Antibodies against alpha-hemolysin and stable formulations thereof

Abstract

The application provides an antibody or fragment thereof binding to Staphylococcus aureus alpha-hemolysin, a composition containing the antibody or fragment thereof binding to Staphylococcus aureus alpha-hemolysin, and the composition is preferably an aqueous injection preparation. The antibody of the application is screened by the strategy of attenuated immunization and virulent screening of alpha-hemolysin, has high affinity to alpha-hemolysin, can effectively block the hemolytic effect of alpha-hemolysin, and is proved to have significant protective or therapeutic effect in alpha-hemolysin sepsis model, MRSA bacteremia model and MRSA lung infection model. On the basis of the antibody or fragment thereof binding to Staphylococcus aureus alpha-hemolysin, the antibody stable preparation provided by further component selection and concentration optimization improves the freeze-thaw stability, oscillation stability and light stability of the antibody, and prolongs the shelf life of the antibody preparation, especially the aqueous injection.

Description

Technical Field

[0001] This invention relates to the field of antibody drugs, and more specifically, to an anti-α-hemolysin antibody and its pharmaceutical use. Background Technology

[0002] Staphylococcus aureus belongs to the genus Staphylococcus and is an important Gram-positive pathogenic bacterium, one of the most common Gram-positive bacteria in humans. + Pathogenic bacteria can cause local infections such as purulent infections, pneumonia, pseudomembranous colitis, and pericarditis, as well as systemic infections such as sepsis and septicemia. Data from the China Antimicrobial Resistance Monitoring Network shows that Staphylococcus aureus ranks fourth among pathogens detected in hospitals, and is the most common Gram-positive bacteria found in hospitals. + The number 1 bacteria.

[0003] Staphylococcus aureus releases large amounts of toxins during infection, damaging tissue cells and inhibiting the body's immune cells from clearing the pathogen. Currently, β-lactam antibiotics are mainly used clinically to treat Staphylococcus aureus infections. However, antibiotics can only inhibit or kill bacteria; they are ineffective against the toxins released by the bacteria. On the contrary, under the pressure of antibiotics, bacteria release more toxins, and the lysis of bacteria killed by antibiotics also releases toxins. When large amounts of toxins enter the bloodstream, they over-activate the host's immune system, releasing excessive inflammatory factors and leading to sepsis.

[0004] Furthermore, in the fight against humans, Staphylococcus aureus has become increasingly insensitive to β-lactam antibiotics. For example, the isolation rate of methicillin-resistant Staphylococcus aureus (MRSA) is rising, with data from the China Antimicrobial Resistance Monitoring Network showing that the MRSA isolation rate reached as high as 38.9% in 2016. Clinically, treatments for MRSA mainly include a limited number of drugs such as vancomycin and linezolid, which are typical examples of "super-resistant" bacteria causing human infections. Therefore, MRSA infections are becoming increasingly serious clinically, with very limited available antimicrobial options, leading to high treatment failure rates and mortality rates. In recent years, vancomycin-resistant VRSA (vancomycin-resistant Staphylococcus aureus) infections have even emerged, threatening a situation where no effective treatments are available.

[0005] Studies have found that pathogenic factors released by Staphylococcus aureus include hemolysins, leukocidins, enterotoxins, and coagulases. Among these, hemolysins are one of the important virulence factors secreted by Staphylococcus aureus, and can be divided into four types: α-hemolysins, β-hemolysins, γ-hemolysins, and δ-hemolysins. α-hemolysin (alpha hemolysis, Hla) is a secreted toxin protein encoded by the HLA gene of Staphylococcus aureus. It is expressed in almost all strains and, compared to other types of hemolysins, is a more critical virulence factor affecting the pathogenicity of Staphylococcus aureus. Studies have shown that Staphylococcus aureus with a deleted HLA gene expressing α-hemolysin exhibits significantly reduced virulence in animals. The full-length α-hemolysin protein is 319 amino acids long, with a relative molecular mass of 33 kDa, and is secreted in monomeric form. As a member of the pore-forming toxin family, α-hemolysin's most well-defined biological characteristic is its ability to rapidly lyse host erythrocytes and other tissue cells. After binding to cholesterol and sphingomyelin on host cells, it aggregates to form a heptamer with a relative molecular mass of approximately 232 kDa. This heptamer then folds to form a β-barrel-shaped transmembrane structure with a diameter of approximately 1.5 nm, causing the leakage of intracellular water, ions, and other small molecules, resulting in hemolysis or target cell death. Furthermore, α-hemolysin can induce smooth muscle contraction and spasm in capillaries, leading to capillary blockage and causing local ischemia and tissue necrosis. It plays a crucial role in diseases caused by Staphylococcus aureus, such as sepsis, pneumonia, breast infections, corneal infections, and severe skin infections. Simultaneously, α-hemolysin can destroy leukocytes in infected tissues, hindering the host's clearance of infected Staphylococcus aureus. Under the pressure of the host's immune system and antibiotics, Staphylococcus aureus at the site of infection releases large amounts of α-hemolysin. Once in the bloodstream, it can activate the host's immune system to release excessive inflammatory factors, leading to sepsis.

[0006] Faced with the continuous emergence of superbugs, antibody drugs have become a powerful tool in the post-antibiotic era. Antibodies are natural proteins produced by the adaptive immune system. When the human body is passively immunized with antibody drugs, they can not only neutralize virulence factors but also enhance the host's immune response to pathogens, accelerating the clearance of infecting pathogens. Alpha-hemolysin has shown potential as a target for anti-infective antibody drugs in the treatment of Staphylococcus aureus infection for the following reasons: α-hemolysin is highly conserved in Staphylococcus aureus and is expressed in almost all strains; α-hemolysin has a well-defined biological function, playing an important role in the formation of host infection and the development of sepsis in Staphylococcus aureus; and there are no homologous proteins of α-hemolysin in mammalian cells, meaning that antibody drugs designed targeting α-hemolysin have a low potential for off-target effects and low toxicity. Therefore, α-hemolysin is an ideal target for anti-Staphylococcus aureus antibody drugs. Effective neutralization of α-hemolysin is beneficial for blocking Staphylococcus aureus infection in humans, preventing severe sepsis after infection, and improving the prognosis of clinically infected patients. Aridis Pharmaceuticals is currently developing a fully human antibody, R-301, targeting α-hemolysin. The drug has entered Phase III clinical trials, with its primary indication being severe pneumonia caused by Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus). AstraZeneca's humanized antibody MEDI4893, targeting α-hemolysin, is currently undergoing Phase II clinical trials, with its primary indication being the prevention and treatment of Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus) pneumonia.

[0007] Currently, antibiotics such as vancomycin remain first-line drugs for treating Staphylococcus aureus infections. However, considering the continued increase in drug resistance and the slowdown in the development of new antibiotics, there is still a need in this field to develop novel and highly effective antibody drugs that directly neutralize toxins to combat Staphylococcus aureus. Our company previously developed the humanized anti-Staphylococcus aureus α-toxin antibody 78D4 H3L3, which specifically binds to α-toxin and blocks its interaction with its receptor, thereby reducing endothelial cell damage in pneumonia and achieving the prevention and treatment of pneumonia caused by Staphylococcus aureus.

[0008] Humanized anti-Staphylococcal α-toxin antibodies are complex biological macromolecules that undergo physical changes such as aggregation, denaturation, and precipitation, as well as chemical changes such as isomerization, deamidation, and oxidation during production and storage. These changes can affect the safety and efficacy of the product, thus requiring a stable formulation to ensure that the antibody retains the necessary biological activity before being used in patients. Currently, there are no suitable stable formulations available on the market. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide an antibody molecule against α-hemolysin, particularly a humanized α-hemolysin monoclonal antibody, said antibody having the ability to bind to Staphylococcus aureus α-hemolysin and inhibit its hemolysis and damage to tissue cells, and can be used alone or in combination with existing antibacterial drugs to treat infections or infection-related diseases caused by α-hemolysin or α-hemolysin-producing microorganisms.

[0010] Another technical problem to be solved by the present invention is that the physicochemical properties of anti-α-hemolysin antibody molecules are easily affected by the environment during production, transportation and storage, which leads to the loss of biological activity such as α-hemolysin neutralizing activity or blocking activity; the present invention provides a stable formulation containing anti-α-hemolysin antibody, and in particular provides a stable anti-α-hemolysin antibody aqueous injection.

[0011] To address the aforementioned technical problems, the purpose of this invention is to provide an antibody or a functional fragment thereof, and to provide its uses based on the antibody or the functional fragment thereof.

[0012] The technical solution of the present invention is as follows.

[0013] On one hand, the present invention provides an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein

[0014] The heavy chain variable region (VH) includes: VH-CDR1 selected from SEQ ID NO:22-24, VH-CDR2 selected from SEQ ID NO:25-28, and VH-CDR3 shown in SEQ ID NO:29;

[0015] The light chain variable region (VL) includes: VL-CDR1 selected from SEQ ID NO:30-31, VL-CDR2 shown in SEQ ID NO:32, and VL-CDR3 shown in SEQ ID NO:33.

[0016] Specifically, the present invention provides an antibody or a fragment thereof, the antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) and the light chain variable region (VL) respectively comprise CDR combinations selected from the following (VH-CDR1, VH-CDR2, VH-CDR3; VL-CDR1, VL-CDR2, VL-CDR3):

[0017] (1) VH-CDR1 as shown in SEQ ID NO:1, VH-CDR2 as shown in SEQ ID NO:4, VH-CDR3 as shown in SEQ ID NO:7; 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;

[0018] (2) VH-CDR1 as shown in SEQ ID NO:2, VH-CDR2 as shown in SEQ ID NO:5, VH-CDR3 as shown in SEQ ID NO:7; 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;

[0019] (3) VH-CDR1 as shown in SEQ ID NO:3, VH-CDR2 as shown in SEQ ID NO:6, VH-CDR3 as shown in SEQ ID NO:7; 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;

[0020] (4) VH-CDR1 as shown in SEQ ID NO:2, VH-CDR2 as shown in SEQ ID NO:6, VH-CDR3 as shown in SEQ ID NO:7; 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;

[0021] (5) VH-CDR1 as shown in SEQ ID NO:2, VH-CDR2 as shown in SEQ ID NO:6, VH-CDR3 as shown in SEQ ID NO:7; VL-CDR1 as shown in SEQ ID NO:8, VL-CDR2 as shown in SEQ ID NO:11, VL-CDR3 as shown in SEQ ID NO:10;

[0022] (6) VH-CDR1 as shown in SEQ ID NO:12, VH-CDR2 as shown in SEQ ID NO:15, VH-CDR3 as shown in SEQ ID NO:18; 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;

[0023] (7) VH-CDR1 as shown in SEQ ID NO:13, VH-CDR2 as shown in SEQ ID NO:16, VH-CDR3 as shown in SEQ ID NO:18; 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;

[0024] (8) VH-CDR1 as shown in SEQ ID NO:14, VH-CDR2 as shown in SEQ ID NO:17, VH-CDR3 as shown in SEQ ID NO:18; 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;

[0025] (9) VH-CDR1 as shown in SEQ ID NO:13, VH-CDR2 as shown in SEQ ID NO:17, VH-CDR3 as shown in SEQ ID NO:18; 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;

[0026] (10) VH-CDR1 as shown in SEQ ID NO:22, VH-CDR2 as shown in SEQ ID NO:25, VH-CDR3 as shown in SEQ ID NO:29; VL-CDR1 as shown in SEQ ID NO:30, VL-CDR2 as shown in SEQ ID NO:32, VL-CDR3 as shown in SEQ ID NO:33;

[0027] (11) VH-CDR1 as shown in SEQ ID NO:23, VH-CDR2 as shown in SEQ ID NO:26, VH-CDR3 as shown in SEQ ID NO:29; VL-CDR1 as shown in SEQ ID NO:30, VL-CDR2 as shown in SEQ ID NO:32, VL-CDR3 as shown in SEQ ID NO:33;

[0028] (12) VH-CDR1 as shown in SEQ ID NO:24, VH-CDR2 as shown in SEQ ID NO:27, VH-CDR3 as shown in SEQ ID NO:29; VL-CDR1 as shown in SEQ ID NO:30, VL-CDR2 as shown in SEQ ID NO:32, VL-CDR3 as shown in SEQ ID NO:33;

[0029] (13) VH-CDR1 as shown in SEQ ID NO:23, VH-CDR2 as shown in SEQ ID NO:27, VH-CDR3 as shown in SEQ ID NO:29; VL-CDR1 as shown in SEQ ID NO:30, VL-CDR2 as shown in SEQ ID NO:32, VL-CDR3 as shown in SEQ ID NO:33;

[0030] (14) VH-CDR1 as shown in SEQ ID NO:23, VH-CDR2 as shown in SEQ ID NO:28, VH-CDR3 as shown in SEQ ID NO:29; VL-CDR1 as shown in SEQ ID NO:31, VL-CDR2 as shown in SEQ ID NO:32, VL-CDR3 as shown in SEQ ID NO:33;

[0031] (15) VH-CDR1 as shown in SEQ ID NO:34, VH-CDR2 as shown in SEQ ID NO:37, VH-CDR3 as shown in SEQ ID NO:40; VL-CDR1 as shown in SEQ ID NO:41, VL-CDR2 as shown in SEQ ID NO:42, VL-CDR3 as shown in SEQ ID NO:43;

[0032] (16) VH-CDR1 as shown in SEQ ID NO:35, VH-CDR2 as shown in SEQ ID NO:38, VH-CDR3 as shown in SEQ ID NO:40; VL-CDR1 as shown in SEQ ID NO:41, VL-CDR2 as shown in SEQ ID NO:42, VL-CDR3 as shown in SEQ ID NO:43;

[0033] (17) VH-CDR1 as shown in SEQ ID NO:36, VH-CDR2 as shown in SEQ ID NO:39, VH-CDR3 as shown in SEQ ID NO:40; VL-CDR1 as shown in SEQ ID NO:41, VL-CDR2 as shown in SEQ ID NO:42, VL-CDR3 as shown in SEQ ID NO:43;

[0034] (18) VH-CDR1 as shown in SEQ ID NO:35, VH-CDR2 as shown in SEQ ID NO:39, VH-CDR3 as shown in SEQ ID NO:40; 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.

[0035] Preferably, in the antibody or fragment thereof provided by the present invention, the heavy chain variable region comprises:

[0036] Such as any one of the amino acid sequences shown in 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 with the shown amino acid sequence; and / or

[0037] The variable region of the light chain contains:

[0038] The amino acid sequence 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 with the shown amino acid sequence.

[0039] According to a specific embodiment of the present invention, the heavy chain variable region and the light chain variable region included in the antibody or its fragment are selected from the following combinations:

[0040] (1) An amino acid sequence as shown in SEQ ID NO:44 or an amino acid sequence having at least 75% identity with an 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 with an amino acid sequence as shown in SEQ ID NO:46.

[0041] (2) An amino acid sequence as shown in SEQ ID NO:48 or an amino acid sequence having at least 75% identity with an 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 with an amino acid sequence as shown in SEQ ID NO:50.

[0042] (3) An amino acid sequence as shown in SEQ ID NO:52 or an amino acid sequence having at least 75% identity with an 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 with an amino acid sequence as shown in SEQ ID NO:54;

[0043] (4) An amino acid sequence as shown in SEQ ID NO:56 or an amino acid sequence having at least 75% identity with an 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 with an amino acid sequence as shown in SEQ ID NO:58.

[0044] (5) An amino acid sequence as shown in SEQ ID NO:60 or an amino acid sequence having at least 75% identity with an 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 with an amino acid sequence as shown in SEQ ID NO:62.

[0045] (6) An amino acid sequence as shown in SEQ ID NO:64 or an amino acid sequence having at least 75% identity with an 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 with an amino acid sequence as shown in SEQ ID NO:66.

[0046] (7) An amino acid sequence as shown in SEQ ID NO:68 or an amino acid sequence having at least 75% identity with an 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 with an amino acid sequence as shown in SEQ ID NO:70.

[0047] (8) An amino acid sequence as shown in SEQ ID NO:72 or an amino acid sequence having at least 75% identity with an 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 with an amino acid sequence as shown in SEQ ID NO:74;

[0048] (9) An amino acid sequence as shown in SEQ ID NO:48 or an amino acid sequence having at least 75% identity with an 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 with an amino acid sequence as shown in SEQ ID NO:76.

[0049] Wherein, the aforementioned at least 75% identity is any percentage of identity of at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%, etc., ≥75%.

[0050] The antibodies or fragments provided by this invention can be in any form, such as monoclonal antibodies, single-chain antibodies, single-domain antibodies, bifunctional antibodies, nanobodies, fully or partially humanized antibodies, or chimeric antibodies; or, the antibodies or fragments thereof are haptens or antigen-binding fragments of haptens, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv; preferably, the fragments of the antibodies provided by this invention are any fragments of the antibody capable of specifically binding to the antigen Staphylococcus aureus α-hemolysin. The antibodies or antigen-binding fragments thereof described in this invention are murine antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, or scFv.

[0051] Alternatively, the antibody of the present invention may be IgA, IgD, IgE, IgG, or IgM, more preferably IgG1. The antibody fragment is selected from the scFv, Fab, F(ab')2, or Fv fragment of the antibody.

[0052] Preferably, the antibody or fragment thereof further comprises a human or mouse constant region, more preferably comprising a human or mouse light chain constant region (CL) and / or heavy chain constant region (CH); more preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD, or IgE and / or a κ or λ type light chain constant region. According to a specific embodiment of the present invention, the antibody is a monoclonal antibody, preferably a mouse-derived, chimeric, or humanized monoclonal antibody; more preferably, the heavy chain constant region of the monoclonal antibody is an IgG1 or IgG4 subtype, and the light chain constant region is κ type.

[0053] Preferably, the antibody or fragment thereof provided by the present invention comprises a heavy chain constant region as shown in SEQ ID NO:86 and / or a light chain constant region as shown in SEQ ID NO:87, or an amino acid sequence having at least 75% identity with the heavy chain constant region or light chain constant region shown.

[0054] On the other hand, the present invention also provides a nucleic acid molecule that encodes any antibody or fragment thereof of the present invention, or encodes a heavy chain CDR, light chain CDR, heavy chain variable region, light chain variable region, heavy chain, or light chain contained in the antibody or fragment thereof.

[0055] According to a specific embodiment of the present invention, the nucleic acid molecule encodes the heavy chain variable region or light chain variable region of the antibody or fragment thereof described in the present invention. For example, the nucleic acid molecule comprises a nucleotide sequence as shown in any one of SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, and SEQ ID NO:77.

[0056] In another aspect, the present invention provides a vector containing the nucleic acid molecule of the present invention. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector, etc.

[0057] The vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells or enter host cells in any way for purposes such as preserving or expressing antibodies. Therefore, in another aspect, the present invention provides a host cell containing the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells.

[0058] Based on the disclosure of this invention, the antibodies or fragments thereof, nucleic acid molecules, vectors, and / or host cells provided by this invention can be obtained using any conventional techniques known in the art. For example, regarding antibodies, the heavy chain variable region and / or light chain variable region of the antibody can be obtained first from the nucleic acid molecule provided by this invention, or the heavy chain and / or light chain of the antibody can be obtained, and then assembled with optional other structural domains of the antibody to form an antibody; or, the host cells can be cultured while allowing the host cells provided by this invention to express the heavy chain variable region and / or light chain variable region of the antibody or the heavy chain and / or light chain of the antibody to assemble the antibody. Optionally, the method further includes the step of recovering the generated antibody.

[0059] The antibodies or fragments thereof provided by this invention can also bind to other components, such as cell surface receptors, small molecule compounds like amino acids and sugars, small molecule polymers, or any other components that modify the antibodies described in this invention, or even active proteins or peptides, such as antimicrobial peptides or antibiotics. Therefore, in another aspect, this invention provides a conjugate or fusion protein comprising the antibodies or fragments thereof provided in this invention. For example, the conjugate or fusion protein may be a bispecific antibody comprising the antibodies or fragments thereof described in this invention.

[0060] On the other hand, the present invention provides an antibody composition comprising an anti-α-hemolysin antibody or an antigen-binding fragment thereof and optionally pharmaceutically acceptable excipients.

[0061] Furthermore, in the antibody composition of the present invention, the pharmaceutically acceptable excipients include one or more selected from the group consisting of buffers, protectants, and surfactants.

[0062] Furthermore, in the antibody composition of the present invention, wherein,

[0063] The concentration of anti-α-hemolysin antibody or its antigen-binding fragment is 10-100 mg / mL;

[0064] The buffer solution has a pH of 5.0-6.5 and a concentration of 1-50 mM.

[0065] The concentration of the protective agent is 1-10%;

[0066] The surfactant concentration is 0.001-0.1%.

[0067] Furthermore, in the antibody composition of the present invention, wherein,

[0068] The buffer solution is selected from citrate buffer, histidine buffer, and acetate buffer, with histidine buffer being preferred.

[0069] The protective agent is selected from one or more of sucrose, trehalose, sorbitol, and mannitol, with sucrose being preferred;

[0070] The surfactant is selected from Tween 20 and Tween 80, with Tween 80 being preferred.

[0071] In one specific embodiment, the antibody composition of the present invention comprises:

[0072]

[0073] Preferably, the composition of the present invention, wherein

[0074] The concentration of anti-α-hemolysin antibody or its antigen-binding fragment is 50 mg / mL;

[0075] The concentration of histidine buffer is 10 mM;

[0076] The concentration of sucrose is 5% (w / v);

[0077] The concentration of polysorbate 80 is 0.005-0.015% (w / v).

[0078] The formulation disclosed in this invention is an aqueous injection formulation.

[0079] In another embodiment, the present invention provides a stable antibody composition which omits the anti-α-hemolysin antibody or its antigen-binding fragment from the antibody composition described in the present invention.

[0080] The present invention also provides the use of compositions for stabilizing antibodies in enhancing antibody stability.

[0081] Furthermore, in the application of the stable antibody composition of the present invention in enhancing antibody stability, the antibody comprises an anti-α-hemolysin antibody or an antigen-binding fragment thereof, preferably the anti-α-hemolysin antibody or an antigen-binding fragment thereof of the present invention.

[0082] Furthermore, in the application of the antibody-stabilizing composition of the present invention to enhance antibody stability, the antibody stability includes freeze-thaw stability, oscillation stability, and light stability.

[0083] In addition, the present invention also provides the use of the antibody composition in the preparation of medicaments for the prevention or treatment of infections and complications caused by α-hemolysin or α-hemolysin-producing microorganisms.

[0084] The antibodies or fragments thereof, nucleic acid molecules, vectors, host cells, conjugates, or fusion proteins provided by this invention can be included in pharmaceutical compositions, and more particularly in pharmaceutical formulations, for use in various purposes as needed. Therefore, in another aspect, this invention also provides a pharmaceutical composition comprising the antibodies or fragments thereof, nucleic acid molecules, vectors, host cells, conjugates, and / or fusion proteins described herein, and optionally pharmaceutically acceptable excipients.

[0085] For any purpose of use, the present invention also provides a kit comprising the antibody molecule or fragment thereof of the present invention, nucleic acid molecule, vector, host cell, conjugate, fusion protein and / or pharmaceutical composition.

[0086] Based on their ability to bind to α-hemolysin and inhibit its hemolysis and damage to tissue cells, the antibodies or fragments thereof of the present invention can be used alone or in combination with other antimicrobial agents to treat or improve infections caused by α-hemolysin or α-hemolysin-producing microorganisms, or other diseases or symptoms resulting from such infections. Therefore, the present invention also provides related applications of the above-mentioned subject matter.

[0087] Specifically, in another aspect, the present invention provides the use of the antibodies or fragments thereof, nucleic acid molecules, vectors, host cells, conjugates, fusion proteins and / or pharmaceutical compositions described herein in the preparation of medicaments for the prevention or treatment of infections and complications caused by α-hemolysin or α-hemolysin-producing microorganisms.

[0088] Furthermore, the present invention provides the use of the antibody or fragment thereof, nucleic acid molecule, carrier, host cell, conjugate, fusion protein and / or pharmaceutical composition in combination with other antimicrobial agents or anti-α-hemolysin antibodies in the preparation of a medicament for the prevention or treatment of infections and complications caused by α-hemolysin or α-hemolysin-producing microorganisms.

[0089] Furthermore, the present invention provides a method for preventing or treating infections and complications caused by α-hemolysin or α-hemolysin-producing microorganisms, the method comprising administering to a subject in need the antibody or a fragment thereof, a nucleic acid molecule, a carrier, a host cell, a conjugate, a fusion protein, and / or a pharmaceutical composition, and optionally an antimicrobial agent. The optional antimicrobial agent may be a drug administered in combination with the antibody or a fragment thereof, the nucleic acid molecule, the carrier, the host cell, the conjugate, the fusion protein, and / or the pharmaceutical composition of the present invention. The combined administration of the two can be carried out in any manner, including simultaneously, continuously, or at intervals.

[0090] In this invention, the microorganism that produces α-hemolysin is preferably Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus.

[0091] In this invention, the infection caused by α-hemolysin or α-hemolysin-producing microorganisms may be one or more selected from upper respiratory tract infection, pneumonia, severe pneumonia, abdominal infection, subcutaneous and soft tissue infection, bacteremia, and infection of various organs; the complication caused by α-hemolysin or α-hemolysin-producing microorganisms may be one or more selected from acute respiratory distress syndrome (ARDS), sepsis, and elevated levels of inflammatory factors in the body.

[0092] In this invention, other antibacterial agents are drugs (including chemical drugs, biological agents, and traditional Chinese medicines) that can be used to treat and prevent Staphylococcus aureus infections, such as methicillin-resistant Staphylococcus aureus (MRSA) infections. Antibiotics, such as β-lactam antibiotics, are preferred. The antibiotics can be drugs listed in the guidelines / treatment strategies issued by the Infectious Diseases Society of America (IDSA) or the Chinese Medical Association for the treatment of MRSA infections, preferably vancomycin, teicoplanin, linezolid, daptomycin, cefepime, fusidic acid, and cefuroxime.

[0093] The present invention also provides a method for diagnosing an infection caused by α-hemolysin or an α-hemolysin-producing microorganism, the method comprising contacting the antibody or a fragment thereof, a nucleic acid molecule, a carrier, a host cell, a conjugate, a fusion protein and / or a pharmaceutical composition with a sample from a subject.

[0094] Compared to existing technologies, this invention obtains Staphylococcus aureus α-toxin and a non-toxic Staphylococcus aureus α-toxin mutant protein (α-Toxin H35L) through prokaryotic expression in Escherichia coli. After successfully immunizing mice with α-Toxin H35L, spleen cells were harvested, and an antibody library was established using hybridoma technology. Monoclonal antibodies with high affinity and biological activity for α-toxin were screened and obtained. These monoclonal antibodies were further humanized to obtain a lead antibody molecule (IgG1 subtype).

[0095] Specifically, this invention utilizes α-Toxin H35L to successfully immunize mice, then harvests spleen cells and establishes an antibody library using hybridoma technology. This library is used to screen and obtain α-hemolysin monoclonal antibodies with high affinity for α-Toxin and biological activity. A total of 16 lead antibody molecules were obtained, which not only have high affinity for α-hemolysin but also exhibit activity in blocking α-hemolysin hemolysis. Particularly noteworthy is the use of a strategy of attenuated immunization followed by strong virulence screening in antigen selection and antibody screening.

[0096] Based on mouse-derived lead antibody molecules, recombinant expression of human-mouse affinity antibodies and humanized antibodies was carried out, ultimately yielding humanized antibody molecules with similar biological activities to mouse-derived antibodies.

[0097] In vitro pharmacodynamic studies have shown that the humanized antibody of this invention can dose-dependently block the hemolytic effect of α-hemolysin on rabbit erythrocytes and the damaging effect of α-hemolysin on lung epithelial cells. Furthermore, this invention has also evaluated the in vivo pharmacodynamics of the lead antibody molecule in mouse models of α-hemolysin sepsis, MRSA bacteremia, and MRSA lung infection. The results show that the humanized antibody of this invention has a significant protective effect against mouse α-hemolysin sepsis; it can significantly prolong the survival time of mouse MRSA bacteremia; and it can significantly reduce the bacterial load in mouse MRSA lung infection. Moreover, the combined use of the humanized antibody of this invention with commonly used antibacterial drugs such as vancomycin and linezolid exhibits a significant synergistic effect in mouse models of α-hemolysin sepsis, MRSA bacteremia, and MRSA lung infection.

[0098] Furthermore, a single-dose pharmacokinetic study (N=3) was conducted on the antibody of the present invention in cynomolgus monkeys at a dose of 20 mg / kg. The results showed that the basic pharmacokinetic parameters met the drug-likeness criteria (Table 1), the elimination half-life (T1 / 2) was 137±36.9 h, and the plasma clearance (CL) was 0.501±0.222 ml / h / kg. In addition, an acute toxicity test was conducted on mice (N=10). The results showed that no mice died when administered a dose of 125 mg / kg within 24 hours, and no adverse reactions were observed in the animals after 14 days of continuous observation. Gross examination of the major organs (heart, liver, spleen, lungs, kidneys, and brain) of the euthanized animals revealed no abnormalities. A single-dose administration of the antibody of the present invention to cynomolgus monkeys at a dose of 10 mg / kg did not result in animal death, and no adverse reactions were observed in the animals after 28 days of continuous observation. The acute toxicity test results indicate that the antibody of the present invention has good safety.

[0099] The antibody of this invention can effectively neutralize toxins, blocking their damage to patient tissue cells, while simultaneously enhancing the patient's immunity. This reduces tissue damage from clinical Staphylococcus aureus infection, promotes faster clearance of infectious bacteria from the patient's body, and prevents or alleviates sepsis. Under the action of this antibody, patients can switch from intravenous infusion to oral therapy more quickly, and the treatment course is shortened. Furthermore, the antibody of this invention has better clinical efficacy and tolerability, making it a valuable supplement to existing antibiotic therapies.

[0100] Antibody compositions were prepared based on the physicochemical properties and biological activities of anti-α-hemolysin antibodies. By optimizing the selection of buffer media, protective agents, and surfactants, the freeze-thaw stability, oscillation stability, and light stability of the antibodies were improved, extending the shelf life of antibody preparations, especially aqueous injections, and preventing changes in the physicochemical properties of the antibodies during transportation and storage, thereby preventing loss of biological activity. Attached Figure Description

[0101] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0102] Figure 1 The construction of a recombinant expression plasmid for Staphylococcus aureus α-toxin protein fused with a His tag is shown.

[0103] Figure 2 This paper demonstrates the construction of a recombinant expression plasmid for a Staphylococcus aureus mutant α-hemolysin (H35Lα-Toxin) protein fused with a His tag.

[0104] Figure 3 The following are 10% SDS-PAGE electrophoresis results of recombinant Staphylococcus aureus mutant α-hemolysin and its mutant H35Lα-Toxin. Figure 3 A is α-Toxin. Figure 3 B is H35Lα-Toxin, and the loading amount is 10 μg.

[0105] Figure 4 This study demonstrates the hemolytic effect of Staphylococcus aureus α-hemolysin and its mutant (H35Lα-Toxin) on sheep blood agar plates.

[0106] Figure 5 The study demonstrates the hemolytic effect of Staphylococcus aureus α-hemolysin and its mutant (H35Lα-Toxin) on rabbit blood. Figure 5 A is α-Toxin. Figure 5 B is H35Lα-Toxin.

[0107] Figure 6 The results of the detection process during the screening of hybridoma cell lines are shown, among which... Figure 6 A represents the ELISA results of antibody-α-hemolysin binding in the supernatant of different cell lines. Figure 6 B represents the inhibition results of antibodies on α-hemolysin in the supernatants of different cell lines.

[0108] Figure 7 The ELISA detection results of the antibody binding to α-hemolysin of the present invention are shown, wherein... Figure 7 A to Figure 7D shows the binding of the screened antibodies 78D4, 16H4, 78F4 and 98G9 to α-hemolysin.

[0109] Figure 8 The Octect binding and dissociation curves of the antibody α-hemolysin of the present invention are shown, wherein... Figure 8 A to Figure 8 D shows the binding of the selected humanized versions of the antibodies 78D4, 16H4, 78F4, and 98G9 to α-hemolysin.

[0110] Figure 9 This invention demonstrates the effect of the antibody against α-hemolysin hemolysis, wherein... Figure 9 A through 9C show the results for different antibody amounts.

[0111] Figure 10 The therapeutic effect of the antibody of the present invention in an animal model of α-hemolysin-induced sepsis is demonstrated.

[0112] Figure 11 This invention demonstrates the therapeutic effect of the antibody in an animal model of bacteremia induced by methicillin-resistant Staphylococcus aureus.

[0113] Figure 12 This demonstrates the therapeutic effect of the antibody of the present invention in an animal model of pneumonia caused by methicillin-resistant Staphylococcus aureus.

[0114] Figure 13 The pharmacokinetic results of a single administration of the antibody of the present invention to cynomolgus monkeys are shown. Detailed Implementation

[0115] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0116] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all medicinal materials and reagents used in the following examples are commercially available products.

[0117] This article uses known antibodies as controls, among which:

[0118] Aridis Pharmaceuticals' fully human antibody R-301 Abbreviated as AR (see US9249215B2), the heavy chain variable region is shown in SEQ ID NO:82, and the light chain variable region is shown in SEQ ID NO:83.

[0119] Astrazeneca Pharmaceuticals' humanized antibody MEDI4893, abbreviated as AZ (see US20140072577A1), has a heavy chain variable region shown in SEQ ID NO:84 and a light chain variable region shown in SEQ ID NO:85.

[0120] The antibody provided by the present invention has 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.

[0121] Example 1 Recombinant expression of Staphylococcus aureus α-toxin fused with His tag

[0122] Using the amino acid sequence of Staphylococcus aureus α-hemolysin as the target sequence, the corresponding base sequence was artificially synthesized and cloned into the Pet-21a plasmid containing a His tag using the NdeI and XhoI restriction enzyme sites. The amino acid sequence of Staphylococcus aureus α-hemolysin is shown in SEQ ID NO:78, and the corresponding base sequence is shown in SEQ ID NO:79. The construction of the recombinant plasmid is described in [link to documentation]. Figure 1 .

[0123] The obtained recombinant plasmid was transformed into competent BL21(DE3)pLysS cells. The following day, single colonies were picked and inoculated into LB broth containing 100 μg / ml ampicillin, and cultured overnight at 37°C with shaking. The overnight culture was then inoculated at a 1:100 volume ratio into LB broth containing 100 μg / ml ampicillin and cultured at 37°C with shaking at 200 rpm until OD500 was reached. 600 The concentration of IPTG was approximately 0.6–0.8. The solution was then added to the bacterial culture to a final concentration of 0.1 mM, and induced at 16°C for 16–18 h. The induced bacterial culture was then collected by centrifugation at 8,000 rpm for 3 min and stored at -80°C.

[0124] Example 2 Recombinant expression of the His-tagged Staphylococcus aureus α-hemolysin mutant (H35Lα-Toxin)

[0125] Based on the amino acid sequence of Staphylococcus aureus α-hemolysin, histidine (His) at position 35 was mutated to leucine (Leu), yielding the mutated amino acid sequence. The corresponding base sequence was artificially synthesized and cloned into the Pet-21a plasmid containing a His tag using the NdeI and XhoI restriction enzyme sites. The amino acid sequence of this Staphylococcus aureus mutated α-hemolysin (H35Lα-Toxin) is shown in SEQ ID NO:80, and the corresponding base sequence is shown in SEQ ID NO:81. The construction of the recombinant plasmid is described in [link to documentation]. Figure 2 .

[0126] The obtained recombinant plasmid was transformed into competent BL21(DE3)pLysS cells. The following day, single colonies were picked and inoculated into LB broth containing 100 μg / ml ampicillin, and cultured overnight at 37°C with shaking. The overnight culture was then inoculated at a 1:100 volume ratio into LB broth containing 100 μg / ml ampicillin and cultured at 37°C with shaking at 200 rpm until OD500 was reached. 600 The concentration of the bacterial culture was approximately 0.6–0.8. IPTG was added to the culture to a final concentration of 0.25 mM, and the culture was induced at 25 °C for 4.5 h. After induction, the bacterial culture was collected by centrifugation at 8,000 rpm for 3 min and stored at -80 °C.

[0127] Example 3 Purification of Staphylococcus aureus α-toxin and its mutant (H35Lα-toxin)

[0128] Escherichia coli induced to express Staphylococcus aureus α-hemolysin and its mutants were disrupted by ultrasonic disruption, with a 3-second interval at 180W for 7-9 minutes; centrifuged at 13,000 rpm for 30 minutes, and the supernatant was collected and filtered through a 0.22 μmL filter for sterilization.

[0129] The Ni column was mixed with the filtered supernatant at room temperature on a rotary mixer for 1 hour, and then the Ni column was loaded into a packed column. Proteins non-specifically bound to the Ni column were eluted with 5 column volumes of BD buffer (containing 30 mM imidazole) until the protein development buffer remained colorless. The target protein was then eluted with 5 column volumes of BB buffer (containing 300 mM imidazole). The elution buffer containing the target protein was then concentrated and replaced using a 10 kDa concentration tube with PBS as the solvent. The electrophoresis results of the obtained protein are shown below. Figure 3 .

[0130] Example 4 Biological activity assays of Staphylococcus aureus α-toxin and its mutant (H35Lα-toxin) (in vitro hemolysis assay and in vivo toxicity in mice)

[0131] Different concentrations (5 μg / ml, 0.5 μg / ml, 0.05 μg / ml) of Staphylococcus aureus α-hemolysin and its mutants were added dropwise to the surface of sheep blood agar plates (Shanghai Kemajia Microbial Technology Co., Ltd.). The blood plates were incubated at 37℃ for 24 h. A clear hemolytic zone was observed around the α-hemolysin, and the diameter of the hemolytic zone was correlated with its concentration; however, no hemolytic zone was observed around the α-hemolysin mutant. The experimental results are shown below. Figure 4 .

[0132] Add 75 μl of 5% rabbit red blood cells (Bio-channel Biotechnology) and different masses of Staphylococcus aureus α-hemolysin or its mutant to each well of a 96-well plate, and add PBS buffer to a final volume of 150 μl. Incubate at 37°C for 1 h, then centrifuge the 96-well plate at 3000 rpm for 3 min. Take 100 μl of the supernatant and measure the absorbance (OD) at 405 nm using a microplate reader. 405 The hemolytic activity of α-hemolysin was evaluated. Results showed that the hemolytic activity of α-hemolysin on rabbit erythrocytes was dose-dependent, while the α-hemolysin mutant showed no hemolytic activity. Experimental results are shown below. Figure 5 .

[0133] Different doses of Staphylococcus aureus α-hemolysin or its mutant were administered to C57 mice via tail vein injection. It was found that the lowest lethal dose of Staphylococcus aureus α-hemolysin recombinantly expressed according to the methods described in Examples 1, 2 and 3 was 3 μg / mouse; the highest dose of α-hemolysin mutant injected was 200 μg / mouse, and no adverse reactions were observed in the mice.

[0134] Example 5 Immunization of Balb / c mice with Staphylococcus aureus α-hemolysin mutant (H35Lα-Toxin)

[0135] α-hemolysin or an α-hemolysin mutant were diluted with PBS to different concentrations and injected via the tail vein into C57 mice. The toxic effects of both on C57 mice were compared to select the appropriate immunogen and dosage for subsequent immunization of animals. The results are shown in Table 1.

[0136] Table 1. Toxicity of different doses of α-hemolysin and α-hemolysin mutants in mice

[0137]

[0138] Example 6 Immunization of Balb / c mice with Staphylococcus aureus α-hemolysin mutant (H35Lα-Toxin)

[0139] Following the guidelines of Antibodies a Laboratory Manual, Second Edition (Edward A. Greenfield 2012), 8-week-old Balb / c mice were immunized over a period of 42 days at 14-day intervals.

[0140] Staphylococcus aureus α-hemolysin mutants were emulsified in complete or incomplete Freund's adjuvant and injected unilaterally into the subcutaneous tissue and peritoneal cavity of mice at three sites: the nape of the neck, the base of the tail, and the groin. Blood was collected from the tail vein on day 35 after immunization, and antibody titers were detected by ELISA. Spleen cells from immunized mice were then fused with myeloma cells.

[0141] Example 7 Screening, identification and antibody sequencing of hybridoma cell lines

[0142] Splenic cells from Balb / c mice immunized with the Staphylococcus aureus α-hemolysin mutant (H35Lα-Toxin) were fused with myeloma cells P3X63Ag8.653 using PEG or electrofusion methods. The fused hybridoma cells were seeded into 30 wells of a 384-well plate. After 24 hours, HAT-containing and HT-containing media were added for hybridoma cell selection. After culturing in the 384-well plates for 10-14 days, the cell supernatant was used for ELISA with α-Toxin to screen hybridoma parent clones that secrete antibodies specifically binding to α-hemolysin (see...). Figure 6 A). Subsequently, 94 wells from each plate were selected from the highest to the lowest ELISA OD values ​​and transferred to 96-well plates for incubation (wells from plate number 30, which had a slightly lower ELISA positivity rate, were not selected for transfer), for a total of 29 96-well plates.

[0143] Under physiological conditions, the presence of α-hemolysin leads to the lysis of red blood cells, and the release of lysates causes a change in the color of the solution supernatant. Anti-α-hemolysin antibodies in the cell secretion supernatant can inhibit the lysis of red blood cells by α-hemolysin. By detecting the absorbance of the supernatant, the degree of cell lysis by α-hemolysin and the inhibition of α-hemolysin by the antibody can be determined.

[0144] Hemolysis was detected in the supernatant of the 96-well plate culture after transfer. The procedure was as follows: WT-α-toxin was diluted to a stock solution of 5 μg / mL. 25 μL of this stock solution was mixed with an equal volume of cell culture supernatant and added to a 96-well plate containing 5% rabbit red blood cells (diluted to 75 μL with PBS). The plate was incubated at 37°C for 1 h. The plate was then centrifuged at 3000 rpm for 3 min. 75 μL of the supernatant was added to a fresh 96-well plate, and the OD405 and OD450 absorbance values ​​were measured using a microplate reader. Some results are shown below. Figure 6 B.

[0145] The selected hybridoma parent clones that secrete anti-α-hemolysin antibodies were added to 96-well plates containing feeder cells using a limiting dilution method. The monoclonal cells were observed and labeled under a microscope after 2-3 days. After 7 days, monoclonal hybridoma cells that could secrete anti-α-hemolysin monoclonal antibodies were screened by ELISA.

[0146] After expanding the culture of monoclonal hybridoma cells secreting anti-α-hemolysin monoclonal antibodies, total RNA was extracted from the cells according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.). The total RNA from the hybridoma cells was reverse transcribed into cDNA using 5×PrimeScript RTMaster Mix (Takara). The antibody light chain variable region IgVL(κ) and heavy chain variable region V were amplified using degenerate primers (Anke Krebber. 1997) and Extaq PCR reagent (Takara). H Sequence. PCR amplification products were purified using a PCR clean-up gel extraction kit (Macherey-Nagel). Following the instructions of the pClone007 Simple Vector Kit (Qingke Biotechnology Co., Ltd.), the amplified PCR products were ligated into a T vector and transformed into competent E. coli cells. After amplification and plasmid extraction, DNA sequencing was performed to obtain the variable region sequence of the monoclonal antibody.

[0147] The variable region sequence of the mouse antibody was obtained and analyzed as follows:

[0148] >98G9 murine antibody

[0149] Heavy chain variable region:

[0150] DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISGGSSTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCASGYPYGLDYWGQGTSVTVSS(SEQ ID NO:44)

[0151] Light chain variable region:

[0152] DIDMTQSPSSMYASLGERVTITCKASQDINWYLSWFQQKPGKSPKTLIYRGNRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPFTFGSGTKLEIK(SEQ ID NO:46)

[0153] The heavy and light chain CDRs of the 98G9 murine antibody were defined using different methods, as shown in Table 2.

[0154] Table 2. CDR sequence of 98G9 murine antibody

[0155] method 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 combination SEQ ID NO:2 SEQ ID NO:6 SEQ ID NO:7 method 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 combination SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:10

[0156] >78F4 murine antibody

[0157] Heavy chain variable region:

[0158] QVQLQQPGAELVRPGASVKLSCKASGYSFTSYWMNWVKQRPGQGLEWIGMIHPSDSETRLSQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRFDWDRAMDYWGQGTSVTVSS(SEQ ID NO:52)

[0159] Light chain variable region:

[0160] DIQMTQSPASSLSASVGETVTITCRASENIFSYLAWYQQKQGKSPQLLVYNTRSLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHYGTPWTFGGGTKLEIK(SEQ ID NO:54)

[0161] The heavy and light chain CDRs of the 78F4 murine antibody were defined using different methods, as shown in Table 3.

[0162] Table 3. CDR sequence of 78F4 murine antibody

[0163] method 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 combination SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:18 method 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 combination SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21

[0164] >78D4 murine antibody

[0165] Heavy chain variable region:

[0166] EVHLQQSGPELMKPGASVKISCKTSGYTFSEYTMHWVKQSHGKSLEWIGSINPNNGGTTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYNCARTRDYDNDGGLFAYWGQGTLVTVSA(SEQ ID NO:60)

[0167] Light chain variable region:

[0168] DVQITQSPSYLAASPGETITINCRASKNISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGNRSGTDFTLTISSLEPEDFAMYYCQQHYEYPFTFGGGTKLEIK(SEQ ID NO:62)

[0169] The heavy and light chain CDRs of the 78D4 murine antibody were defined using different methods, as shown in Table 4.

[0170] Table 4. CDR sequence of 78D4 murine antibody

[0171] method 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 combination SEQ ID NO:23 SEQ ID NO:27 SEQ ID NO:29 method 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 combination SEQ ID NO:30 SEQ ID NO:32 SEQ ID NO:33

[0172] >16H4 murine antibody

[0173] Heavy chain variable region:

[0174] EVQLQQSGAELVKPGASVTLSCTVSGFNIKDTYMHWVKQRPEQGLEWIGKIDPASGNTKYDPQFQGKATITADTSSNTAYLHLSSLTSEDSAVYFCASPYGNDYAMNYWGQGTSVTVSS(SEQ ID NO:68)

[0175] Light chain variable region:

[0176] DIQMTQSPASSLSASVGETVTITCRASEKIYSFLAWYQQKQEKSPQLLVYNAETLAEGVPSRFSGTGSGIQFSLKIISLQPEDFGIYYCQHHYGTPYTFGGGTKLEIK(SEQ ID NO:70)

[0177] The heavy and light chain CDRs of 16H4 murine antibodies were defined using different methods, as shown in Table 5.

[0178] Table 5. CDR sequences of 16H4 murine antibodies

[0179]

[0180]

[0181] Example 8 The binding of the antibody to α-toxin in this invention

[0182] Dilute α-hemolysin to 1 μg / ml with PBS buffer, and coat each well with 100 μl of the solution in a 96-well plate (Microwell 96F167008, Thermo). Incubate overnight at 4°C. The next day, remove the 96-well plate and wash it with PBST (containing 0.5% PBS), soaking for 1 min each time and then thoroughly drying off any remaining water. Add 200 μl of PBST containing 5% BSA to each sample well and block at 37°C for 1 h. Then wash the plate with PBST and dry the wells.

[0183] Add 100 μl of recombinant anti-α-hemolysin monoclonal antibody at serially diluted concentrations to each well of a 96-well plate (antibody concentrations are shown in the image). Figure 7 (x-axis) Incubate overnight at 4℃. After removing the 96-well plate, wash with PBST, then add 100 μl of anti-mouse IgG secondary antibody (IH-0031, Beijing Dingguo Changsheng Biotechnology) to each well and incubate at 37℃ for 1 h. Wash 5 more times with PBST, add 100 μl of Substrate Solution (Invitrogen) to each well, and incubate at 37℃ for 10 min; stop the reaction by adding 50 μl of 2N sulfuric acid to each well, and then measure the absorbance at 450 nm using a microplate reader (Multiskcin FC, Thermo).

[0184] See results Figure 7 .

[0185] Example 9 The Acquisition of Chimeric Antibodies and Humanized Antibodies in this Invention

[0186] First, the complete light and heavy chain variable regions of the murine antibody were combined with the human light and heavy chain constant regions to obtain a chimeric antibody as a control. The resulting chimeric antibody was named "mice-derived antibody (abbreviation -xi)".

[0187] After comprehensive analysis of the murine antibody heavy chain sequence to identify the antigen complementarity determinant (CDR) region for antibody-antigen binding and the framework region supporting the conserved three-dimensional conformation of the antibody, a search of known human antibody sequences was conducted. The most similar human antibody heavy chain sequence to the murine antibody, such as IGHV1-3*01, was selected, and its antibody framework region sequence was chosen as a template. The murine antibody heavy chain CDR was embedded into the human antibody framework region to generate a humanized antibody heavy chain sequence (heavy chain version 0). Subsequently, individual amino acid sites in the murine framework region that might be involved in antigen-antibody binding were reconstituted to generate humanized antibody heavy chain sequences (versions 1, 2, 3, etc.). The same process was used to generate humanized antibody light chain sequences (versions 0, 1, 2, etc.). The designed and synthesized humanized antibody light and heavy chains were co-transfected into 293 cells for recombinant expression of the humanized antibody (version naming convention, for example: heavy chain version 0 + light chain version 0 co-expression, i.e., H0L0, which can be further abbreviated to version 00). Experiments have shown that the purified humanized antibody and the mouse maternal antibody exhibit the same specific binding activity to α-hemolysin protein.

[0188] The antigen-binding power of the finally obtained humanized antibody version and chimeric antibody xi version was compared using the Octect instrument. Figure 8 These are the results of partial antibody assays. Based on the binding and dissociation curves of the antibody and antigen, specific humanized antibody versions exhibit properties similar to or better than control antibodies (including the chimeric antibody or AZ / AR antibody of this invention) during the antigen-antibody binding and dissociation phases.

[0189] The following humanized antibodies were obtained through screening.

[0190] Humanized antibody 98G9-02 (98G9-H0L2)

[0191] Heavy chain variable region (H0):

[0192] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWVSYISGGSSTIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASGYPYGLDYWGQGTLVTVSS(SEQ ID NO:48)

[0193] Light chain variable region (L2):

[0194] DIQMTQSPSSSLSASVGDRVTITCKASQDINWYLSWFQQKPGKAPKTLIYRGNRLVDGVPSRFSGSGSGQDYTFTISSLQPEDMATYYCLQYDEFPFTFGQGTKVEIK(SEQ ID NO:76)

[0195] Humanized antibody 98G9-03 (98G9-H0L3)

[0196] Heavy chain variable region (H0):

[0197] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWVSYISGGSSTIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASGYPYGLDYWGQGTLVTVSS(SEQ ID NO:48)

[0198] Light chain variable region (L3):

[0199] DIQMTQSPSSSLSASVGDRVTITCKASQDINWYLSWFQQKPGKAPKTLIYRGNRLVEGVPSRFSGSGSGQDYTFTISSLQPEDMATYYCLQYDEFPFTFGQGTKVEIK(SEQ ID NO:50)

[0200] Humanized antibody 78F4-00 (78F4-H0L0)

[0201] Heavy chain variable region (H0):

[0202] QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMNWVRQAPGQGLEWMGMIHPSDSETRLSQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARFDWDRAMDYWGQGTLVTVSS(SEQ ID NO:56)

[0203] Light chain variable region (L0):

[0204] DIQMTQSPSSSLSASVGDRVTITCRASENIFSYLAWYQQKPGKAPKLLIYNTRSLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPWTFGQGTKLEIK(SEQ ID NO:58)

[0205] Humanized antibody 78D4-33 (78D4-H3L3)

[0206] Heavy chain variable region (H3):

[0207] QVQLVQSGAEVKKPGASVKVSCKTSGYTFSEYTMHWVRQAPGQRLEWMGSINPNQGGTTYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCARTRDYDNDGGLFAYWGQGTLVTVSS(SEQ ID NO:64)

[0208] Light chain variable region (L3):

[0209] DVQITQSPSFLSASVGDRVTITCRASKNILKYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSRSGTEFTLTISSLQPEDFATYYCQQHYEYPFTFGQGTKLEIK(SEQ ID NO:66)

[0210] Humanized antibody 16H4-11 (16H4-H1L1)

[0211] Heavy chain variable region (H1):

[0212] EVQLVQSGAEVKKPGATVKISCKVSGFNIKDTYMHWVQQAPGKGLEWMGKIDPASGNTKYDPQFQGRVTITADTSNTAYMELSSLRSEDTAVYYCATPYGNDYAMNYWGQGTLVTVSS(SEQ ID NO:72)

[0213] Light chain variable region (L1):

[0214] DIQMTQSPSSSLSASVGDRVTITCRASEKIYSFLAWYQQKPGKAPKLLLYNAETLAEGVPSRFSGSGSGIDFTLTISSLQPEDFATYYCQHHYGTPYTFGGGTKVEIK(SEQ ID NO:74)

[0215] The heavy and light chain CDRs of humanized antibodies are shown in Table 6.

[0216] Table 6. CDR sequences of humanized antibodies

[0217]

[0218]

[0219] Example 10 Detection of the hemolytic activity of the antibody against α-toxin in this invention

[0220] Add 75 μl of 5% rabbit red blood cells (Bio-channel Biotechnology), 12.5 ng of Staphylococcus aureus α-hemolysin, and different weights of anti-α-hemolysin antibody (12.5 ng, 25 ng, 50 ng) to each well of a 96-well plate, and add PBS buffer to a final volume of 150 μl. Incubate at 37°C for 1 h, then centrifuge the 96-well plate at 3000 rpm for 3 min. Take 100 μl of the supernatant and measure the absorbance (OD) at 405 nm using a microplate reader. 405 To evaluate its hemolytic activity.

[0221] Experimental results show that the chimeric and humanized antibodies of this invention exhibit significant anti-α-hemolysin hemolytic activity in a dose-dependent manner, with some antibody activities comparable to the control antibodies AR and AZ. See results below. Figure 9 .

[0222] Example 11 The binding kinetics (K-toxin) of the antibody in this invention with α-toxin on K off and affinity constant K D Detection

[0223] Antibody-antigen interaction was determined using a GE BIAcore S200 instrument.

[0224] Following the instructions of the GE Human Antibody Capture Kit (catalog number BR-1008-39, Lot 10261753), the maximum amount of anti-human Fc antibody was first saturated and coupled to both the analytical and control sample channels of the CM5 sensor chip. Then, 7.5 μg / ml of the antibody to be tested was flowed through the analytical channel to ensure uniform antibody distribution. Finally, a serially diluted antigen sample (initial concentration 20 nM, 1:3 dilution to 8 concentration points, with repetition at a 0.741 nm concentration point) was flowed through both the analytical and sample channels. The photoreaction value after antibody-antigen binding was measured. Subsequently, the binding constant Kon, dissociation constant Koff, and affinity constant KD of the antibody were obtained through instrument software fitting (1:1) analysis.

[0225] The results are shown in Table 7.

[0226] Table 7. Binding kinetics and affinity constants of the antibodies and antigens of the present invention

[0227] Antibody ka(1 / Ms) kd(1 / s) KD(M) Rmax(RU) <![CDATA[Chi 2 (RU 2 )]]> 78D4 xiIgG 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 xiIgG 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

[0228] Example 12 Replication of an animal model of sepsis induced by α-toxin and detection of the therapeutic effect of the antibody of this invention.

[0229] C57BL / 6J mice were randomly divided into a model control group and a monoclonal antibody treatment group based on body weight. Thirty minutes before the experiment, the treatment group received a tail vein injection of anti-α-hemolysin monoclonal antibody (6 μg / mouse), while the control group received the same dose of PBS. Then, all mice received a tail vein injection of α-hemolysin (3 μg / mouse) to establish a mouse model of sepsis infection. The survival time of the experimental animals was observed and recorded. Results are shown below. Figure 10 .

[0230] Example 13 Replication of an animal model of bacteremia induced by methicillin-resistant Staphylococcus aureus and detection of the therapeutic effect of the antibody of this invention.

[0231] Methicillin-resistant Staphylococcus aureus USA300 was activated for two generations on TSB solid medium plates, inoculated into TSB liquid medium and cultured overnight. The cells were collected by centrifugation at 12,000 rpm and resuspended in physiological saline for later use.

[0232] C57BL / 6J mice were infected with USA300 via the tail vein at a rate of 6 × 10⁶ mm. 7Mice were randomly divided into a control group (CFU / mouse) and different anti-α-hemolysin monoclonal antibody treatment groups based on body weight. Two hours after infection, mice were injected via tail vein. The monoclonal antibody treatment groups received 15 mg / kg of the corresponding antibody, while the control group received the same dose of PBS. Survival time was observed and recorded for each group. Results are shown below. Figure 11 .

[0233] Example 14 Replication of an animal model of pneumonia caused by methicillin-resistant Staphylococcus aureus and detection of the therapeutic effect of the antibody of this invention.

[0234] Methicillin-resistant Staphylococcus aureus USA300 was activated for two generations on TSB solid medium plates, inoculated into TSB liquid medium and cultured overnight. The cells were collected by centrifugation at 12,000 rpm and resuspended in physiological saline for later use.

[0235] C57BL / 6J mice were infected with USA300 1.8×10⁻⁶ via tracheal inoculation. 8 Animals were randomly divided into four groups based on body weight: a model control group, a monoclonal antibody treatment group, a vancomycin treatment group, and a vancomycin + monoclonal antibody treatment group. Two hours after infection, animals received the corresponding drug treatment via tail vein injection. The monoclonal antibody dose was 15 mg / kg, and the vancomycin dose was 1.25 mg / kg. The control group received the same dose of PBS. Animals were sacrificed 24 hours after infection, and lung tissue was homogenized, weighed, homogenized, and spread on TSB solid medium to detect the bacterial load. The experimental results showed that the anti-α-hemolysin antibody in this invention can enhance the pharmacodynamic effect of vancomycin in treating methicillin-resistant Staphylococcus aureus pneumonia.

[0236] See results Figure 12 .

[0237] Example 15 Acute toxicity study of the antibody of this invention

[0238] Acute toxicity studies of the 78D4 H3L3 antibody molecule of this invention were conducted in mice and cynomolgus monkeys (N=10 in mice and N=3 in cynomolgus monkeys).

[0239] C57BL / 6 mice (18-20g), half male and half female, were injected intravenously with 125mg / kg of 78D4 H3L3 antibody molecules within 24 hours. Results showed that no mice died when given the maximum dose of 125mg / kg within 24 hours. No adverse effects were observed in the animals after 14 days of continuous observation. Gross examination of the major organs (heart, liver, spleen, lungs, kidneys, and brain) of euthanized animals revealed no abnormalities.

[0240] Each male cynomolgus macaque was administered a single intravenous infusion of 10 mg / kg of 78D4 H3L3 antibody molecules via the limbs. Results showed that no animal deaths occurred at the 10 mg / kg dose of 78D4 H3L3 antibody molecules, and no adverse effects were observed in the animals during a 28-day observation period.

[0241] Acute toxicity studies have shown that the 78D4 H3L3 antibody molecule has good safety.

[0242] Example 16 Pharmacokinetic study of the antibody of this invention

[0243] The pharmacokinetic study of the 78D4 H3L3 antibody molecule was conducted in cynomolgus monkeys at a single dose of 10 mg / kg (N=3).

[0244] Male cynomolgus macaques were administered a single intravenous infusion of 10 mg / kg of the 78D4 H3L3 antibody molecule via limb veins. Blood samples were collected at 0 h before administration (pre-dose), and at 0.25 h (15 min), 0.5 h (needle removal point), 4 h, 24 h (D2), 48 h (D3), 96 h (D5), 168 h (D8), 336 h (D15), 504 h (D22), and 672 h (D29). Blood was collected from peripheral veins of the limbs (excluding the administration limb) or the inguinal vein. Approximately 1 mL of whole blood was collected per animal at each time point. Antibody concentrations in cynomolgus macaque serum were determined using ELISA. Pharmacokinetic parameters, including AUClast, CL, and T1 / 2, were calculated using a non-compartmental model analysis with WinNonlin Phoenix (v6.4, Phrasight) software.

[0245] The concentration of 78D4 H3L3 antibody in the serum of cynomolgus monkeys was determined by ELISA. Individual serum drug concentrations are shown in the figure. Figure 13 The pharmacokinetic parameters are summarized in Table 8. Drug exposure was observed in all animals after administration.

[0246] Table 8. Pharmacokinetic results of antibody 78D4 H3L3

[0247]

[0248] Example 17 Optimization of the formulation of the antibody stabilizing agent of the present invention

[0249] Taking the 78D4 H3L3 antibody molecule as an example, we screened a stable formulation of humanized anti-Staphylococcus aureus α-toxin antibody.

[0250] Antibody preparation method: The antibody was replaced into the target buffer, aliquoted, and placed at 4℃ and 40℃ for stability study. Samples were taken for testing at 0 days, 7 days, and 14 days. The test items included SEC and CEX.

[0251] The buffer solution composition is as follows:

[0252]

[0253] Buffer system and pH selection criteria:

[0254] The changes in the acidic peak content of 78D4 H3L3 at 40℃ were investigated using the CEX method to determine the optimal buffer system and pH for this antibody. The results are shown in Table 9. The CEX acidic peak content was fitted with straight lines at the start of the experiment (0 days), 7 days at 40℃, and 14 days, and the rate of increase of the acidic peak (% / day) was calculated.

[0255] Table 9. Results of CEX acid peak investigation on high-temperature stability of different buffer systems

[0256]

[0257] As shown in Table 9, the rate of increase of the acid peak in the histidine buffer system at pH 5.0-6.0 is lower than that in the citric acid and acetic acid systems. Therefore, the histidine buffer system was selected for the next screening step.

[0258] Criteria for selecting protein concentration:

[0259] After determining the optimal buffer composition, the effect of different protein concentrations on stability was further investigated. 78D4H3L3 samples were displaced into different formulations and subjected to accelerated stability testing at 40°C. Samples were taken at 0, 7, 14, and 28 days for SEC detection. Linear values ​​of the SEC peak content at the start of the experiment (0 days), 7 days, 14 days, and 28 days at 40°C were fitted, and the rate of decline of the peak (% / day) was calculated. The results are shown in Table 10.

[0260] Table 10. Results of SEC main peak assay for high-temperature stability at different protein concentrations

[0261]

[0262]

[0263] As can be seen from Table 10, the rate of decrease of the SEC peak content tends to increase with the increase of concentration. In view of this result, and considering future clinical medication plans, further investigation will be conducted around a concentration of 50 mg / ml.

[0264] Criteria for selecting buffer salt concentration:

[0265] The 78D4 H3L3 sample was displaced into a non-concentration buffer solution and then placed at 40°C for accelerated stability testing. Samples were taken at 0, 7, 14, and 28 days for CEX detection. Linear fitting of CEX acid peak content at the start of the experiment (0 days), 7 days, 14 days, and 28 days at 40°C was performed, and the increase rate of the acid peak (% / day) was calculated. The results are shown in Table 11.

[0266] Table 11. Results of CEX acid peak investigation on high-temperature stability at different buffer concentrations

[0267]

[0268] Table 11 shows that as the buffer concentration increases, the rate of increase of the CEX acid peak of 78D4 H3L3 increases. There is no significant difference between 5 mM and 10 mM on the effect of the CEX acid peak. In order to maintain the buffering capacity of the buffer, 10 mM was selected as the final buffer concentration.

[0269] Criteria for selecting protective agents:

[0270] After determining the above conditions, further research and comparison were conducted on the addition of protective agents to select the excipients that could make the antibody most stable. The results are shown in Tables 12 and 13.

[0271] Table 12. Results of SEC main peak investigation on high-temperature stability of different protective agents

[0272]

[0273]

[0274] Table 13. Results of CEX acid peak investigation on high-temperature stability of different protective agents

[0275]

[0276] Tables 12 and 13 show that the rate of decrease in SEC purity and the rate of increase in CEX acid peak were not significantly different among different protective agents. Therefore, sucrose, which is commonly used in biopharmaceuticals, was chosen as the additive.

[0277] Basis for selecting the concentration of protective agent:

[0278] After determining sucrose as the preservative, the effects of different concentrations of sucrose on the storage period of 78D4 H3L3 antibody protein were investigated. The results are shown in Tables 14 and 15.

[0279] Table 14. Results of high-temperature stability study of the SEC main peak at different protective agent concentrations

[0280]

[0281] Table 15. Results of CEX acid peak investigation on high-temperature stability of different protective agents

[0282]

[0283] Tables 14 and 15 show that the antibody is relatively stable at different sucrose concentrations. Based on the principle of minimizing the amount of excipients added and considering both the need to fully protect the protein, 5% sucrose was selected as the protective agent for 78D4 H3L3.

[0284] Criteria for surfactant selection:

[0285] The 78D4 H3L3 antibody sample was displaced into a 10mM histidine, 6% sucrose buffer solution, resulting in a protein concentration of 50 mg / ml. Different final concentrations of Tween 20 and 80 were added to the displaced sample. The prepared samples were subjected to 1, 3, 5, and 10 freeze-thaw cycles before the insoluble microparticles were measured. The results are shown in Tables 16 and 17.

[0286] Table 16. Results of insoluble particulate matter in Tween 20 freeze-thaw samples with different proportions

[0287]

[0288] Table 17. Results of insoluble particulate matter in Tween 80 freeze-thaw samples with different proportions

[0289]

[0290] Table 16 shows a significant increase in insoluble particles in samples without Tween 20, while the sample containing 0.01% Tween 20 showed a greater increase in insoluble particles after 10 freeze-thaw cycles, indicating that 0.01% Tween 20 cannot effectively inhibit the aggregation of 78D4 H3L3 protein; ≥0.02% Tween 20 showed no significant difference in anti-aggregation effect. Table 17 shows that ≥0.005% Tween 80 effectively inhibited aggregation, and there was no significant difference in the change of insoluble particles compared to Tween 20, indicating that for 78D4 H3L3 samples, Tween 80 has a better anti-aggregation effect than Tween 20. Therefore, 0.01% Tween 80 was selected as the surfactant for 78D4 H3L3.

[0291] Example 18 Efficacy verification of the antibody stabilizing agent formulation of the present invention

[0292] The formulation validation tests included freeze-thaw stability tests, 10°C shaking stability tests, and light exposure tests to examine the stability of the 78D4H3L3 antibody in the final formulation.

[0293] Oscillation stability test:

[0294] The 78D4 H3L3 protein used in the experiment was a three-step purified sample, concentrated to the required concentration using a preferred antibody formulation, and subjected to a shaking stability test at 10°C for 5 days. The study focused on the 78D4 H3L3 monomer content (SEC), charge isomer peak content (CEX), and antibody activity (binding ELISA). The results are summarized in Table 18.

[0295] Table 18. Results of physicochemical property investigation on the oscillation stability of 78D4 H3L3

[0296]

[0297] Freeze-thaw stability test:

[0298] The 78D4 H3L3 protein used in the experiment was a three-step purified sample, concentrated to the required concentration using a preferred antibody formulation, and subjected to repeated freeze-thaw cycles of 1, 3, and 5. The study focused on the 78D4 H3L3 monomer content (SEC), charge isomer peak content (CEX), and antibody activity (binding ELISA). The results are summarized in Table 19.

[0299] Table 19. Results of the physicochemical properties investigation of freeze-thaw stability of 78D4 H3L3

[0300]

[0301] Light stability test:

[0302] The 78D4 H3L3 protein used in the experiment was a three-step purified sample, concentrated to the required concentration using a preferred antibody formulation, and subjected to a photostability test. The sample was exposed to light at 4500 lx ± 500 lx for 5 and 10 days, while the same sample was placed in its packaging box as a light-protected control. The study focused on the 78D4 H3L3 monomer content (SEC), charge isomer peak content (CEX), and antibody activity (binding ELISA). The results are summarized in Table 20.

[0303] Table 20. Results of the physicochemical properties investigation of freeze-thaw stability of 78D4 H3L3

[0304]

[0305]

[0306] The above results indicate that, under the defined formulation, shaking for 5 days, freeze-thaw cycles for 5 days, and light exposure for 10 days had no significant effect on the physicochemical properties of the 78D4 H3L3 protein. This suggests that the 78D4 H3L3 antibody is relatively stable under this formulation.

[0307] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims. sequence list <110> Maiwei (Shanghai) Biotechnology Co., Ltd. <120> Anti-α-hemolysin antibodies and their stabilizing agents <160> 87 <170> SIPOSequenceListing 1.0 <210> 1 <211> 7 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR1 <400> 1 Gly Phe Thr Phe Ser Thr Phe 1 5 <210> 2 <211> 10 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR1 <400> 2 Gly Phe Thr Phe Ser Thr Phe Gly Met His 1 5 10 <210> 3 <211> 5 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR1 <400> 3 Thr Phe Gly Met His 1 5 <210> 4 <211> 6 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR2 <400> 4 Ser Gly Gly Ser Ser Thr 1 5 <210> 5 <211> 10 <212> PRT <213> 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Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR3 <400> 18 Phe Asp Trp Asp Arg Ala Met Asp Tyr 1 5 <210> 19 <211> 11 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain, CDR1 <400> 19 Arg Ala Ser Glu Asn Ile Phe Ser Tyr Leu Ala 1 5 10 <210> 20 <211> 7 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain, CDR2 <400> 20 Asn Thr Arg Ser Leu Ala Glu 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain, CDR3 <400> twenty one Gln His His Tyr Gly Thr Pro Trp Thr 1 5 <210> twenty two <211> 7 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR1 <400> twenty two Gly Tyr Thr Phe Ser Glu Tyr 1 5 <210> twenty three <211> 10 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR1 <400> twenty three Gly Tyr Thr Phe Ser Glu Tyr Thr Met His 1 5 10 <210> twenty four <211> 5 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR1 <400> twenty four Glu Tyr Thr Met His 1 5 <210> 25 <211> 6 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR2 <400> 25 Asn Pro Asn Asn Gly Gly 1 5 <210> 26 <211> 10 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR2 <400> 26 Ser Ile Asn Pro Asn Asn Gly Gly Thr Thr 1 5 10 <210> 27 <211> 17 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR2 <400> 27 Ser Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 28 <211> 17 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR2 <400> 28 Ser Ile Asn Pro Asn Gln Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 29 <211> 13 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR3 <400> 29 Thr Arg Asp Tyr Asp Asn Asp Gly Gly Leu Phe Ala Tyr 1 5 10 <210> 30 <211> 11 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain, CDR1 <400> 30 Arg Ala Ser Lys Asn Ile Ser Lys Tyr Leu Ala 1 5 10 <210> 31 <211> 11 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain, CDR1 <400> 31 Arg Ala Ser Lys Asn Ile Leu Lys Tyr Leu Ala 1 5 10 <210> 32 <211> 7 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain, CDR2 <400> 32 Ser Gly Ser Thr Leu Gln Ser 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain, CDR3 <400> 33 Gln Gln His Tyr Glu Tyr Pro Phe Thr 1 5 <210> 34 <211> 7 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR1 <400> 34 Gly Phe Asn Ile Lys Asp Thr 1 5 <210> 35 <211> 10 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR1 <400> 35 Gly Phe Asn Ile Lys Asp Thr Tyr Met His 1 5 10 <210> 36 <211> 5 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR1 <400> 36 Asp Thr Tyr Met His 1 5 <210> 37 <211> 6 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR2 <400> 37 Asp Pro Ala Ser Gly Asn 1 5 <210> 38 <211> 10 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR2 <400> 38 Lys Ile Asp Pro Ala Ser Gly Asn Thr Lys 1 5 10 <210> 39 <211> 17 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR2 <400> 39 Lys Ile Asp Pro Ala Ser Gly Asn Thr Lys Tyr Asp Pro Gln Phe Gln 1 5 10 15 Gly <210> 40 <211> 10 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain, CDR3 <400> 40 Pro Tyr Gly Asn Asp Tyr Ala Met Asn Tyr 1 5 10 <210> 41 <211> 11 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain, CDR1 <400> 41 Arg Ala Ser Glu Lys Ile Tyr Ser Phe Leu Ala 1 5 10 <210> 42 <211> 7 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain, CDR2 <400> 42 Asn Ala Glu Thr Leu Ala Glu 1 5 <210> 43 <211> 9 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain, CDR3 <400> 43 Gln His His Tyr Gly Thr Pro Tyr Thr 1 5 <210> 44 <211> 117 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain variable region <400> 44 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Gly Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ser Gly Tyr Pro Tyr Gly Leu Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 45 <211> 351 [[ID=2,0]]<212> DNA <213> artificial <220> <221> gene <222> ()..() <223> Heavy chain variable region<0,001\399><400> 45 gatgtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc 60 tcctgtgcag cctctggatt cactttcagt acctttggaa tgcactgggt tcgtcaggct 120 ccagagaagg ggctggagtg ggtcgcatac attagtggtg gcagtagtac catctactat 180 gcagacacag tgaagggccg attcaccatc tccagagaca atcccaagaa caccctgttc 240 ctgcaaatga ccagtctaag gtctgaggac acggccatgt attactgtgc aagcggctac 300 It should be noted that in the original text, there is a tag <0,001\399> which seems to be an incorrect format. It is likely a typo and should probably be . The translation has been done as accurately as possible based on the provided text.ccctatggtt tggactactg gggtcaagga acctcagtca ccgtctcctc a 351 <210> 46 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain variable region <400> 46 Asp Ile Asp Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Trp Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Gly Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 47 <211> 321 <212> DNA <213> Artificial <220> <221> gene <222> ()..() <223> Light chain variable region <400> 47 gacatcgaca tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacattaat tggtatttaa gttggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt ggaaacagat tggtagatgg ggtcccatca 180 aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 240 gaagatatgg gaatttatta ttgtctacag tatgatgagt ttccattcac gttcggctcg 300 gggacaaagt tggaaataaa a 321 <210> 48 <211> 117 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain variable region <400> 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Gly Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Gly Tyr Pro Tyr Gly Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 49 <211> 351 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> heavy chain variable region <400> 49 gaggtgcagc tggtggagag cgggggggga ctggtgcagc caggaggaag cctgagactg 60 agctgtgccg caagcgggtt cacatttagt acctttggaa tgcactgggt gaggcaggcc 120 cccggcaaag ggctggagtg ggtgtcttat atttccggcg gaagtagcac catatactac 180 gctgatacag tgaagggcag attcaccata agcagggaca acgccaagaa cagcctgtac 240 ctgcagatga acagcctgag agccgaagac accgctgtgt actactgcgc cagcggctac 300 ccctacggcc tggattactg gggacaagga acactggtga ccgtgagcag c 351 <210> 50 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> light chain variable region <400> 50 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Trp Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Gly Asn Arg Leu Val Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 51 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> light chain variable region <400> 51 gacattcaga tgacccagag ccccagcagc ctgagcgcca gcgtgggaga cagagtgacc 60 ataacctgca aagccagcca agacatcaac tggtatctgt cctggtttca gcagaagccc 120<00015​​​​​​​​​​​​​​​​​​​​​​<223> Heavy chain variable region <400> 52 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Ser Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Asp Trp Asp Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 53 <211> 354 <212> DNA <213> Artificial <220> <221> gene <222> ()..() <223> Heavy chain variable region <400> 53 caggtccaac t tgcagcagcc tggggctgag ctggtgaggc ctggagcttc agtgaagctg 60 tcctgcaagg cttctggcta ctccttcacc agctactgga tgaactgggt gaagcagagg 120 cctggacaag gccttgagtg gattggcatg attcatcctt ccgatagtga aactaggtta 180 agtcagaagt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240 atgcaactca gcagcccgac atctgaggac tctgcggtct attactgtac aagattcgac 300 tgggaccggg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354 <210> 54 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain variable region <400> 54 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Phe Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Thr Arg Ser Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 55 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> light chain variable region <400> 55 gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtga gaatattttc agttatttag catggtatca acagaaacag 120 ggaaaatctc ctcagctcct ggtctataat acaagatcct tagcagaagg tgtgccatca 180 aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240 gaagattttg ggacttatta ctgtcaacat cattatggta ctccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 56 <211> 118 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> heavy chain variable region <400> 56 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Ser Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Asp Trp Asp Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 ​ <211> 354 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> heavy chain variable region <400> 57 caggtccaac tcgtccaaag cggcgcagaa gtcaaaaagc ccggcgcatc agtcaaagtt 60 agctgcaagg ccagcggcta cagcttcaca tcatactgga tgaactgggt gcggcaagcc 120 cccggccaag gtctcgaatg gatgggaatg atccacccca gcgacagcga gaccaggctg 180 agccagaaat ttaaagacag agtcaccatg accagagaca cctccacctc aaccgtctat 240 atggaactga gcagcctcag aagcgaggac accgccgtgt attactgcgc ccggttcgac 300 tgggacagag ccatggacta ctggggccaa ggcaccctcg ttaccgtgag cagc 354 <210> 58 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> light chain variable region <400> 58 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Phe Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Thr Arg Ser Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 59 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> light chain variable region <400> 59 gacattcaga tgacccagag ccccagcagc ctgagcgcca gcgtgggaga cagagtgacc 60 ataacctgca gagccagcga gaacatattc tcatacctcg cctggtacca gcagaaaccc 120 ggcaaagccc caaaactgct catctacaac acaagaagcc tggctgaagg agtgcccagc 180 agattcagcg ggtcaggcag cggcaccgac ttcaccctga ccatcagcag cctgcaacca 240 gaagacttcg ccacctacta ctgccaacac cactacggca ccccctggac cttcggacaa 300 ggcaccaaac tcgagatcaa a 321 <210> 60 <211> 122 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain variable region <400> 60 Glu Val His Leu Gln Gln Ser Gly Pro Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Ser Glu Tyr 20 25 30 Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Ser Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Asn Cys 85 90 95 Ala Arg Thr Arg Asp Tyr Asp Asn Asp Gly Gly Leu Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 61 <211> 366 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> heavy chain variable region <400> 61 gaggtccacc tgcaacagtc tggacctgag ctgatgaagc ctggggcttc agtgaagata 60 tcctgcaaga cttctggata cacattcagt gaatacacca tgcactgggt gaagcagagc 120 catggaaaga gccttgagtg gattggaagt attaatccta acaatggtgg tactacctac 180 aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac 240 atggagctcc gcagcctgac atctgaggat tctgcagtct ataactgtgc aagaactagg 300 gactatgata acgacggggg tctttttgct tactggggcc aagggactct ggtcactgtc 360 tctgca 366 <210> 62 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> light chain variable region <400> 62 Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Asn Ile Ser Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Asn Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Tyr Glu Tyr Pro Phe 85 90 95 [[ID=<222> ()..() <223> Light chain variable region <400> 63 gatgtccaga taacccagtc tccatcttat cttgctgcat ctcctggaga aaccattact 60 attaattgca gggcaagtaa gaacattagc aaatatttag cctggtatca agagaaacct 120 gggaaaacta ataagcttct tatctactct ggatccactt tgcaatctgg aattccatca 180 aggttcagtg gcaatagatc tggtacagat ttcactctca ccatcagtag cctggagcct 240 gaagattttg caatgtatta ctgtcaacaa cattatgaat acccgttcac gttcggaggg 300 gggaccaagc tggaaataaa a 321 <210> 64 <211> 122 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain variable region <400> 64 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Ser Glu Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Ser Ile Asn Pro Asn Gln Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Arg Asp Tyr Asp Asn Asp Gly Gly Leu Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 65 <211> 366 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> heavy chain variable region <400> 65 caggtccaac tcgtccaaag cggcgcagaa gtcaaaaagc ccggcgcatc agtcaaagtt 60 agctgcaaga catccggcta caccttcagc gagtacacca tgcactgggt gagacaagcc 120 ccaggccaaa gactggagtg gatgggaagc atcaacccca accaaggcgg caccacctac 180 aaccaaaaat tcaagggcag agtgacaatt accgtggaca agagcgccag caccgcctac 240 atggagctgt ctagcctgag aagcgaggac accgccgtgt actattgcgc cagaaccaga 300 gactacgaca acgatggagg actgttcgcc tattggggcc agggaaccct cgtgaccgtg 360 agcagc 366 <210> 66 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain variable region <400> 66 Asp Val Gln Ile Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Asn Ile Leu Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Glu Tyr Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 67 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> light chain variable region <400> 67 gacgtgcaga ttacccaaag ccccagcttc ctgtccgcca gcgtgggcga cagagtgaca 60 attacatgca gagccagcaa gaacatactg aagtacctgg catggtacca acaaaaaccc 120 ggcaaggccc ccaaactgct catctactcc ggcagtaccc tgcagagcgg cgtgcccagc 180 agattcagcg gaagcagaag cggcaccgag ttcactctga ccatcagcag cctccaacca 240 gaggacttcg ccacctacta ctgccagcag cactacgaat accccttcac cttcggccag 300 ggcaccaagc tggagatcaa a 321 <210> 68 <211> 119 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain variable region <400> 68 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Thr Val Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Lys Ile Asp Pro Ala Ser Gly Asn Thr Lys Tyr Asp Pro Gln Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Asn Asp Tyr Ala Met Asn Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 69 <211> 357 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> Heavy chain variable region <400> 69 gaggttcagc tgcagcagtc tggggcagaa cttgtgaagc caggggcctc agtcacgttg 60 tcctgcacag tttctggctt caacattaaa gacacctata tgcactgggt gaaacagagg 120 cctgaacagg gcctggagtg gattggaaag attgatcctg cgagtggtaa tactaaatat 180 gacccgcagt tccagggcaa ggccactata acagcagaca catcctccaa cacagcctac 240 ctgcacctca gcagcctgac atctgaggac agtgccgtct atttctgtgc tagcccctat 300 ggtaacgact atgctatgaa ctactgggga caaggaacct cagtcaccgt ctcctca 357 <210> 70 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> light chain variable region <400> 70 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Lys Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Glu Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Glu Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Thr Gly Ser Gly Ile Gln Phe Ser Leu Lys Ile Ile Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ile Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 71 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> light chain variable region <400> 71 gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtga gaaaatttac agttttttag catggtatca gcagaaacag 120 gaaaaatctc ctcaactcct ggtctataat gcagaaacct tagcagaagg tgtgccatca 180 aggttcagtg gcactggatc gggcatccag ttttctctga agattatcag cctgcagcct 240 ​​gggaccaagt tggaaataaa a 321 <210> 72 <211> 119 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> heavy chain variable region <400> 72 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Lys Ile Asp Pro Ala Ser Gly Asn Thr Lys Tyr Asp Pro Gln Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Tyr Gly Asn Asp Tyr Ala Met Asn Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 73 <211> 357 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> heavy chain variable region <400> 73 gaggtgcagc tggtgcagag cggagcagag gtgaagaagc caggggccac agtgaagata 60 agctgtaagg tgagcggatt caacattaag gacacatata tgcactgggt gcagcaggca 120 cccggcaaag gactggagtg gatgggaaag atcgaccccg ccagtggcaa taccaagtac 180 gacccccagt tccagggccg agtgaccatc accgcagaca ccagcaccaa cacagcctac 240 atggagctga gcagcctccg cagcgaagac acagccgtgt actactgcgc caccccctat 300 ggcaacgact acgctatgaa ttactggggc cagggaacac tggtcaccgt gtccagc 357 <210> 74 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> light chain variable region <400> 74 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Lys Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Asn Ala Glu Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ile Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 75 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> light chain variable region <400> 75 gacattcaga tgacccagag ccccagcagc ctgagcgcca gcgtgggaga cagagtgacc 60 ataacctgca gagccagcga gaaaatatat agctttctgg cctggtatca gcagaagccc 120 ggaaaagccc caaaactgct gctgtacaac gcagaaaccc tggcagaggg agtgcccagc 180 agattcagcg gatcaggaag cggcatcgac ttcaccctga ccatcagcag cctgcaaccc 240 gaagacttcg ccacctacta ctgccaacac cactacggca ccccatacac cttcggagga 300 ggcacaaagg tcgaaatcaa a 321 <210> 76 <211> 107 <212> PRT​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 77 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> light chain variable region <400> 77 gacattcaga tgacccagag ccccagcagc ctgagcgcca gcgtgggaga cagagtgacc 60 ataacctgca aagccagcca agacatcaac tggtatctgt cctggtttca gcagaagccc 120 ggcaaagccc caaaaaccct catctaccgg ggaaacagac tggtggacgg agtgcccagc 180 agattcagcg gaagcggcag cgggcaagac tacaccttca ccatatcaag cctgcagccc 240 gaagacatgg ccacctacta ctgcctgcag tacgacgaat tcccctttac cttcggccaa 300 gggaccaagg tggagatcaa g . 321 <210> 78 <211> 294 <212> PRT <213> Staphylococcus aureus <400> 78 Met Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly 1 5 10 15 Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu 20 25 30 Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn 35 40 45 His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly 50 55 60 Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala 65 70 75 80 Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val 85 90 95 Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu 100 105 110 Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp 115 120 125 Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly 130 135 140 His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser 145 150 155 160 Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val 165 170 175 Asn Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr 180 185 190 Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala 195 200 205 Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly 210 215 220 Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser 225 230 235 240 Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp 245 250 255 Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys 260 265 270 Asp Lys Trp Thr Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu 275 280 285 Lys Glu Glu Met Thr Asn 290 <210> 79 <211> 891 <212> DNA <213> Staphylococcus aureus <400> 79 CATGGcag acagcgacat taacattaaa accggcacca ccgatatcgg aagcaatacc 60 accgtaaaaa ccggagatct ggtcacctat gataaagaaa acggcatgct gaaaaaggtg 120 ttttatagct ttatcgacga taaaaaccac aataaaaagc tgttagtgat ccgcaccaaa 180 ggtacaatcg caggtcaata cagagtttat agcgaagaag gtgccaataa aagcggtctg 240 gcatggccga gcgcatttaa agtgcagctg cagctgccgg acaatgaagt tgcacagata 300 agcgactatt atccacgtaa tagtattgac acaaaggaat atatgagcac cctgacctat 360 ggttttaacg gtaatgttac cggtgatgac accggtaaga ttgggggatt aattggtgca 420 aatgtttcta ttggtcacac cctgaaatat gttcaaccgg attttaagac catcctggaa 480 tctccgacag ataaaaaggt ggggtggaaa gttatcttta ataatatggt taaccagaac 540 tggggcccgt atgatcgcga tagctggaat ccagtttatg gtaatcagct gttcatgaaa 600 acacgcaatg gcagcatgaa agcagcagat aactttctgg acccgaataa agcaagcagc 660 ctgctgagca gcggtttttc accggacttt gcaaccgtta tcaccatgga tcgcaaagcc 720 tcaaaacagc agaccaacat tgatgttatc tacgaaagag tacgcgatga ttaccagtta 780 cattggacaa gcaccaattg gaaagggacc aataccaaag acaaatggac cgatagaagc 840 agcgaacggt ataaaattga ttgggagaaa gaagaaatga ccaatctcga 891 <210> 80 <211> 294 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Mutant α - hemolysin <400> 80 Met Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly 1 5 10 15 Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu 20 25 30 Asn Gly Met Leu Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn 35 40 45 His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly 50 55 60 Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala 65 70 75 80 Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val 85 90 95 Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu 100 105 110 Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp 115 120 125 Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly 130 135 140 His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser 145 150 155 160 Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val 165 170 175 Asn Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr 180 185 190 Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala 195 200 205 Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly 210 215 220 Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser 225 230 235 240 Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp 245 250 255 Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys 260 265 270 Asp Lys Trp Thr Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu 275 280 285 Lys Glu Glu Met Thr Asn 290 <210> 81 <211> 891 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> mutant α - hemolysin <400> 81 catatggcag acagcgacat taacattaaa accggcacca ccgatatcgg aagcaatacc 60 accgtaaaaa ccggagatct ggtcacctat gataaagaaa acggcatgct gaaaaaggtg 120 ttttatagct ttatcgacga taaaaaccac aataaaaagc tgttagtgat ccgcaccaaa 180 ggtacaatcg caggtcaata cagagtttat agcgaagaag gtgccaataa aagcggtctg 240 gcatggccga gcgcatttaa agtgcagctg cagctgccgg acaatgaagt tgcacagata 300 agcgactatt atccacgtaa tagtattgac acaaaggaat atatgagcac cctgacctat 360 ggttttaacg gtaatgttac cggtgatgac accggtaaga ttgggggatt aattggtgca 420 aatgtttcta ttggtcacac cctgaaatat gttcaaccgg attttaagac catcctggaa 480 tctccgacag ataaaaaggt ggggtggaaa gttatcttta ataatatggt taaccagaac 540 tggggcccgt atgatcgcga tagctggaat ccagtttatg gtaatcagct gttcatgaaa 600 acacgcaatg gcagcatgaa agcagcagat aactttctgg acccgaataa agcaagcagc 660 ctgctgagca gcggtttttc accggacttt gcaaccgtta tcaccatgga tcgcaaagcc 720 tcaaaacagc agaccaacat tgatgttatc tacgaaagag tacgcgatga ttaccagtta 780 cattggacaa gcaccaattg gaaagggacc aataccaaag acaaatggac cgatagaagc 840 agcgaacggt ataaaattga ttgggagaaa gaagaaatga ccaatctcga 891 <210> 82 <211> 129 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain variable region <400> 82 Glu Val Gln Met Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Pro Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Lys Phe Gly Thr His 20 25 30 Trp Ile Gly Trp Val Arg Gln Arg Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile His Pro Ala Asp Ser Glu Thr Lys Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Ser Phe Ser Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu His Trp Ser Thr Leu Arg Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Ser Gly Ser Ser Ser Trp Tyr Ala Leu Asp Phe Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val <210> 83 <211> 123 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain variable region <400> 83 Gln Ser Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Phe Pro Gly Ala Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Thr Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu 85 90 95 Asn Gly Leu Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe 115 120 <210> 84 <211> 122 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> Heavy chain variable region <400> 84 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Tyr Ser Pro Thr Gly His Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 85 <211> 106 <​​​​​​​​​​​​​Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Lys Gln Tyr Ala Asp Tyr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 86 <211> 330 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> heavy chain constant region <400> 86 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 87 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> Light chain constant region <400> 87 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims

1. A composition comprising an antibody against α-hemolysin or an antigen-binding fragment thereof, and a pharmaceutically acceptable excipient, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region (VH) and light chain variable region (VL) each comprise a combination of CDRs selected from: VH-CDR1, VH-CDR2, VH-CDR3; VL-CDR1, VL-CDR2, VL-CDR3, wherein said α-hemolysin is derived from Staphylococcus aureus: (1) VH-CDR1 as shown in SEQ ID NO:22, VH-CDR2 as shown in SEQ ID NO:25, VH-CDR3 as shown in SEQ ID NO:29; VL-CDR1 as shown in SEQ ID NO:30, VL-CDR2 as shown in SEQ ID NO:32, VL-CDR3 as shown in SEQ ID NO:33; (2) VH-CDR1 as shown in SEQ ID NO:23, VH-CDR2 as shown in SEQ ID NO:26, VH-CDR3 as shown in SEQ ID NO:29; VL-CDR1 as shown in SEQ ID NO:30, VL-CDR2 as shown in SEQ ID NO:32, VL-CDR3 as shown in SEQ ID NO:33; (3) VH-CDR1 as shown in SEQ ID NO:24, VH-CDR2 as shown in SEQ ID NO:27, VH-CDR3 as shown in SEQ ID NO:29; VL-CDR1 as shown in SEQ ID NO:30, VL-CDR2 as shown in SEQ ID NO:32, VL-CDR3 as shown in SEQ ID NO:33; (4) VH-CDR1 as shown in SEQ ID NO:23, VH-CDR2 as shown in SEQ ID NO:27, VH-CDR3 as shown in SEQ ID NO:29; VL-CDR1 as shown in SEQ ID NO:30, VL-CDR2 as shown in SEQ ID NO:32, VL-CDR3 as shown in SEQ ID NO:33; (5) VH-CDR1 as shown in SEQ ID NO:23, VH-CDR2 as shown in SEQ ID NO:28, VH-CDR3 as shown in SEQ ID NO:29; VL-CDR1 as shown in SEQ ID NO:31, VL-CDR2 as shown in SEQ ID NO:32, VL-CDR3 as shown in SEQ ID NO:33; The pharmaceutically acceptable excipients include the group consisting of buffer solutions, protectants, and surfactants; in, The concentration of the antibody or its antigen-binding fragment is 10-100 mg / mL; The buffer solution has a pH of 5.0-6.5 and a concentration of 1-50 mM. The concentration of the protective agent is 1-10%, and the protective agent is selected from one or more of sucrose, trehalose, sorbitol, and mannitol; The surfactant concentration is 0.001-0.1%.

2. The composition of claim 1, wherein, The buffer solution is selected from citrate buffer, histidine buffer, and acetate buffer; The surfactants were selected from Tween 20 and Tween 80.

3. The composition of claim 2, wherein, The buffer solution is histidine buffer.

4. The composition of claim 1, wherein, The protective agent is sucrose.

5. The composition of claim 2, wherein, The surfactant is Tween 80.

6. The composition of claim 2, wherein, The buffer solution is histidine buffer, the protectant is sucrose, and the surfactant is Tween 80.

7. The composition of claim 1, wherein, The antibody or its antigen-binding fragment contains heavy chain variable regions and light chain variable regions selected from the following combinations: (1) The amino acid sequence as shown in SEQ ID NO:60; and the amino acid sequence as shown in SEQ ID NO:62; (2) The amino acid sequence as shown in SEQ ID NO:64; and the amino acid sequence as shown in SEQ ID NO:

66.

8. The composition of claim 1, wherein, The antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, Fab, Fab', F(ab')2, Fv, or scFv.

9. The composition according to any one of claims 1-8, comprising:

10. The composition of claim 9, wherein The concentration of the antibody or its antigen-binding fragment is 50 mg / mL; The concentration of histidine buffer is 10 mM; The concentration of sucrose is 5% (w / v); The concentration of polysorbate 80 is 0.005-0.015% (w / v).

11. Use of the composition of any one of claims 1-10 in the preparation of a medicament for the prevention or treatment of sepsis or bacteremia.

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