ANTI-ALPHA-HEMOLYSIN ANTIBODY AND USE THEREOF

Abstract

Provided are an antibody that binds to Staphylococcus aureus alpha-hemolysin or a fragment thereof, and the use of the antibody or the fragment thereof for preventing or treating Staphylococcus aureus infections. The antibody is obtained through screening by means of the strategies of attenuated immunity and virulent screening of alpha-hemolysin, and the antibody has high affinity to alpha-hemolysin, can effectively block the hemolysis effect of alpha-hemolysin, has proved significant protective or therapeutic effects in the alpha-hemolysin sepsis model, MRSA bacteremia model and MRSA lung infection model, and has a synergistic effect with antibiotics, which is a beneficial supplement to existing antibiotic therapy for Staphylococcus aureus.

Claims

1. A method for preparing an anti-alpha-hemolysin monoclonal antibody, including steps as follows: (1) immunizing animals with an attenuated alpha-hemolysin or an alpha-hemolysin without virulence; (2) taking splenocytes and preparing hybridomas; (3) screening an antibody having high affinity to virulent alpha-hemolysin or wild-type alpha-hemolysin using the same; (4) selecting an antibody which has an activity of inhibiting erythrocyte lysis caused by virulent alpha-hemolysin or wild-type alpha-hemolysin through an assay of inhibition of erythrocyte lysis.

2. The method for preparing an anti-alpha-hemolysin monoclonal antibody according to claim 1, wherein the virulent alpha-hemolysin or wild-type alpha-hemolysin is Staphylococcus aureus alpha-hemolysin, and preferably has an amino acid sequence as shown in SEQ ID NO: 78; and the attenuated alpha-hemolysin or alpha-hemolysin without virulence is alpha-hemolysin with H35L, and preferably has an amino acid sequence as shown in SEQ ID NO: 80.

3. The method for preparing an anti-alpha-hemolysin monoclonal antibody according to claim 1, wherein the virulent alpha-hemolysin or wild-type alpha-hemolysin, the attenuated alpha-hemolysin, or the alpha-hemolysin without virulence is independently linked to a purification tag, a carrier protein, and/or an adjuvant molecule; and the linkage includes coupling, cross-linking, conjugation, and/or fusion.

4. An anti-alpha-hemolysin monoclonal antibody prepared by the method according to claim 1, wherein the antibody has at least one activity selected from the group consisting of blocking hemolytic effect of alpha-hemolysin, blocking damage of alpha-hemolysin to lung epithelial cells, reducing bacterial load in MRSA infected lung tissue, prolonging survival time of a patient suffering from MRSA bacteremia, and preventing or alleviating sepsis caused by alpha-hemolysin.

5. An anti-alpha-hemolysin monoclonal antibody prepared by the method according to any one of claims 1 to 3 claim 1, wherein the antibody has VH-CDR1: selected from the group consisting of SEQ ID NOs: 1, 2, 3, 12, 13, 14, 22, 23, 24, 34, 35, and 36; VH-CDR2: selected from the group consisting of SEQ ID NOs: 4, 5, 6, 15, 16, 17, 25, 26, 27, 28, 37, 38, and 39; VH-CDR3: selected from the group consisting of SEQ ID NOs: 7, 18, 29, and 40; VL-CDR1: selected from the group consisting of SEQ ID NOs: 8, 19, 30, 31, and 41; VL-CDR2: selected from the group consisting of SEQ ID NOs: 9, 11, 20, 32, and 42; and VL-CDR3: selected from the group consisting of SEQ ID NOs: 10, 21, 33, and 43.

6. An antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of CDRs (VH-CDR1, VH-CDR2, VH-CDR3; and VL-CDR1, VL-CDR2, VL-CDR3) selected from the group consisting of: (1) VH-CDR1 as shown in SEQ ID NO: 1, VH-CDR2 as shown in SEQ ID NO: 4, and VH-CDR3 as shown in SEQ ID NO: 7; and, VL-CDR1 as shown in SEQ ID NO: 8, VL-CDR2 as shown in SEQ ID NO: 9, and VL-CDR3 as shown in SEQ ID NO: 10; (2) VH-CDR1 as shown in SEQ ID NO: 2, VH-CDR2 as shown in SEQ ID NO: 5, and VH-CDR3 as shown in SEQ ID NO: 7; and, VL-CDR1 as shown in SEQ ID NO: 8, VL-CDR2 as shown in SEQ ID NO: 9, and VL-CDR3 as shown in SEQ ID NO: 10; (3) VH-CDR1 as shown in SEQ ID NO: 3, VH-CDR2 as shown in SEQ ID NO: 6, and VH-CDR3 as shown in SEQ ID NO: 7; and, VL-CDR1 as shown in SEQ ID NO: 8, VL-CDR2 as shown in SEQ ID NO: 9, and VL-CDR3 as shown in SEQ ID NO: 10; (4) VH-CDR1 as shown in SEQ ID NO: 2, VH-CDR2 as shown in SEQ ID NO: 6, and VH-CDR3 as shown in SEQ ID NO: 7; and, VL-CDR1 as shown in SEQ ID NO: 8, VL-CDR2 as shown in SEQ ID NO: 9, and VL-CDR3 as shown in SEQ ID NO: 10; (5) VH-CDR1 as shown in SEQ ID NO: 2, VH-CDR2 as shown in SEQ ID NO: 6, and VH-CDR3 as shown in SEQ ID NO: 7; and, VL-CDR1 as shown in SEQ ID NO: 8, VL-CDR2 as shown in SEQ ID NO: 11, and VL-CDR3 as shown in SEQ ID NO: 10; (6) VH-CDR1 as shown in SEQ ID NO: 12, VH-CDR2 as shown in SEQ ID NO: 15, and VH-CDR3 as shown in SEQ ID NO: 18; and, VL-CDR1 as shown in SEQ ID NO: 19, VL-CDR2 as shown in SEQ ID NO: 20, and VL-CDR3 as shown in SEQ ID NO: 21; (7) VH-CDR1 as shown in SEQ ID NO: 13, VH-CDR2 as shown in SEQ ID NO: 16, and VH-CDR3 as shown in SEQ ID NO: 18; and, VL-CDR1 as shown in SEQ ID NO: 19, VL-CDR2 as shown in SEQ ID NO: 20, and VL-CDR3 as shown in SEQ ID NO: 21; (8) VH-CDR1 as shown in SEQ ID NO: 14, VH-CDR2 as shown in SEQ ID NO: 17, and VH-CDR3 as shown in SEQ ID NO: 18; and, VL-CDR1 as shown in SEQ ID NO: 19, VL-CDR2 as shown in SEQ ID NO: 20, and VL-CDR3 as shown in SEQ ID NO: 21; (9) VH-CDR1 as shown in SEQ ID NO: 13, VH-CDR2 as shown in SEQ ID NO: 17, and VH-CDR3 as shown in SEQ ID NO: 18; and, VL-CDR1 as shown in SEQ ID NO: 19, VL-CDR2 as shown in SEQ ID NO: 20, and VL-CDR3 as shown in SEQ ID NO: 21; (10) VH-CDR1 as shown in SEQ ID NO: 22, VH-CDR2 as shown in SEQ ID NO: 25, and VH-CDR3 as shown in SEQ ID NO: 29; and, VL-CDR1 as shown in SEQ ID NO: 30, VL-CDR2 as shown in SEQ ID NO: 32, and VL-CDR3 as shown in SEQ ID NO: 33; (11) VH-CDR1 as shown in SEQ ID NO: 23, VH-CDR2 as shown in SEQ ID NO: 26, and VH-CDR3 as shown in SEQ ID NO: 29; and, VL-CDR1 as shown in SEQ ID NO: 30, VL-CDR2 as shown in SEQ ID NO: 32, and VL-CDR3 as shown in SEQ ID NO: 33; (12) VH-CDR1 as shown in SEQ ID NO: 24, VH-CDR2 as shown in SEQ ID NO: 27, and VH-CDR3 as shown in SEQ ID NO: 29; and, VL-CDR1 as shown in SEQ ID NO: 30, VL-CDR2 as shown in SEQ ID NO: 32, and VL-CDR3 as shown in SEQ ID NO: 33; (13) VH-CDR1 as shown in SEQ ID NO: 23, VH-CDR2 as shown in SEQ ID NO: 27, and VH-CDR3 as shown in SEQ ID NO: 29; and, VL-CDR1 as shown in SEQ ID NO: 30, VL-CDR2 as shown in SEQ ID NO: 32, and VL-CDR3 as shown in SEQ ID NO: 33; (14) VH-CDR1 as shown in SEQ ID NO: 23, VH-CDR2 as shown in SEQ ID NO: 28, and VH-CDR3 as shown in SEQ ID NO: 29; and, VL-CDR1 as shown in SEQ ID NO: 31, VL-CDR2 as shown in SEQ ID NO: 32, and VL-CDR3 as shown in SEQ ID NO: 33; (15) VH-CDR1 as shown in SEQ ID NO: 34, VH-CDR2 as shown in SEQ ID NO: 37, and VH-CDR3 as shown in SEQ ID NO: 40; and, VL-CDR1 as shown in SEQ ID NO: 41, VL-CDR2 as shown in SEQ ID NO: 42, and VL-CDR3 as shown in SEQ ID NO: 43; (16) VH-CDR1 as shown in SEQ ID NO: 35, VH-CDR2 as shown in SEQ ID NO: 38, and VH-CDR3 as shown in SEQ ID NO: 40; and, VL-CDR1 as shown in SEQ ID NO: 41, VL-CDR2 as shown in SEQ ID NO: 42, and VL-CDR3 as shown in SEQ ID NO: 43; (17) VH-CDR1 as shown in SEQ ID NO: 36, VH-CDR2 as shown in SEQ ID NO: 39, and VH-CDR3 as shown in SEQ ID NO: 40; and, VL-CDR1 as shown in SEQ ID NO: 41, VL-CDR2 as shown in SEQ ID NO: 42, and VL-CDR3 as shown in SEQ ID NO: 43; and (18) VH-CDR1 as shown in SEQ ID NO: 35, VH-CDR2 as shown in SEQ ID NO: 39, and VH-CDR3 as shown in SEQ ID NO: 40; and, VL-CDR1 as shown in SEQ ID NO: 41, VL-CDR2 as shown in SEQ ID NO: 42, and VL-CDR3 as shown in SEQ ID NO: 43.

7. The antibody or fragment thereof according to claim 6, wherein the heavy chain variable region comprises: an amino acid sequence as shown in any one of SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68 and SEQ ID NO: 72, or an amino acid sequence having at least 75% identity to the amino acid sequence as shown; and/or the light chain variable region comprises: an amino acid sequence as shown in any one of SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74 and SEQ ID NO: 76 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown.

8. The antibody or fragment thereof according to claim 7, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of following combinations: (1) an amino acid sequence as shown in SEQ ID NO: 44 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 44; and, an amino acid sequence as shown in SEQ ID NO: 46 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 46; (2) an amino acid sequence as shown in SEQ ID NO: 48 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 48; and, an amino acid sequence as shown in SEQ ID NO: 50 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 50; (3) an amino acid sequence as shown in SEQ ID NO: 52 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 52; and, an amino acid sequence as shown in SEQ ID NO: 54 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 54; (4) an amino acid sequence as shown in SEQ ID NO: 56 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 56; and, an amino acid sequence as shown in SEQ ID NO: 58 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 58; (5) an amino acid sequence as shown in SEQ ID NO: 60 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 60; and, an amino acid sequence as shown in SEQ ID NO: 62 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 62; (6) an amino acid sequence as shown in SEQ ID NO: 64 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 64; and, an amino acid sequence as shown in SEQ ID NO: 66 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 66; (7) an amino acid sequence as shown in SEQ ID NO: 68 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 68; and, an amino acid sequence as shown in SEQ ID NO: 70 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 70; (8) an amino acid sequence as shown in SEQ ID NO: 72 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 72; and, an amino acid sequence as shown in SEQ ID NO: 74 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 74; and (9) an amino acid sequence as shown in SEQ ID NO: 48 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 48; and, an amino acid sequence as shown in SEQ ID NO: 76 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 76.

9. The antibody or fragment thereof according to claim 8, wherein the at least 75% identity is any percent identity greater than or equal to 75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91° A, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity; preferably, the antibody or fragment thereof is in any form, for example, a monoclonal antibody, a single-chain antibody, a single-domain antibody, a bifunctional antibody, a nanobody, a fully or partially humanized antibody or a chimeric antibody and the like; alternatively, the antibody or fragment thereof is a half-antibody or an antigen-binding fragment of a half-antibody, for example, scFv, BsFv, dsFv, (dsFv).sub.2, Fab, Fab′, F(ab′).sub.2, or Fv; alternatively, the antibody is IgA, IgD, IgE, IgG or IgM, more preferably IgG1; or, the fragment of the antibody is selected from the group consisting of scFv, Fab, F(ab′).sub.2 and Fv fragments of the antibody.

10. The antibody or fragment thereof according to claim 9, wherein the antibody or fragment thereof further comprises a human or murine light chain constant region (CL) and/or heavy chain constant region (CH); preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD and IgE and/or a kappa or lambda type light chain constant region; more preferably, the heavy chain constant region is of IgG1 or IgG4 subtype and the light chain constant region is of kappa type.

11. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or fragment thereof according to claim 10, or encoding a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain or a light chain comprised in the antibody or fragment thereof.

12. A vector comprising the nucleic acid molecule according to claim 11.

13. A host cell comprising the nucleic acid molecule and/or the vector according to claim 12, or transformed or transfected with the nucleic acid molecule and/or the vector.

14. A conjugate or fusion protein comprising the antibody or fragment thereof according to claim 6.

15. The conjugate or fusion protein according to claim 14, wherein the conjugate or fusion protein comprises an additional moiety, for example, a cell surface receptor, a small molecule compound, a small molecule polymer, an active protein or a polypeptide, directly or indirectly linked to the antibody or fragment thereof.

16. A pharmaceutical composition comprising the antibody or fragment thereof according to claim 6, and optionally a pharmaceutically acceptable excipient.

17. A kit comprising the antibody or fragment thereof according to claim 6.

18. Use of the antibody or fragment thereof according to claim 4, in the manufacture of a medicament for preventing or treating infection and complication caused by alpha-hemolysin or an alpha-hemolysin producing microorganism.

19. Use of the antibody or fragment thereof according to claim 4, in combination with another antibacterial agent or anti-alpha-hemolysin antibody in the manufacture of a medicament for preventing or treating infection and complication caused by alpha-hemolysin or an alpha-hemolysin producing microorganism.

20. Use according to claim 18, wherein the microorganism is Staphylococcus aureus; preferably, the Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus; preferably, the infection includes upper respiratory tract infection, pneumonia, severe pneumonia, abdominal infection, subcutaneous and soft tissue infection, bacteremia, and infection in various organs; preferably, the complication includes Acute Respiratory Distress Syndrome (ARDS), sepsis, and a rise in inflammatory factors in body; preferably, the another antibacterial agent is an agent (including chemical agents, biological agents and Chinese medicines) useful for the treatment and prevention of infection caused by Staphylococcus aureus, e.g. methicillin-resistant Staphylococcus aureus, preferably is an antibiotic, such as beta-lactam antibiotics, more preferably vancomycin, norvancomycin, teicoplanin, linezolid, daptomycin, cephapiprazole, fusidic acid, or ceftaroline.

21. A method for preventing or treating infection and complication caused by alpha-hemolysin or an alpha-hemolysin producing microorganism, including administering to a subject in need thereof the antibody or fragment thereof according to claim 4, and optionally an antibacterial agent.

22. A method for diagnosing infection caused by alpha-hemolysin or an alpha-hemolysin producing microorganism, including contacting the antibody or fragment thereof according to claim 4, with a sample from a subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Embodiments of the present invention are described in detail below with reference to the attached drawing figures, in which:

[0103] FIG. 1 shows the construction of a recombinant expression plasmid for His-tag fused Staphylococcus aureus alpha-hemolysin (alpha-Toxin) protein.

[0104] FIG. 2 shows the construction of a recombinant expression plasmid for His-tag fused Staphylococcus aureus alpha-hemolysin mutant (H35L alpha-Toxin) protein.

[0105] FIG. 3 shows the SDS-PAGE electrophoresis (10% gels) results of recombinantly expressed Staphylococcus aureus alpha-hemolysin and its mutant (H35L alpha-Toxin), in which panel 3A shows alpha-Toxin, and panel 3shows H35L alpha-Toxin, and the loading amounts are 10

[0106] FIG. 4 shows the hemolytic effect of Staphylococcus aureus alpha-hemolysin and its mutant (H35L alpha-Toxin) on sheep blood agar plates.

[0107] FIG. 5 shows the hemlolytic effect of Staphylococcus aureus alpha-hemolysin and its mutant (H35L alpha-Toxin) on rabbit blood, in which panel 5A shows alpha-Toxin and panel 5B shows H35L alpha-Toxin.

[0108] FIG. 6 shows the detection results obtained during screening of hybridorna cell strains, in which panel 6A shows the ELISA detection results of the binding of antibodies in the supernatants of different cell strains to alpha-hemolysin, and panel 6B shows the inhibition results of antibodies in the supernatants of different cell strains on alpha-hemolysin, respectively.

[0109] FIG. 7 shows the ELISA detection results of the binding of antibodies of the present invention to alpha-hemolysin, in which panels 7A to 7D show the binding of the screened antibodies 78D4, 16H4, 78F4 and 98G9 to alpha-hemolysin, respectively.

[0110] FIG. 8 shows the curves of association and dissociation as detected by Octct of antibodies of the present invention and alpha-hemolysin, in which panels 8A to 8D show the binding of the screened humanized versions of antibodies 78D4, 16H4, 78F4 and 98G9 to alpha-hemolysin, respectively.

[0111] FIG. 9 shows the effects of antibodies of the present invention on the hemolytic activity of alpha-hemolysin, in which panels 9A to 9C show the results at different antibody amounts, respectively.

[0112] FIG. 10 shows the therapeutic effects of antibodies of the present invention in animal model of alpha-hemolysin-induced sepsis.

[0113] FIG. 11 shows the therapeutic effects of antibodies of the present invention in animal model of methicillin-resistant Staphylococcus aureus-induced bacteremia.

[0114] FIG. 12 shows the therapeutic effects of antibodies of the present invention in animal model of methicillin-resistant Staphylococcus aureus pneumonia.

[0115] FIG. 13 shows the results of a pharmacokinetic study of antibody of the present invention after a single administration in cynomolgus monkeys.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0116] The present invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way.

[0117] Experimental procedures in the following examples are all conventional ones, unless otherwise specified. Raw materials and reagents used in the following examples are all commercially available products, unless otherwise specified.

[0118] Known antibodies as follows are used herein as controls:

[0119] Fully human antibody R-301 (Salvecin®), abbreviated as AR (see U.S. Pat. No. 9,249,215B2), from Aridis Pharmaceuticals, Inc., of which heavy chain variable region is as shown in SEQ ID NO: 82 and light chain variable region is as shown in SEQ ID NO: 83.

[0120] Humanized antibody MEDI4893, abbreviated as AZ (see US20140072577A1), from Astrazeneca Pharmaceuticals Inc., of which heavy chain variable region is as shown in SEQ ID NO: 84 and light chain variable region is as shown in SEQ ID NO: 85.

[0121] The antibodies provided by the present invention as follows have a heavy chain constant region as shown in SEQ ID NO: 86 and a light chain constant region as shown in SEQ ID NO: 87.

EXAMPLE 1

Recombinant Expression of His-Tag Fused Staphylococcus aureus Alpha-Hemolysin (Alpha-Toxin)

[0122] A nucleotide sequence corresponding to the amino acid sequence of Staphylococcus aureus alpha-hemolysin which was used as a target sequence was artificially synthesized, and was cloned into plasmid pET-21a containing His tag utilizing sites for restriction enzymes NdeI and XhoI. The amino acid sequence of Staphylococcus aureus alpha-hemolysin is as shown in SEQ ID NO: 78, the corresponding nucleotide sequence is as shown in SEQ ID NO: 79, and the construction of the recombinant plasmid is shown in FIG. 1.

[0123] The obtained recombinant plasmid was transformed into competent BL21(DE3) pLysS cells, and single colonies were picked up next day and inoculated into LB liquid medium containing 100 μg/mL ampicillin, followed by shaking culture at 37° C. overnight. The overnight culture was inoculated into LB liquid medium containing 100 μg/mL ampicillin in a volume ratio of 1:100, and a shaking culture was performed at 37° C. at 200 rpm till OD.sub.600 of about 0.6-0.8. IPTG was added into the culture to reach a final concentration of 0.1 mM, and induction was performed at 16° C. for 16-18 h. After induction, the bacterial culture was taken and centrifuged at 8,000 rpm for 3 min, and bacterial cells were collected and preserved at −80° C.

EXAMPLE 2

Recombinant Expression of His-Tag Fused Staphylococcus aureus Alpha-Hemolysin Mutant (H35L Alpha-Toxin)

[0124] Based on the amino acid sequence of Staphylococcus aureus alpha-hemolysin, a mutated amino acid sequence was obtained by mutating histidine (His) at position 35 to leucine (Leu). A nucleotide sequence corresponding to the mutated amino acid sequence was artificially synthesized, and was cloned into plasmid Pet-21a containing His tag utilizing sites for restriction enzymes NdeI and XhoI. The amino acid sequence of Staphylococcus aureus alpha-hemolysin mutant (H35L alpha-Toxin) is as shown in SEQ ID NO: 80 (H35L is at position 36 in that sequence), the corresponding nucleotide sequence is as shown in SEQ ID NO: 81, and the construction of the recombinant plasmid is shown in FIG. 2.

[0125] The obtained recombinant plasmid was transformed into competent BL21(DE3) pLysS cells, and single colonies were picked up next day and inoculated into LB liquid medium containing 100 μg/mL ampicillin, followed by shaking culture at 37° C. overnight. The overnight culture was inoculated into LB liquid medium containing 100 μg/mL ampicillin in a volume ratio of 1:100, and a shaking culture was performed at 37° C. at 200 rpm till OD.sub.600 of about 0.6-0.8. IPTG was added into the culture to reach a final concentration of 0.25 mM, and induction was performed at 25° C. for 4.5 h. After induction, the bacterial culture was taken and centrifuged at 8,000 rpm for 3 min, and bacterial cells were collected and preserved at −80° C.

EXAMPLE 3

Purification of Staphylococcus aureus Alpha-Hemolysin (Alpha-Toxin) and its Mutant (H35L Alpha-Toxin)

[0126] Escherichia coli cells with Staphylococcus aureus alpha-hemolysin and its mutant inducibly expressed therein were sonicated respectively with an ultrasonic system at 180 W with a mode of operating for 3 sec, and pausing for 3 sec, 7-9 min in total. After centrifugation at 13,000 rpm for 30 min, supernatant was collected and sterilized by filtration through a 0.22 μm filter.

[0127] A Ni column was packed with filler and then was mixed with the filtered supernatant on a rotary mixer for 1 h at room temperature. The Ni column was firstly eluted with 5 column volumes of Buffer 1 (containing imidazole at a concentration of 30 mM) till the color of developing solution did not change, to remove proteins non-specifically bound to the column, and then was eluted with 5 column volumes of Buffer 2 (containing imidazole at a concentration of 300 mM) to obtain target proteins. Then, eluates containing target proteins were concentrated and displaced using a 10 KD concentrator in PBS as a solvent. The electrophoresis results of the obtained protein are shown in FIG. 3.

EXAMPLE 4

Detection of Biological Activity of Staphylococcus aureus Alpha-Hemolysin (Alpha-Toxin) and its Mutant (H35L Alpha-Toxin) (In Vitro Hemolysis Assay and In Vivo Toxicity Assay in Mice)

[0128] Respectively, 10 μL of each of Staphylococcus aureus alpha-hemolysin and its mutant in different concentrations (5 μg/mL, 0.5 μg/mL and 0.05 μg/mL) was dripped on the surface of a sheep blood agar plate (Shanghai Comagai Microbial Technology CO., LTD), and the plates were placed in an incubator at 37° C. for incubation for 24 h. Then hemolytic rings were obviously visible around the alpha-hemolysin dripped, having diameters related to concentrations of the alpha-hemolysin dripped; while no hemolytic ring was found around the alpha-hemolysin mutant dripped. The results of the assay are shown in FIG. 4.

[0129] 75 μL of 5% rabbit erythrocytes (Bio-channel Biotechnology) and different amounts of Staphylococcus aureus alpha-hemolysin or its mutant were added to the wells of a 96-well plate, and PBS was added to obtain a final volume of 150 μL. The 96-well plate was incubated in an incubator at 37° C. for 1 h, and then was centrifuged at 3000 rpm for 3 min. 100 μL of the supernatant from each well was evaluated for hemolytic activity by absorbance at 405 nm (OD405) measured using a microplate reader. The results showed that alpha-hemolysin had a dose-dependent hemolytic activity on rabbit erythrocytes, whereas the alpha-hemolysin mutant had no hemolytic activity, The results of the assay are shown in FIG. 5.

[0130] Different dosages of Staphylococcus aureus alpha-hemolysin or its mutant recombinantly expressed according to the methods described in Examples 1, 2 and 3 were administered to C57 mice by tail vein injection. It was found that the lowest lethal dosage of Staphylococcus aureus alpha-hemolysin in C57 mice was 3 μg/mouse; while no any discomfort was seen with the mice administered with the highest dosage 200 μg/mouse of the alpha-hemolysin mutant.

EXAMPLE 5

Immunization of C57 Mice With Staphylococcus aureus Alpha-Hemolysin Mutant (H35L Alpha-Toxin)

[0131] Alpha-hemolysin or alpha-hemolysin mutant was diluted to different concentrations with PBS and injected via tail vein into C57 mice. Toxic effects of the two on C57 mice were compared, to choose an immunogen and dosage thereof suitable for the subsequent immunization of animals. The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Toxic effects of different dosages of alpha-hemolysin and alpha-hemolysin mutant on mice Protein Dosage Performance of the mice WT α-toxin 50 μg/mouse (2.5 mg/kg) Instantly died 10 μg/mouse (0.5 mg/kg) Instantly died 3 μg/mouse (0.15 mg/kg) Died within 30 min 1 μg/mouse (0.05 mg/kg) Discomforts including tremble, fidget, and the like exhibited; survived; and recovered 16 hours later (overnight) 0.1 μg/mouse (0.005 mg/kg) No obvious discomfort shown H35L α-toxin 200 μg/mouse (10 mg/kg) No obvious discomfort shown Unrelated 200 μg/mouse (10 mg/kg) No obvious discomfort shown protein BSA

EXAMPLE 6

Immunization of Balb/c Mice With Staphylococcus aureus Alpha-Hemolysin Mutant (H35L Alpha-Toxin)

[0132] 8 week old Balblc mice were immunized using a procedure of 42 days in total at 14 day interval, with reference to Antibodies a Laboratory Manual, Second Edition (Edward A. Greenfield 2012).

[0133] The Staphylococcus aureus alpha-hemolysin mutant was emulsified in complete or incomplete Freund's adjuvant and injected into the subcutaneous tissue and peritoneal cavity of the nape of neck, tail root and groin of each mouse in a unilateral manner. On the 35th day of immunization, tail vein blood was collected, antibody titer was detected by ELISA, and spleen cells of the immunized mice were collected and fused with myeloma cells.

EXAMPLE 7

Screening and Identification of Hybridoma Cell Strains and Sequencing of Antibodies

[0134] Spleen cells of Balb/c mice immunized with Staphylococcus aureus alpha-hemolysin mutant (H35L alpha-Toxin) were taken and fused with myeloma cells P3X63Ag8.653 using PEG or electrofusion method. The fused hybridoma cells were inoculated in 30 384-well plates, and 24 hours later, HAT-containing medium and HT-containing medium were added to select hybridoma cells, After cultured in 384-well plates for 10-14 days, supernatants of cell cultures were taken and subjected to ELISA assay with α-Toxin to screen hybridoma clones capable of secreting antibodies that specifically bind to alpha-hemolysin (see panel 6A). Subsequently, OD values from the wells detected by ELISA were ordered from the highest to the lowest, and wells per plate with values in the top 94 were selected and cells therein were transferred to a 96-well plate for culture (among the 384-well plates, plate NO. 30 was less positive for ELISA detection, so no wells of the plate were selected for transferring); and 29 96-well plates in total were transferred.

[0135] Under physiological conditions, the presence of alpha-hemolysin leads to lysis of red blood cells, and the release of lysates leads to a change in the color of the supernatant. An anti-alpha-hemolysin antibody in the supernatant secreted by cells can inhibit alpha-hemolysin from lysing red blood cells. With the detection of absorbance of the supernatant, the degree of lysis of the cells caused by alpha-hemolysin and the inhibition of the antibody on alpha-hemolysin can be detected.

[0136] Hemolysis detection was performed on the culture supernatants of the 96-well plates after plate transferring, comprising following steps: the WT alpha-Toxin was diluted to obtain a stock solution with a concentration of 5 μg/mL; 25 μL of the stock solution was taken and mixed with an equal volume of cell culture supernatant, and the mixture was added into a 96-well plate containing 5% rabbit erythrocytes per well (diluted to 75 μL in PBS); and the 96-well plate was placed in an incubator at 37° C. for incubation for 1 h. Subsequently, the 96-well plate was centrifuged in a centrifuge at 3000 rpm for 3 min. 75 μL of the supernatant was added to a new 96-well plate and OD405 and OD450 were measured using a microplate reader. Part of the results is shown in panel 6B.

[0137] The screened hybridoma clones secreting anti-alpha-hemolysin antibodies were added into a 96-well plate which was plated with feeder cells by Limiting Dilution. Monoclonal cells were observed and marked under a microscope after 2-3 days, and monoclonal hybridoma cells capable of secreting anti-alpha-hemolysin monoclonal antibodies were screened by an ELISA assay after 7 days.

[0138] The monoclonal hybridoma cells secreting anti-alpha-hemolysin monoclonal antibodies were subjected to expanded culture, and total RNA of the cells was extracted using RNAfast200 Kit (Shanghai Flytech Biotechnology Co., Ltd.) according to the steps described in Kit instructions; the total RNA of the hybridoma cells obtained was reverse transcribed to cDNA using 5×primeScript RT Master Mix (Takara); and sequences of antibody light chain variable region, IgVL (κ) and heavy chain variable region V.sub.H were amplified using degenerate primers (Anke Krebber., 199) and Extaq PCR reagents (Takara). PCR amplification products were purified using PCR Clean-up Gel Extraction Kit (Macherey-Nagel GmbH & Co.); and linked to T-vector using pClone007 Simple Vector Kit (Tsingke Biotechnology Co., Ltd.) according to Kit instructions, and transformed into competent Escherichia coli cells. Variable region sequences of the monoclonal antibodies were obtained by DNA sequencing after strain amplification and plasmid extraction.

[0139] Variable region sequences of murine antibodies obtained were analyzed as follows:

TABLE-US-00002 > murine antibody 98G9 Heavy chain variable region: (SEQ ID NO: 44) DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEW VAYISGGSSTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMY YCASGYPYGLDYWGQGTSVTVSS  Light chain variable region: (SEQ ID NO: 46) DIDMTQSPSSMYASLGERVTITCKASQDINWYLSWFQQKPGKSPKTL IYRGNRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEF  PFTFGSGTKLEIK

[0140] Heavy and light chain CDRs were defined for murine antibody 98G9 using different definition tools, and are shown in Table 2.

TABLE-US-00003 TABLE 2 CDR sequences of murine antibody 98G9 Definition Heavy chain CDR1 Heavy chain CDR2 Heavy chain CDR3 Chothia SEQ ID NO: 1 SEQ ID NO: 4 SEQ ID NO: 7 AbM SEQ ID NO: 2 SEQ ID NO: 5 SEQ ID NO: 7 Kabat SEQ ID NO: 3 SEQ ID NO: 6 SEQ ID NO: 7 Contact SEQ ID NO: 2 SEQ ID NO: 6 SEQ ID NO: 7 Definition Light chain CDR1 Light chain CDR2 Light chain CDR3 Chothia SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 AbM SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 Kabat SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 Contact SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10

TABLE-US-00004 > murine antibody 78F4 Heavy chain variable region: (SEQ ID NO: 52) QVQLQQPGAELVRPGASVKLSCKASGYSFTSYWMNWVKQRPGQGLEW IGMIHPSDSETRLSQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVY YCTRFDWDRAMDYWGQGTSVTVSS  Light chain variable region: (SEQ ID NO: 54) DIQMTQSPASLSASVGETVTITCRASENIFSYLAWYQQKQGKSPQLL VYNTRSLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHYGT  PWTFGGGTKLEIK

[0141] Heavy and light chain CDRs were defined for murine antibody 78F4 using different definition tools, and are shown in Table 3.

TABLE-US-00005 TABLE 3 CDR sequences of murine antibody 78F4 Definition Heavy chain CDR1 Heavy chain CDR2 Heavy chain CDR3 Chothia SEQ ID NO: 12 SEQ ID NO: 15 SEQ ID NO: 18 AbM SEQ ID NO: 13 SEQ ID NO: 16 SEQ ID NO: 18 Kabat SEQ ID NO: 14 SEQ ID NO: 17 SEQ ID NO: 18 Contact SEQ ID NO: 13 SEQ ID NO: 17 SEQ ID NO: 18 Definition Light chain CDR1 Light chain CDR2 Light chain CDR3 Chothia SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 AbM SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 Kabat SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 Contact SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21

TABLE-US-00006 > murine antibody 78D4 Heavy chain variable region: (SEQ ID NO: 60) EVHLQQSGPELMKPGASVKISCKTSGYTFSEYTMHWVKQSHGKSLEW IGSINPNNGGTTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVY NCARTRDYDNDGGLFAYWGQGTLVTVSA  Light chain variable region: (SEQ ID NO: 62) DVQITQSPSYLAASPGETITINCRASKNISKYLAWYQEKPGKTNKLL IYSGSTLQSGIPSRFSGNRSGTDFTLTISSLEPEDFAMYYCQQHYEY  PFTFGGGTKLEIK

[0142] Heavy and light chain CDRs were defined for murine antibody 78D4 using different definition tools, and are shown in Table 4.

TABLE-US-00007 TABLE 4 CDR sequences of murine antibody 78D4 Definition Heavy chain CDR1 Heavy chain CDR2 Heavy chain CDR3 Chothia SEQ ID NO: 22 SEQ ID NO: 25 SEQ ID NO: 29 AbM SEQ ID NO: 23 SEQ ID NO: 26 SEQ ID NO: 29 Kabat SEQ ID NO: 24 SEQ ID NO: 27 SEQ ID NO: 29 Contact SEQ ID NO: 23 SEQ ID NO: 27 SEQ ID NO: 29 Definition Light chain CDR1 Light chain CDR2 Light chain CDR3 Chothia SEQ ID NO: 30 SEQ ID NO: 32 SEQ ID NO: 33 AbM SEQ ID NO: 30 SEQ ID NO: 32 SEQ ID NO: 33 Kabat SEQ ID NO: 30 SEQ ID NO: 32 SEQ ID NO: 33 Contact SEQ ID NO: 30 SEQ ID NO: 32 SEQ ID NO: 33

TABLE-US-00008 > murine antibody 16H4 Heavy chain variable region: (SEQ ID NO: 68) EVQLQQSGAELVKPGASVTLSCTVSGFNIKDTYMHWVKQRPEQGLEW IGKIDPASGNTKYDPQFQGKATITADTSSNTAYLHLSSLTSEDSAVY FCASPYGNDYAMNYWGQGTSVTVSS  Light chain variable region: (SEQ ID NO: 70) DIQMTQSPASLSASVGETVTITCRASEKIYSFLAWYQQKQEKSPQLL VYNAETLAEGVPSRFSGTGSGIQFSLKIISLQPEDFGIYYCQHHYGT  PYTFGGGTKLEIK

[0143] Heavy and light chain CDRs were defined for murine antibody 16H4 using different definition tools, and are shown in Table 5.

TABLE-US-00009 TABLE 5 CDR sequences of murine antibody 16H4 Definition Heavy chain CDR1 Heavy chain CDR2 Heavy chain CDR3 Chothia SEQ ID NO: 34 SEQ ID NO: 37 SEQ ID NO: 40 AbM SEQ ID NO: 35 SEQ ID NO: 38 SEQ ID NO: 40 Kabat SEQ ID NO: 36 SEQ ID NO: 39 SEQ ID NO: 40 Contact SEQ ID NO: 35 SEQ ID NO: 39 SEQ ID NO: 40 Definition Light chain CDR1 Light chain CDR2 Light chain CDR3 Chothia SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43 AbM SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43 Kabat SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43 Contact SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43

EXAMPLE 8

Binding Affinity of Antibodies of the Present Invention to Alpha-Hemolysin (Alpha-Toxin)

[0144] Alpha-hemolysin was diluted to 1 μg/mL with PBS, then 100 μL per well was added into a 96-well plate (Microwell 96F 167008, Thermo) and incubated at 4° C. overnight for coating. Next day, the 96-well plate was washed with PBST (containing 0.5% PBS), for which the wells were soaked with PBST for 1 min each time, and then residual water was thoroughly spun off. 200 μL of PBST containing 5% BSA was added into the wells of the plate respectively, which then was incubated at 37° C. for 1 h for blocking; subsequently, the plate was washed with PBST and then residual water in the wells was thoroughly spun off.

[0145] 100 μL of each of recombinantly expressed anti-alpha-hemolysin monoclonal antibodies of different concentrations obtained through serial dilution (see the abscissae in FIG. 7 for antibody concentrations) was added to the 96-well plate per well, which then was incubated at 4° C. overnight. Then the 96-well plate was washed with PBST, before 100 μL of anti-mouse IgG secondary antibody (IH-0031, Beijing Dingguo Changsheng Biotechnology CO. LTD.) was added into each well, and the plate was incubated for 1 h at 37° C. Washed with PBST for 5 times, the plate was added with 100 μL of Substrate Solution (Invitrogen) per well, and incubated at 37° C. for 10 min. The reaction was stopped by the addition of 50 μL of 2 N sulfuric acid per well, and absorbance at a wavelength of 450 nm was measured in a microplate reader (Multiskein FC, Thermo).

[0146] The results are shown in FIG. 7.

EXAMPLE 9

Obtainment of Chimeric Antibodies and Humanized Antibodies of the Present Invention

[0147] First, complete light and heavy chain variable regions of the murine antibodies were combined with the human light and heavy chain constant regions to obtain chimeric antibodies as controls. Each obtained chimeric antibody was named following a format “murine antibody abbreviation-xi”.

[0148] With a comprehensive analysis of the heavy chain sequence of each murine antibody, Complementarity Determining Regions (CDRs) of the antibody binding to the antigen as well as Framework Regions (FRs) supporting the conserved three-dimensional conformation of the antibody were determined. Sequences of known human antibodies were searched and analyzed, and a heavy chain sequence of human antibody which was most similar and closest to the murine antibody was selected, such as IGHV1-3*01, and the sequences of framework regions in it were selected as templates. The heavy chain CDRs of the murine antibody were grafted into the framework regions of the human antibody to generate a humanized antibody heavy chain sequence (heavy chain version 0). Subsequently, residues at individual amino acid positions corresponding to positions in the murine framework regions that may be involved in antigen-antibody binding were back mutated to original murine residues, to generate humanized antibody heavy chain sequences (versions 1, 2, 3, . . . ). Humanized antibody light chain sequences (version 0, 1, 2, . . . ) were generated by applying the same procedure. Humanized antibody light and heavy chain sequences were designed, synthesized, and co-transfected into HEK 293 cells and humanized antibodies were expressed recombinantly (versions were named as follows: e.g., H0L0 was obtained by co-expressing heavy chain version 0+light chain version 0, and could be further abbreviated as version 00). Experiments proved that purified humanized antibodies showed activities of specifically binding to alpha-hemolysin protein consistent to those of murine parent antibodies respectively.

[0149] Those finally obtained humanized antibody versions and chimeric antibody xi versions were compared for antigen binding using an Octet instrument, and FIG. 8 shows measurement results of certain antibodies. From the curves of association and dissociation phases of binding of the antibodies to the antigen, certain humanized antibody versions shows similar or better properties in the antibody-antigen association and dissociation phases than the control antibodies ('including chimeric antibodies of the present invention or antibodies AZ, and AR).

[0150] Humanized antibodies having following sequences were obtained by screening.

TABLE-US-00010 > humanized antibody 98G9-02(98G9-H0L2) Heavy chain variable region (H0): (SEQ ID NO: 48) EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMHWVRQAPGKGLEW VSYISGGSSTIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCASGYPYGLDYWGQGTLVTVSS  Light chain variable region (L2): (SEQ ID NO: 76) DIQMTQSPSSLSASVGDRVTITCKASQDINWYLSWFQQKPGKAPKTL IYRGNRLVDGVPSRFSGSGSGQDYTFTISSLQPEDMATYYCLQYDEF PFTFGQGTKVEIK  > humanized antibody98G9-03 (98G9-H0L3) Heavy chain variable region (H0): (SEQ ID NO: 48) EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMHWVRQAPGKGLEW VSYISGGSSTIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCASGYPYGLDYWGQGTLVTVSS  Light chain variable region (L3): (SEQ ID NO: 50) DIQMTQSPSSLSASVGDRVTITCKASQDINWYLSWFQQKPGKAPKTL IYRGNRLVEGVPSRFSGSGSGQDYTFTISSLQPEDMATYYCLQYDEF PFTFGQGTKVEIK > humanized antibody78F4-00 (78F4-H0L0) Heavy chain variable region (H0): (SEQ ID NO: 56) QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMNWVRQAPGQGLEW MGMIHPSDSETRLSQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVY YCARFDWDRAMDYWGQGTLVTVSS  Light chain variable region (L0): (SEQ ID NO: 58) DIQMTQSPSSLSASVGDRVTITCRASENIFSYLAWYQQKPGKAPKLL IYNTRSLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGT PWTFGQGTKLEIK  > humanized antibody78D4-33 (78D4-H3L3) Heavy chain variable region (H3): (SEQ ID NO: 64) QVQLVQSGAEVKKPGASVKVSCKTSGYTFSEYTMHWVRQAPGQRLEW MGSINPNQGGTTYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVY YCARTRDYDNDGGLFAYWGQGTLVTVSS  Light chain variable region (L3): (SEQ ID NO: 66) DVQITQSPSFLSASVGDRVTITCRASKNILKYLAWYQQKPGKAPKLL IYSGSTLQSGVPSRFSGSRSGTEFTLTISSLQPEDFATYYCQQHYEY PFTFGQGTKLEIK  > humanized antibody16H14-11 (16H4-H1L1) Heavy chain variable region (H1): (SEQ ID NO: 72) EVQLVQSGAEVKKPGATVKISCKVSGFNIKDTYMHWVQQAPGKGLEW MGKIDPASGNTKYDPQFQGRVTITADTSTNTAYMELSSLRSEDTAVY YCATPYGNDYAMNYWGQGTLVTVSS  Light chain variable region (L1): (SEQ ID NO: 74) DIQMTQSPSSLSASVGDRVTITCRASEKIYSFLAWYQQKPGKAPKLL LYNAETLAEGVPSRFSGSGSGIDFTLTISSLQPEDFATYYCQHHYGT  PYTFGGGTKVEIK

[0151] Heavy and light chain CDRs of the humanized antibodies are shown in Table 6.

TABLE-US-00011 TABLE 6 CDR sequences of humanized antibodies 98G9 H0L2 heavy chain CDR1 heavy chain CDR2 heavy chain CDR3 SEQ ID NO: 2 SEQ ID NO: 6 SEQ ID NO: 7 light chain CDR1 light chain CDR2 light chain CDR3 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 98G9 H0L3 heavy chain CDR1 heavy chain CDR2 heavy chain CDR3 SEQ ID NO: 2 SEQ ID NO: 6 SEQ ID NO: 7 light chain CDR1 light chain CDR2 light chain CDR3 SEQ ID NO: 8 SEQ ID NO: 11 SEQ ID NO: 10 78F4 H0L0 heavy chain CDR1 heavy chain CDR2 heavy chain CDR3 SEQ ID NO: 13 SEQ ID NO. 17 SEQ ID NO: 18 light chain CDR1 light chain CDR2 light chain CDR3 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 78D4 H3L3 heavy chain CDR1 heavy chain CDR2 heavy chain CDR3 SEQ ID NO: 23 SEQ ID NO: 28 SEQ ID NO: 29 light chain CDR1 light chain CDR2 light chain CDR3 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 16H4 H1L1 heavy chain CDR1 heavy chain CDR2 heavy chain CDR3 SEQ ID NO: 35 SEQ ID NO: 39 SEQ ID NO: 40 light chain CDR1 light chain CDR2 light chain CDR3 SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43

EXAMPLE 10

Detection of Antihemolytic Activity of Antibodies of the Present Invention Against Alpha-Hemolysin (Alpha-Toxin)

[0152] 75 μL of 5% rabbit erythrocytes (Bio-channel Biotechnology) and 12.5 ng Staphylococcus aureus alpha-hemolysin and different amounts of anti-alpha-hemolysin antibodies (12.5 ng, 25 ng, and 50 ng) were added to the wells of a 96-well plate, and PBS was added to obtain a final volume of 150 μL. The 96-well plate was incubated in an incubator at 37° C. for 1 h, and then was centrifuged at 3000 rpm for 3 min. 100 μL of the supernatant from each well was evaluated for hemolytic activity by absorbance at 405 nm (OD405) measured using a microplate reader.

[0153] The experimental results showed that the chimeric antibodies and humanized antibodies of the present invention had obvious antihemolytic activities against alpha-hemolysin, which were dose dependent, and certain antibodies had comparable activities to control antibodies AR and AZ. The results are shown in FIG. 9.

EXAMPLE 11

Detection of Binding Kinetics (K.SUB.on., K.SUB.off.) and Affinity Constant KD of Antibodies of the Present Invention to Alpha-Hemolysin (Alpha-Toxin)

[0154] Antibody-antigen interactions were measured using BIAcore S200 from GE Healthcare.

[0155] Referring to the instructions provided in Human Antibody Capture Kit (cat No. BR-1008-39, Lot 10261753) from GE Healthcare, the analytical channel and the control sample channel on CM5 sensor chip were first saturated and coupled with maximum amount of anti-human Fc antibody, then the antibody to be detected at 7.5 ug/ml was allowed to flow through the analytical channel and uniformly distribute; and finally antigen samples gradiently diluted (the initial concentration was 20 nM and diluted by 1:3 to get 8 concentrations, and the concentration 0.741 nM was set to be repeated) were allowed to flow through both the analytical channel and the sample channel, and the photoreactions upon the binding of antibody to antigen were measured. Subsequently, the association constant Kon and dissociation constant Koff and affinity constant KD of the antibody were finally obtained by instrument software fitting (1:1) analysis.

[0156] The results are shown in Table 7.

TABLE-US-00012 TABLE 7 Binding kinetics and affinity constants of antibodies of the present invention to antigen Antibody ka (1/Ms) kd (1/s) KD (M) Rmax (RU) Chi.sup.2 (RU.sup.2) 78D4 xilgG 1 40E+06 3.36E−04 2.39E−10 67.1 0.198 78D4-H3L3 1.25E+06 3.02E−04 2.40E−10 37.2 0.0303 98G9 xilgG 8.84E+05 3.77E−04 4.26E−10 47.3 0.46 98G9-H0L2 7.76E+05 5.24E−04 6.74E−10 18.9 0.0677 98G9-H0L3 7.19E+05 5.12E−04 7.13E−10 22.5 0.0481 AZ IgG 1.60E+06 2.11E−04 1.32E−10 48.8 0.0552 AR IgG 2.76E+05 6.77E−05 2.45E−10 48.7 0.48

EXAMPLE 12

Establishment of Animal Model of Alpha-Hemolysin-Induced Sepsis and Detection of Therapeutic Effects of Antibodies of the Present Invention

[0157] C57BL/6J mice were randomly divided into a model control group and monoclonal antibody drug treatment groups according to body weight. 30 min before experiment, mice in the treatment groups were injected with a corresponding anti-alpha-hemolysin monoclonal antibody (6 μg/mouse) via tail vein respectively, and mice in the control group were injected with the same dose of PBS; and then all the mice were injected with alpha-hemolysin (3 μg/mouse) via tail vein to establish a sepsis infected mouse model. The survival time of the experimental animals was observed and recorded, and the results are shown in FIG. 10.

EXAMPLE 13

Establishment of Animal Model of Methicillin-Resistant Staphylococcus aureus-Induced Bacteremia and Detection of Therapeutic Effects of Antibodies of the Present Invention

[0158] A methicillin-resistant Staphylococcus aureus USA300 strain was activated for 2 generations on a solid TSB plate, and then inoculated into a liquid TSB medium for overnight culture. The bacterial cells were collected by being centrifuged at 12,000 rpm, and resuspended in physiological saline for later use

[0159] C257BL/6J mice were infected with 6×10.sup.7 CFU/mouse USA300 via tail vein and were randomly divided into a model control group (Control) and anti-alpha-hemolysin monoclonal antibody drug treatment groups according to body weight. 2 h after infection, the mice in the monoclonal antibody drug treatment groups were administered by tail vein injection a dosage of 5 mg/kg of a corresponding antibody respectively, and the mice in the control group were injected the same dose of PBS. The survival time of the mice in the groups was observed and recorded, and the results are shown in FIG. 11.

EXAMPLE 14

Establishment of Animal Model of Methicillin-Resistant Staphylococcus aureus Pneumonia and Detection of Therapeutic Effects of Antibodies of the Present Invention

[0160] A methicillin-resistant Staphylococcus aureus USA300 strain was activated for 2 generations on a solid TSB plate, and then inoculated into a liquid TSB medium for overnight culture. The bacterial cells were collected by being centrifuged at 12,000 rpm, and resuspended in physiological saline for later use.

[0161] C57BL/6J mice were infected with 1.8×10.sup.8 CFU/mouse USA300 via trachea and were randomly divided into a model control group, a monoclonal antibody drug treatment group, a vancomycin treatment group, and vancomycin plus monoclonal antibody treatment groups according to body weight. 2 h after infection, the mice in the groups were administered by tail vein injection corresponding drug for treatment (the dosage of the corresponding monoclonal antibody was 15 mg/kg, and the dosage of vancomycin was 1.25 mg/kg), or the same volume of PBS. The animals were killed 24 h after infection, and lung tissues were taken, weighted and homogenized, and coated on a solid TSB medium to detect bacterial load in the tissues. The experimental results showed that the anti-alpha-hemolysin antibodies of the present invention were capable of enhancing the pharmacodynamic effect of vancomycin in the treatment of methicillin-resistant Staphylococcus aureus pneumonia.

[0162] The results are shown in FIG. 12

EXAMPLE 15

Acute Toxicity Study of Antibodies of the Present Invention

[0163] Acute toxicity study on antibody molecule 78D4 H3L3 of the present invention was performed in mice and cynomolgus monkeys (N=10 for the experiment in mice, and N=3 for the experiment in cynomolgus monkeys).

[0164] C57BL/6 mice (18-20 g), half male and half female, were injected with 125 mg/kg of antibody molecule 78D4 H3L3 per mouse via tail vein within 24 hours. The results showed that when a dosage of 125 mg/kg at the maximum was administered in 24 hours, no mice died, and no discomfort was shown by the animals during continuous observation for 14 days, and no abnormality was seen in main organs (heart, liver, spleen, lung, kidney and brain) taken for histology observation from sacrificed animals.

[0165] Male cynomolgus monkeys were administered 10 mg/kg of antibody molecule 78D4 H3L3 per monkey via intravenous drip in limbs, in a single dose. The results showed that when a dosage of 10 mg/kg of antibody molecule 78D4 H3L3 was administrated, no death of the animals occurred, and no discomfort was shown by the animals during continuous observation for 28 days.

[0166] Acute toxicity test research showed that antibody molecule 78D4 H3L3 had good safety.

EXAMPLE 16

Pharmacokinetic Study of Antibodies of the Present Invention

[0167] Antibody molecule 78D4 H3L3, at a dose of 10 mg/kg, was subjected to a single dose pharmacokinetic study in cynomolgus monkeys (N=3).

[0168] Male cynomolgus monkeys were administered 10 mg/kg of antibody molecule 78D4 H3L3 per monkey via intravenous drip in limbs, in a single dose. Blood was sampled at 0 h before administration (pre-dose, 0 h), and at 0.25 h (15 min), 0.5 h (time point when needle was withdrawn), 4 h, 24 h (D2), 48 h (D3), 96 h (D5), 168 h (D8), 336 h (D15), 504 h (D22), and 672 h (D29) after administration started. Blood sampling sites were in peripheral veins in limbs of the animals (not the administered limb), or inguinal veins of the animals. The blood samples were about 1 mL whole blood/animal/time point. Antibody concentrations in the sera from the cynomolgus monkeys were determined using ELISA, and pharmacokinetic parameters such as AUClast, CL, T1/2 and the like were calculated by Phoenix WinNonlin (v6.4, Pharsight, Inc.) software using non-compartmental analysis methods.

[0169] The concentrations of antibody 78D4 H3L3 in sera from the cynomolgus monkeys were determined using ELISA. Serum drug levels of individual animals are shown in FIG. 13, and pharmacokinetic parameters are summarized in Table 8. Drug exposure can be seen with each animal after administration.

TABLE-US-00013 TABLE 8 Pharmacokinetic results of antibody 78D4 H3L3 Estimate Animal ID Parameter Unit 111 112 113 N Mean SD CV % AUC.sub.INF_obs h*ng/mL 26900000 44700000 64300000 3 45300000 18700000 41.3 AUC.sub.last h*ng/mL 26800000 43200000 60700000 3 43600000 17000000 38.9 C.sub.0 ng/mL 431000 387000 592000 3 470000 108000 22.9 Cl_.sub.obs mL/h/kg 0.744 0.447 0.311 3 0.501 0.222 44.2 MRT.sub.last h 83.8 160 170 3 138 47.1 34.2 T.sub.1/2z h 99.2 140 173 3 137 36.9 26.9 V.sub.z _obs mL/kg 107 90.4 77.6 3 91.5 14.5 15.8

[0170] The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications to the present invention without departing from the spirit of the present invention, which should fall within the scope of the appended claims.