y, Y954Y, Y969K. The encoding gene of membrane protein of novel coronavirus Omikou BA.2.12.1 strain is recorded in GISAID: EPI_ISL_12560123.

The membrane protein of the novel coronavirus amikau strain BA.2.75 has the following mutation relative to the membrane protein of a wild strain: T19I, del-26 (LPP) a 27I, del 142I, del 147I, del 152I, del 157I, del 213I, del 257I, del 339I, del 371I, del 373I, del 375I, del 376I, del 52405I, del 408I, del 417I, del 440I, del 446I, del 460I, del 477I, del 478I, del 484I, del 498I, del 501I, del 505I, del 655I, del 679I, del 681I, del 764I, del 796 5297 954I, del 969K. The encoding gene of membrane protein of novel coronavirus Omikou BA.2.75 strain is recorded in GISAID: EPI_ISL_14393635.

The membrane protein of the novel coronavirus amikau strain ba.3 (Pango linear ba.3) was mutated as follows relative to the membrane protein of the wild strain: a67V, del69-70 (HV), T95I, G142D, del143-145 (VYY), 211del (N), L212I, G339D, S371F, S373P, S375F, D405 676767N, N440K, G446, 477N, T4735K, E484A, Q493R, Q498R, N Y, Y614Y, Y655Y, Y679Y, Y681 764Y, Y796 5297 954Y, Y969K. The encoding gene of the membrane protein of the novel coronavirus Omikovia BA.3 strain is described in GISAID: EPI_ISL_7740765.

The membrane protein of the novel coronavirus Omikou BA.4/5 strain (Pango linear BA.4) has the following mutation relative to the membrane protein of the wild strain: T19I, 24-26del (LPP), A27S, del69-70 (HV) G142D, V213G, G339D, S371 54373 63373F, S373F, T376A, D79405 79408 405-417 3575 417N, N3546S, L452R, S477N, T478K, E484K, E486K, E498K, E501K, E505K, E614K, E655K, E679K, E681K, E764K, E796 5297 969K. The encoding gene of membrane protein of novel coronavirus Omikou BA.4/5 strain is recorded in EPI_ISL_12559461.

2. Detection of neutralizing Activity of antibodies

Test virus liquid: the virus solutions of pseudoviruses of the 12 novel coronavirus strains prepared in the step one respectively.

1. The P5-1H1 antibody solution prepared in example 1 was diluted with PBS buffer (pH 7.2, 10 mM) to obtain various concentrations of antibody dilutions.

2. A96-well cell culture plate was prepared, and 100. Mu.l of the antibody diluent and 50. Mu.l of the test virus liquid (50. Mu.l of the virus liquid had a virus concentration of 1X 10) were added to each well ⁴ TCID 50/ml), and incubating at 37℃for 1 hour. An equal volume of PBS buffer (pH 7.2, 10 mM) was used instead of the antibody dilution as a virus control. An equal volume of DMEM medium containing 10% fetal bovine serum was used as a cell control instead of the test virus solution.

3. After completion of step 2, the cell culture plate was taken and 100. Mu.l of hACE2-hela cell suspension (the solvent used for preparing the cell suspension was DMEM medium containing 10% fetal bovine serum, and the concentration of hACE2-hela cells in the cell suspension was 2X 10) was inoculated per well ⁵ Individual cells/ml), and incubated at 37℃for 64 hours.

4. After completion of step 3, the cell culture plate was removed by pipetting the supernatant, 150 μl of lysate (microgella biotechnology, cat No. T003) was added to each well, and incubated at 37 ℃ for 5 minutes by standing.

5. After step 4 is completed, the cell culture plate is taken and luciferase activity is detected.

A plurality of duplicate wells are provided for each process.

Neutralization activity (%) = [1- (fluorescence intensity of test group-fluorescence intensity of cell control)/(fluorescence intensity of virus control-fluorescence intensity of cell control) ] ×100%.

The neutralization activity results are shown in FIG. 1. In fig. 1: the ordinate is neutralization activity (%); the abscissa is the base 10 log of the antibody concentration (μg/ml), which refers to the concentration of the antibody in the mixed system of 100 μl of the antibody dilution and 50 μl of the test virus liquid in step 2.

The concentration of the antibody at which the neutralizing activity was 50%, i.e., the IC50 value of the antibody, was calculated using Prism 5 software.

IC50 values (in ng/ml) of the P5-1H1 antibody against pseudoviruses of the 12 novel coronavirus strains are shown in Table 1.

TABLE 1

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. An IgG antibody consisting of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region in the heavy chain are set forth in SEQ ID NO:1, 26 th to 33 th, 51 th to 57 th and 96 th to 106 th are shown; CDR1, CDR2 and CDR3 in the light chain variable region in the light chain are set forth in SEQ ID NO:4, 27 th to 32 th, 50 th to 52 th and 89 th to 96 th.

2. The IgG antibody of claim 1 wherein:

the heavy chain variable region is shown in SEQ ID NO:1 is shown in the specification;

the light chain variable region is shown in SEQ ID NO: 4.

3. The IgG antibody of claim 2 wherein:

the heavy chain is as follows (a) or (b): (a) SEQ ID NO:2 from position 20 to 466; (b) SEQ ID NO: 2;

the light chain is as follows (c) or (d): (c) SEQ ID NO:5 from position 20 to position 232; (d) SEQ ID NO: 5.

4. A gene encoding the IgG antibody of any one of claims 1 to 3.

5. Use of an IgG antibody as claimed in claim 1 or 2 or 3 in the manufacture of a medicament for inhibiting novel coronaviruses.

6. A medicament for inhibiting novel coronaviruses, which comprises the IgG antibody of claim 1, 2 or 3 as an active ingredient.

7. Use of an IgG antibody as claimed in claim 1 or 2 or 3 in the manufacture of a medicament for neutralising a novel coronavirus.

8. A medicament for neutralizing a novel coronavirus, which comprises the IgG antibody of claim 1 or 2 or 3 as an active ingredient.

9. Use of an IgG antibody according to claim 1 or 2 or 3 for the manufacture of a medicament for the prevention and/or treatment of a neocrown infection.

10. A medicament for preventing and/or treating a neocrown infection, which comprises the IgG antibody of claim 1 or 2 or 3 as an active ingredient.