Anti-alpha-hemolysin antibody and application thereof
阅读说明：本技术 抗α-溶血素的抗体及其应用 (Anti-alpha-hemolysin antibody and application thereof ) 是由 安毛毛 王骊淳 高攀 林鉴 姜远英 慎慧 陈思敏 郭诗雨 方玮 于 2019-09-20 设计创作，主要内容包括：本发明提供了一种结合金黄色葡萄球菌α-溶血素的抗体或其片段,以及所述抗体或其片段用于预防或治疗金黄色葡萄球菌感染的用途。本发明的抗体通过α-溶血素的弱毒免疫、强毒筛选的策略筛选得到,对α-溶血素具有高亲和力,能够有效阻断α-溶血素的溶血作用,且在α-溶血素脓毒症模型、MRSA菌血症模型和MRSA肺部感染模型中均证明了显著的保护或治疗作用,且与抗生素具有协同作用,是对金黄色葡萄球菌现有抗生素疗法的有利补充。(The invention provides an antibody or fragment thereof which binds to staphylococcus aureus alpha-hemolysin and the use of the antibody or fragment thereof for preventing or treating staphylococcus aureus infections. The antibody is obtained by screening the strategy of weak-toxic immunity and strong-toxic screening of alpha-hemolysin, has high affinity to the alpha-hemolysin, can effectively block the hemolytic action of the alpha-hemolysin, has obvious protection or treatment effects in an alpha-hemolysin sepsis model, an MRSA bacteremia model and an MRSA lung infection model, has a synergistic effect with antibiotics, and is favorable for supplementing the conventional antibiotic therapy of staphylococcus aureus.)
1. An antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) and light chain variable region (VL) comprise a combination of CDRs (VH-CDR1, VH-CDR2, VH-CDR 3; VL-CDR1, VL-CDR2, VL-CDR3), respectively, selected from the group consisting of:
(1) VH-CDR1 shown in SEQ ID NO. 1, VH-CDR2 shown in SEQ ID NO. 4, VH-CDR3 shown in SEQ ID NO. 7; VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO. 9, VL-CDR3 shown in SEQ ID NO. 10;
(2) VH-CDR1 shown in SEQ ID NO. 2, VH-CDR2 shown in SEQ ID NO. 5, VH-CDR3 shown in SEQ ID NO. 7; VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO. 9, VL-CDR3 shown in SEQ ID NO. 10;
(3) VH-CDR1 shown in SEQ ID NO. 3, VH-CDR2 shown in SEQ ID NO. 6, VH-CDR3 shown in SEQ ID NO. 7; VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO. 9, VL-CDR3 shown in SEQ ID NO. 10;
(4) VH-CDR1 shown in SEQ ID NO. 2, VH-CDR2 shown in SEQ ID NO. 6, VH-CDR3 shown in SEQ ID NO. 7; VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO. 9, VL-CDR3 shown in SEQ ID NO. 10;
(5) VH-CDR1 shown in SEQ ID NO. 2, VH-CDR2 shown in SEQ ID NO. 6, VH-CDR3 shown in SEQ ID NO. 7; VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO. 11, VL-CDR3 shown in SEQ ID NO. 10;
(6) VH-CDR1 shown in SEQ ID NO. 12, VH-CDR2 shown in SEQ ID NO. 15, VH-CDR3 shown in SEQ ID NO. 18; VL-CDR1 shown in SEQ ID NO. 19, VL-CDR2 shown in SEQ ID NO. 20, VL-CDR3 shown in SEQ ID NO. 21;
(7) VH-CDR1 shown in SEQ ID NO. 13, VH-CDR2 shown in SEQ ID NO. 16, VH-CDR3 shown in SEQ ID NO. 18; VL-CDR1 shown in SEQ ID NO. 19, VL-CDR2 shown in SEQ ID NO. 20, VL-CDR3 shown in SEQ ID NO. 21;
(8) VH-CDR1 shown in SEQ ID NO. 14, VH-CDR2 shown in SEQ ID NO. 17, VH-CDR3 shown in SEQ ID NO. 18; VL-CDR1 shown in SEQ ID NO. 19, VL-CDR2 shown in SEQ ID NO. 20, VL-CDR3 shown in SEQ ID NO. 21;
(9) VH-CDR1 shown in SEQ ID NO 13, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 18; VL-CDR1 shown in SEQ ID NO. 19, VL-CDR2 shown in SEQ ID NO. 20, VL-CDR3 shown in SEQ ID NO. 21;
(10) VH-CDR1 shown in SEQ ID NO. 22, VH-CDR2 shown in SEQ ID NO. 25, VH-CDR3 shown in SEQ ID NO. 29; VL-CDR1 shown in SEQ ID NO. 30, VL-CDR2 shown in SEQ ID NO. 32, VL-CDR3 shown in SEQ ID NO. 33;
(11) VH-CDR1 shown in SEQ ID NO. 23, VH-CDR2 shown in SEQ ID NO. 26, VH-CDR3 shown in SEQ ID NO. 29; VL-CDR1 shown in SEQ ID NO. 30, VL-CDR2 shown in SEQ ID NO. 32, VL-CDR3 shown in SEQ ID NO. 33;
(12) VH-CDR1 shown in SEQ ID NO. 24, VH-CDR2 shown in SEQ ID NO. 27, VH-CDR3 shown in SEQ ID NO. 29; VL-CDR1 shown in SEQ ID NO. 30, VL-CDR2 shown in SEQ ID NO. 32, VL-CDR3 shown in SEQ ID NO. 33;
(13) VH-CDR1 shown in SEQ ID NO. 23, VH-CDR2 shown in SEQ ID NO. 27, VH-CDR3 shown in SEQ ID NO. 29; VL-CDR1 shown in SEQ ID NO. 30, VL-CDR2 shown in SEQ ID NO. 32, VL-CDR3 shown in SEQ ID NO. 33;
(14) VH-CDR1 shown in SEQ ID NO. 23, VH-CDR2 shown in SEQ ID NO. 28, VH-CDR3 shown in SEQ ID NO. 29; VL-CDR1 shown in SEQ ID NO. 31, VL-CDR2 shown in SEQ ID NO. 32, VL-CDR3 shown in SEQ ID NO. 33;
(15) VH-CDR1 shown in SEQ ID NO:34, VH-CDR2 shown in SEQ ID NO:37, VH-CDR3 shown in SEQ ID NO: 40; VL-CDR1 shown in SEQ ID NO:41, VL-CDR2 shown in SEQ ID NO:42, VL-CDR3 shown in SEQ ID NO: 43;
(16) VH-CDR1 shown in SEQ ID NO:35, VH-CDR2 shown in SEQ ID NO:38, VH-CDR3 shown in SEQ ID NO: 40; VL-CDR1 shown in SEQ ID NO:41, VL-CDR2 shown in SEQ ID NO:42, VL-CDR3 shown in SEQ ID NO: 43;
(17) VH-CDR1 shown in SEQ ID NO:36, VH-CDR2 shown in SEQ ID NO:39, VH-CDR3 shown in SEQ ID NO: 40; VL-CDR1 shown in SEQ ID NO:41, VL-CDR2 shown in SEQ ID NO:42, VL-CDR3 shown in SEQ ID NO: 43;
(18) VH-CDR1 shown in SEQ ID NO:35, VH-CDR2 shown in SEQ ID NO:39, VH-CDR3 shown in SEQ ID NO: 40; VL-CDR1 shown in SEQ ID NO. 41, VL-CDR2 shown in SEQ ID NO. 42, and VL-CDR3 shown in SEQ ID NO. 43.
2. The antibody or fragment thereof of claim 1, wherein the heavy chain variable region comprises:
an amino acid sequence as set forth in any one of SEQ ID NO 44, SEQ ID NO 48, SEQ ID NO 52, SEQ ID NO 56, SEQ ID NO 60, SEQ ID NO 64, SEQ ID NO 68 and SEQ ID NO 72 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth; and/or
The light chain variable region comprises:
an amino acid sequence as shown in any one of SEQ ID NO 46, SEQ ID NO 50, SEQ ID NO 54, SEQ ID NO 58, SEQ ID NO 62, SEQ ID NO 66, SEQ ID NO 70, SEQ ID NO 74 and SEQ ID NO 76 or an amino acid sequence having at least 75% identity to the amino acid sequence shown.
3. The antibody or fragment thereof of claim 1 or 2, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
(1) an amino acid sequence as shown in SEQ ID NO. 44 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 44; and, the amino acid sequence as set forth in SEQ ID NO. 46 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 46;
(2) an amino acid sequence as shown in SEQ ID NO. 48 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 48; and, an amino acid sequence as set forth in SEQ ID NO. 50 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 50;
(3) an amino acid sequence as shown in SEQ ID NO. 52 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 52; and, the amino acid sequence as set forth in SEQ ID NO:54 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 54;
(4) an amino acid sequence as shown in SEQ ID NO. 56 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 56; and, the amino acid sequence as set forth in SEQ ID NO:58 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 58;
(5) an amino acid sequence as set forth in SEQ ID NO:60 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 60; and, the amino acid sequence as set forth in SEQ ID NO:62 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 62;
(6) an amino acid sequence as shown in SEQ ID NO. 64 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 64; and, the amino acid sequence as set forth in SEQ ID NO 66 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 66;
(7) an amino acid sequence as shown in SEQ ID NO. 68 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 68; and, an amino acid sequence as set forth in SEQ ID NO:70 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 70;
(8) an amino acid sequence as set forth in SEQ ID NO. 72 or an amino acid sequence with at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 72; and, the amino acid sequence shown as SEQ ID NO. 74 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID NO. 74;
(9) an amino acid sequence as shown in SEQ ID NO. 48 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 48; and, an amino acid sequence as set forth in SEQ ID NO:76 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 76.
4. The antibody or fragment thereof of any one of claims 1 to 3, wherein the at least 75% identity is any percentage identity of at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity or the like ≧ 75%;
preferably, the antibody or fragment thereof is in any form of monoclonal antibody, single chain antibody, single domain antibody, bifunctional antibody, nanobody, fully or partially humanized antibody, or chimeric antibody; alternatively, the antibody or fragment thereof is a half-antibody or antigen-binding fragment of a half-antibody, e.g., scFv, BsFv, dsFv, (dsFv)2、Fab、Fab'、F(ab')2Or Fv;
alternatively, the antibody is IgA, IgD, IgE, IgG or IgM, more preferably IgG 1; the fragment of said antibody is selected from the group consisting of scFv, Fab, F (ab')2Or an Fv fragment.
5. The antibody or fragment thereof of any one of claims 1 to 4, wherein the antibody or fragment thereof further comprises a human or murine light chain constant region (CL) and/or heavy chain constant region (CH); preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of the kappa or lambda type; more preferably, the heavy chain constant region is of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type.
6. A nucleic acid molecule encoding the antibody or fragment thereof of any one of claims 1 to 5 or encoding a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain or a light chain comprised in said antibody or fragment thereof.
7. A vector comprising the nucleic acid molecule of claim 6.
8. A host cell comprising or transformed or transfected with the nucleic acid molecule of claim 6 and/or the vector of claim 7.
9. A conjugate or fusion protein comprising the antibody or fragment thereof of any one of claims 1 to 5.
10. The conjugate or fusion protein of claim 9, wherein the conjugate or fusion protein comprises an additional moiety, such as a cell surface receptor, a small molecule compound, a small molecule polymer, an active protein or a polypeptide, directly or indirectly linked to the antibody or fragment thereof.
11. A pharmaceutical composition comprising the antibody or fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, and/or the conjugate or fusion protein of claim 9 or 10, and optionally a pharmaceutically acceptable excipient.
12. A kit comprising the antibody or fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the conjugate or fusion protein of claim 9 or 10, and/or the pharmaceutical composition of claim 11.
13. Use of the antibody or fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the conjugate or fusion protein of claim 9 or 10, and/or the pharmaceutical composition of claim 11 in the manufacture of a medicament for preventing or treating infections and complications caused by α -hemolysin or a α -hemolysin producing microorganism.
14. Use of the antibody or fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the conjugate or fusion protein of claim 9 or 10, and/or the pharmaceutical composition of claim 11 in combination with another antibacterial agent or an anti- α -hemolysin antibody for the manufacture of a medicament for the prevention or treatment of infections and complications caused by α -hemolysin or a α -hemolysin producing microorganism.
15. Use according to claim 13 or 14, characterized in that the microorganism is staphylococcus aureus; preferably, the staphylococcus aureus comprises methicillin-resistant staphylococcus aureus;
the infection comprises upper respiratory tract infection, pneumonia, severe pneumonia, abdominal cavity infection, subcutaneous and soft tissue infection, bacteremia and infection of various organs;
the complications include Acute Respiratory Distress Syndrome (ARDS), sepsis, and elevated body inflammatory factors;
preferably, the further antibacterial agent is a drug (including chemical, biological and chinese drugs) useful for the treatment and prevention of infection by staphylococcus aureus, e.g. methicillin-resistant staphylococcus aureus, preferably an antibiotic, e.g. a beta-lactam antibiotic, more preferably vancomycin, norvancomycin, teicoplanin, linezolid, daptomycin, cephaplopram, fusidic acid, ceftaroline.
Technical Field
The invention relates to the field of antibody medicines, in particular to an anti-alpha-hemolysin antibody and pharmaceutical application thereof.
Background
Staphylococcus aureus (Staphylococcus aureus) belongs to the genus Staphylococcus, is an important gram-positive pathogenic bacterium, and is the leading G of human+Pathogenic bacteria can cause local infection such as pyogenic infection, pneumonia, pseudomembranous enteritis, pericarditis and the like, and systemic infection such as septicemia, sepsis and the like. The Chinese drug resistance monitoring network data shows that the staphylococcus aureus is located at the 4 th site of pathogenic bacteria detected in a hospital and the 1 st site of G + bacteria detected in the hospital.
Staphylococcus aureus releases a large amount of toxins to destroy tissue cells during the process of infecting a human body, and inhibits immune cells of the body from removing pathogens. At present, beta-lactam antibiotics are mainly adopted clinically to treat staphylococcus aureus infection. However, antibiotics only inhibit or kill bacteria, but do not have the ability to release toxins from bacteria. On the contrary, under the pressure of antibiotics, bacteria release more toxins, and the bacteria killed by the antibiotics also release toxins after lysis, and after a large amount of toxins enter blood, the host immune system is over-activated, and excessive inflammatory factors are released, so that sepsis is formed.
Moreover, in the process of fighting with human beings, staphylococcus aureus is increasingly insensitive to beta-lactam antibiotics, for example, the separation rate of methicillin-resistant staphylococcus aureus (MRSA) is higher and higher at present, and the data of the Chinese drug-resistant monitoring network show that the separation rate of MRSA is even as high as 38.9% in 2016. For MRSA, the therapeutic drugs mainly comprise limited drugs such as vancomycin and linezolid, which are typical representatives of the "super-drug-resistant" bacteria causing human infection. Therefore, clinically, MRSA infection becomes increasingly severe, the choice of antibacterial drugs is very limited, and clinical patients have failed treatment and have a high mortality rate. In recent years, vancomycin-resistant VRSA (vancomycin-resistant staphylococcus aureus) infection has even occurred, and humans are about to face the situation where no drug is available.
The pathogenic factors released by staphylococcus aureus are found to include hemolysin, leukocidin, enterotoxin, coagulase and the like. The hemolysin is one of important virulence factors secreted by staphylococcus aureus and can be divided into four types of alpha-hemolysin, beta-hemolysin, gamma-hemolysin and delta-hemolysin. Alpha-hemolysin (Hla) is a secreted toxin protein coded by HLA genes of staphylococcus aureus, is expressed in almost all strains, is a more key virulence factor influencing the pathogenicity of the staphylococcus aureus compared with other types of hemolysin, and researches show that the toxicity of animals infected by the staphylococcus aureus with alpha-hemolysin expression and HLA gene deletion is obviously reduced. The protein total length of the alpha-hemolysin is 319 amino acids, the relative molecular mass is 33KD, and the alpha-hemolysin is secreted in a monomer form. The most well-defined biological property of α -hemolysin, one of the members of the pore forming toxin (pore forming toxin) family, is its ability to rapidly lyse host red blood cells and other tissue cells, which, when bound to cholesterol and sphingomyelin on host cells, aggregate to form a heptamer with a relative molecular mass of about 232KD, and then fold to form a β -barrel-shaped transmembrane structure with a diameter of about 1.5nm, allowing intracellular water, ions and other small molecular substances to escape, thereby causing hemolysis or death of target cells. In addition, alpha-hemolysin can also cause the contraction and the twitch spasm of the smooth muscle of the capillary vessel, thereby causing the blockage of the capillary vessel and causing ischemia and tissue necrosis, and plays an important role in the process that staphylococcus aureus causes diseases such as sepsis, pneumonia, mammary gland infection, cornea infection, serious skin infection and the like. At the same time, alpha-hemolysin can also destroy leukocytes in infected tissues, preventing the host from clearing infected staphylococcus aureus. Under the pressure of host immune system and antibiotics, a large amount of alpha-hemolysin is released by staphylococcus aureus at the infected part, and after the staphylococcus aureus enters blood, the host immune system can be activated to release excessive inflammatory factors to form sepsis.
In the face of the continuous emergence of superbacteria, antibody drugs have become the leading edge of the post-antibiotic era. The antibody is natural protein produced by adaptive immune system, and the human body is given antibody medicine by passive immunity, so that the antibody not only can neutralize virulence factor, but also can strengthen the immune response capability of host to pathogen and accelerate the elimination of infectious pathogen. Alpha-hemolysin, in turn, shows its potential as an anti-infective antibody drug target for the treatment of staphylococcus aureus infections for the following reasons:
alpha-hemolysin is highly conserved among staphylococcus aureus and is expressed in almost all staphylococcus aureus; the alpha-hemolysin has definite biological function and plays an important role in the process of forming host infection and sepsis by staphylococcus aureus; the homologous protein of the alpha-hemolysin does not exist in mammalian cells, the antibody drug is designed by targeting the alpha-hemolysin, the potential off-target possibility is low, and the toxic and side effects are low.
Therefore, the alpha-hemolysin is an ideal target of the anti-staphylococcus aureus infection antibody drug, and the effective neutralization of the alpha-hemolysin is beneficial to blocking staphylococcus aureus infection of a human body, avoiding sepsis after severe infection and improving the prognosis of a clinical infection patient. Fully human antibody R-301 developed by Aridis pharmaceutical company at present according to alpha-hemolysin targetHas entered phase III clinical studies, and the main indications are severe pneumonia caused by staphylococcus aureus (including methicillin-resistant staphylococcus aureus); humanized antibody MEDI4893 developed by Aslicon pharmaceutical Co., Ltd based on the target of alpha-hemolysin is developing phase II clinical research, and the main indication is the prevention and treatment of pneumonia of Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus).
Antibiotics such as vancomycin and the like are still the first line drugs for the treatment of staphylococcus aureus infection at present, but in view of the continuous increase in drug resistance and the slow development of new antibiotics, there is still a need in the art to develop novel, highly effective antibody drugs against staphylococcus aureus by directly neutralizing toxins.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an anti-alpha-hemolysin antibody molecule, in particular a humanized alpha-hemolysin monoclonal antibody, which has the capacity of binding with staphylococcus aureus alpha-hemolysin and inhibiting hemolysis and damaging tissue cells, and can be used for treating infection or infection-related diseases caused by alpha-hemolysin or microorganisms producing alpha-hemolysin alone or in combination with the existing antibacterial drugs.
In view of the above-mentioned problems, it is an object of the present invention to provide an antibody or a functional fragment thereof, and uses thereof based on the antibody or the functional fragment thereof.
The technical scheme of the invention is as follows.
In one aspect, the invention provides an antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) and light chain variable region (VL) comprise a combination of CDRs (VH-CDR1, VH-CDR2, VH-CDR 3; VL-CDR1, VL-CDR2, VL-CDR3), respectively, selected from the group consisting of:
(1) VH-CDR1 shown in SEQ ID NO. 1, VH-CDR2 shown in SEQ ID NO. 4, VH-CDR3 shown in SEQ ID NO. 7; VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO. 9, VL-CDR3 shown in SEQ ID NO. 10;
(2) VH-CDR1 shown in SEQ ID NO. 2, VH-CDR2 shown in SEQ ID NO. 5, VH-CDR3 shown in SEQ ID NO. 7; VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO. 9, VL-CDR3 shown in SEQ ID NO. 10;
(3) VH-CDR1 shown in SEQ ID NO. 3, VH-CDR2 shown in SEQ ID NO. 6, VH-CDR3 shown in SEQ ID NO. 7; VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO. 9, VL-CDR3 shown in SEQ ID NO. 10;
(4) VH-CDR1 shown in SEQ ID NO. 2, VH-CDR2 shown in SEQ ID NO. 6, VH-CDR3 shown in SEQ ID NO. 7; VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO. 9, VL-CDR3 shown in SEQ ID NO. 10;
(5) VH-CDR1 shown in SEQ ID NO. 2, VH-CDR2 shown in SEQ ID NO. 6, VH-CDR3 shown in SEQ ID NO. 7; VL-CDR1 shown in SEQ ID NO. 8, VL-CDR2 shown in SEQ ID NO. 11, VL-CDR3 shown in SEQ ID NO. 10;
(6) VH-CDR1 shown in SEQ ID NO. 12, VH-CDR2 shown in SEQ ID NO. 15, VH-CDR3 shown in SEQ ID NO. 18; VL-CDR1 shown in SEQ ID NO. 19, VL-CDR2 shown in SEQ ID NO. 20, VL-CDR3 shown in SEQ ID NO. 21;
(7) VH-CDR1 shown in SEQ ID NO. 13, VH-CDR2 shown in SEQ ID NO. 16, VH-CDR3 shown in SEQ ID NO. 18; VL-CDR1 shown in SEQ ID NO. 19, VL-CDR2 shown in SEQ ID NO. 20, VL-CDR3 shown in SEQ ID NO. 21;
(8) VH-CDR1 shown in SEQ ID NO. 14, VH-CDR2 shown in SEQ ID NO. 17, VH-CDR3 shown in SEQ ID NO. 18; VL-CDR1 shown in SEQ ID NO. 19, VL-CDR2 shown in SEQ ID NO. 20, VL-CDR3 shown in SEQ ID NO. 21;
(9) VH-CDR1 shown in SEQ ID NO 13, VH-CDR2 shown in SEQ ID NO 17, VH-CDR3 shown in SEQ ID NO 18; VL-CDR1 shown in SEQ ID NO. 19, VL-CDR2 shown in SEQ ID NO. 20, VL-CDR3 shown in SEQ ID NO. 21;
(10) VH-CDR1 shown in SEQ ID NO. 22, VH-CDR2 shown in SEQ ID NO. 25, VH-CDR3 shown in SEQ ID NO. 29; VL-CDR1 shown in SEQ ID NO. 30, VL-CDR2 shown in SEQ ID NO. 32, VL-CDR3 shown in SEQ ID NO. 33;
(11) VH-CDR1 shown in SEQ ID NO. 23, VH-CDR2 shown in SEQ ID NO. 26, VH-CDR3 shown in SEQ ID NO. 29; VL-CDR1 shown in SEQ ID NO. 30, VL-CDR2 shown in SEQ ID NO. 32, VL-CDR3 shown in SEQ ID NO. 33;
(12) VH-CDR1 shown in SEQ ID NO. 24, VH-CDR2 shown in SEQ ID NO. 27, VH-CDR3 shown in SEQ ID NO. 29; VL-CDR1 shown in SEQ ID NO. 30, VL-CDR2 shown in SEQ ID NO. 32, VL-CDR3 shown in SEQ ID NO. 33;
(13) VH-CDR1 shown in SEQ ID NO. 23, VH-CDR2 shown in SEQ ID NO. 27, VH-CDR3 shown in SEQ ID NO. 29; VL-CDR1 shown in SEQ ID NO. 30, VL-CDR2 shown in SEQ ID NO. 32, VL-CDR3 shown in SEQ ID NO. 33;
(14) VH-CDR1 shown in SEQ ID NO. 23, VH-CDR2 shown in SEQ ID NO. 28, VH-CDR3 shown in SEQ ID NO. 29; VL-CDR1 shown in SEQ ID NO. 31, VL-CDR2 shown in SEQ ID NO. 32, VL-CDR3 shown in SEQ ID NO. 33;
(15) VH-CDR1 shown in SEQ ID NO:34, VH-CDR2 shown in SEQ ID NO:37, VH-CDR3 shown in SEQ ID NO: 40; VL-CDR1 shown in SEQ ID NO:41, VL-CDR2 shown in SEQ ID NO:42, VL-CDR3 shown in SEQ ID NO: 43;
(16) VH-CDR1 shown in SEQ ID NO:35, VH-CDR2 shown in SEQ ID NO:38, VH-CDR3 shown in SEQ ID NO: 40; VL-CDR1 shown in SEQ ID NO:41, VL-CDR2 shown in SEQ ID NO:42, VL-CDR3 shown in SEQ ID NO: 43;
(17) VH-CDR1 shown in SEQ ID NO:36, VH-CDR2 shown in SEQ ID NO:39, VH-CDR3 shown in SEQ ID NO: 40; VL-CDR1 shown in SEQ ID NO:41, VL-CDR2 shown in SEQ ID NO:42, VL-CDR3 shown in SEQ ID NO: 43;
(18) VH-CDR1 shown in SEQ ID NO:35, VH-CDR2 shown in SEQ ID NO:39, VH-CDR3 shown in SEQ ID NO: 40; VL-CDR1 shown in SEQ ID NO. 41, VL-CDR2 shown in SEQ ID NO. 42, and VL-CDR3 shown in SEQ ID NO. 43.
Preferably, in the antibody or fragment thereof provided by the present invention, the heavy chain variable region comprises:
an amino acid sequence as set forth in any one of SEQ ID NO 44, SEQ ID NO 48, SEQ ID NO 52, SEQ ID NO 56, SEQ ID NO 60, SEQ ID NO 64, SEQ ID NO 68 and SEQ ID NO 72 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth; and/or
The light chain variable region comprises:
an amino acid sequence as shown in any one of SEQ ID NO 46, SEQ ID NO 50, SEQ ID NO 54, SEQ ID NO 58, SEQ ID NO 62, SEQ ID NO 66, SEQ ID NO 70, SEQ ID NO 74 and SEQ ID NO 76 or an amino acid sequence having at least 75% identity to the amino acid sequence shown.
According to a particular embodiment of the invention, the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
(1) an amino acid sequence as shown in SEQ ID NO. 44 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 44; and, the amino acid sequence as set forth in SEQ ID NO. 46 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO. 46;
(2) an amino acid sequence as shown in SEQ ID NO. 48 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 48; and, an amino acid sequence as set forth in SEQ ID NO. 50 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 50;
(3) an amino acid sequence as shown in SEQ ID NO. 52 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 52; and, the amino acid sequence as set forth in SEQ ID NO:54 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 54;
(4) an amino acid sequence as shown in SEQ ID NO. 56 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 56; and, the amino acid sequence as set forth in SEQ ID NO:58 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 58;
(5) an amino acid sequence as set forth in SEQ ID NO:60 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 60; and, the amino acid sequence as set forth in SEQ ID NO:62 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 62;
(6) an amino acid sequence as shown in SEQ ID NO. 64 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 64; and, the amino acid sequence as set forth in SEQ ID NO 66 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO 66;
(7) an amino acid sequence as shown in SEQ ID NO. 68 or an amino acid sequence with at least 75% identity with the amino acid sequence as shown in SEQ ID NO. 68; and, an amino acid sequence as set forth in SEQ ID NO:70 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 70;
(8) an amino acid sequence as set forth in SEQ ID NO. 72 or an amino acid sequence with at least 75% identity to an amino acid sequence as set forth in SEQ ID NO. 72; and, the amino acid sequence shown as SEQ ID NO. 74 or an amino acid sequence having at least 75% identity to the amino acid sequence shown as SEQ ID NO. 74;
(9) an amino acid sequence as shown in SEQ ID NO. 48 or an amino acid sequence with at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 48; and, an amino acid sequence as set forth in SEQ ID NO:76 or an amino acid sequence having at least 75% identity to an amino acid sequence as set forth in SEQ ID NO: 76.
Wherein the at least 75% identity is any percentage identity of at least 75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity.
The antibody or the fragment thereof provided by the invention can be in any form such as a monoclonal antibody, a single-chain antibody, a single-domain antibody, a bifunctional antibody, a nano antibody, a fully or partially humanized antibody or a chimeric antibody; alternatively, the antibody or fragment thereof is a half-antibody or antigen-binding fragment of a half-antibody, e.g., scFv, BsFv, dsFv, (dsFv)2、Fab、Fab'、F(ab')2Or Fv; regarding the fragments of the antibodies provided by the present invention, preferably, the fragments are any fragments of the antibodies capable of specifically binding to the antigen staphylococcus aureus alpha-hemolysin.
Alternatively, the antibody of the invention is IgA, IgD, IgE, IgG or IgM, more preferably IgG 1. The fragment of the antibody is selected from the group consisting of scFv, Fab, F (ab')2Or an Fv fragment.
Preferably, the antibody or fragment thereof further comprises a human or murine constant region, preferably a human or murine 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 light chain constant region of the kappa or lambda type. According to a particular embodiment of the invention, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; more preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype and the light chain constant region is of the kappa type.
Preferably, the antibodies or fragments thereof provided herein comprise a heavy chain constant region as set forth in SEQ ID NO 86 and/or a light chain constant region as set forth in SEQ ID NO 87, or an amino acid sequence at least 75% identical to a heavy chain constant region or a light chain constant region as set forth herein.
In another aspect, the invention also provides a nucleic acid molecule encoding any of the antibodies or fragments thereof of the invention or encoding a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain or a light chain comprised in said antibody or fragment thereof.
According to a specific embodiment of the invention, the nucleic acid molecule encoding the heavy chain variable region or the light chain variable region of the antibody or fragment thereof according to the invention, e.g.the nucleic acid molecule comprises a nucleotide sequence as set forth 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.
In yet another aspect, the invention provides a vector comprising a nucleic acid molecule of the invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector and the like.
The vectors or nucleic acid molecules of the invention may be used to transform or transfect host cells or in any way into host cells for the purpose of preserving or expressing antibodies, etc. Thus, in a further aspect, the present invention provides a host cell comprising a nucleic acid molecule and/or vector of the invention, or transformed or transfected with a nucleic acid molecule and/or vector of the invention. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
Based on the present disclosure, the antibodies or fragments thereof, nucleic acid molecules, vectors, and/or host cells provided herein can be obtained using any conventional techniques known in the art. For example, with respect to an antibody, the heavy chain variable region and/or the light chain variable region of the antibody may be obtained from the nucleic acid molecule provided herein, or the heavy chain and/or the light chain of the antibody may be obtained and then assembled into an antibody with optional additional domains of the antibody; alternatively, the host cell provided by the present invention is cultured under conditions that allow the host cell to express the heavy chain variable region and/or the light chain variable region of the antibody or the heavy chain and/or the light chain of the antibody to assemble into the antibody. Optionally, the method further comprises the step of recovering the produced antibody.
The antibodies or fragments thereof provided herein may also bind to other moieties, such as cell surface receptors, small molecule compounds such as amino acids and carbohydrates, small molecule polymers or any other moiety that modifies the antibodies of the invention, or even active proteins or polypeptides, such as antimicrobial peptides or antibiotics. Thus, in another aspect, the invention provides a conjugate or fusion protein comprising an antibody or fragment thereof provided by the invention. For example, the conjugate or fusion protein can be a bispecific antibody comprising an antibody or fragment thereof of the invention.
The antibody or a fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a fusion protein, or the like provided by the present invention may be contained in a pharmaceutical composition, more particularly, a pharmaceutical preparation, so as to be used for various purposes according to actual needs. Thus, in a further aspect, the invention also provides a pharmaceutical composition comprising an antibody or fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate and/or a fusion protein according to the invention, and optionally a pharmaceutically acceptable excipient.
For any purpose of use, the invention also provides a kit comprising an antibody molecule or fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a fusion protein and/or a pharmaceutical composition of the invention.
The antibody or fragment thereof of the present invention can be used alone or in combination with other antibacterial agents for treating or ameliorating infection by α -hemolysin or α -hemolysin-producing microorganism or other diseases or symptoms caused by infection thereof, based on the ability to bind to α -hemolysin and inhibit its hemolysis and damage tissue cells. Accordingly, the present invention also provides related applications of the above subject matter.
In particular, in a further aspect, the invention provides the use of an antibody or fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a fusion protein and/or a pharmaceutical composition according to the invention in the manufacture of a medicament for the prevention or treatment of infections and complications caused by α -hemolysin or a microorganism producing α -hemolysin.
Furthermore, the present invention provides the use of said antibodies or fragments thereof, nucleic acid molecules, vectors, host cells, conjugates, fusion proteins and/or pharmaceutical compositions in combination with other antibacterial agents or anti- α -hemolysin antibodies for the preparation of a medicament for the prevention or treatment of infections and complications caused by α -hemolysin or a microorganism producing α -hemolysin.
In addition, the present invention provides a method for preventing or treating an infection and complications caused by α -hemolysin or a microorganism producing α -hemolysin, said method comprising administering to a subject in need thereof said antibody or fragment thereof, nucleic acid molecule, vector, host cell, conjugate, fusion protein and/or pharmaceutical composition thereof, and optionally an antibacterial drug. The optional antibacterial agent may be an agent administered in combination with the antibody or fragment thereof, nucleic acid molecule, vector, host cell, conjugate, fusion protein and/or pharmaceutical composition of the invention. The combined administration of the two may take any form, including simultaneous, sequential or spaced apart.
In the present invention, the α -hemolysin producing microorganism is preferably staphylococcus aureus, including methicillin-resistant staphylococcus aureus.
In the present invention, the infection caused by α -hemolysin or a microorganism producing α -hemolysin may be one or more selected from the group consisting of upper respiratory infection, pneumonia, severe pneumonia, abdominal infection, subcutaneous and soft tissue infection, bacteremia, and infection of various organs; the complication may be one or more selected from Acute Respiratory Distress Syndrome (ARDS), sepsis, and an increase in body inflammatory factors.
In the present invention, the other antibacterial agents are agents (including chemical agents, biological agents and Chinese medicines) useful for the treatment and prevention of infection with staphylococcus aureus, e.g., methicillin-resistant staphylococcus aureus, preferably antibiotics, e.g., β -lactam antibiotics. The antibiotic may be a drug listed in the guidelines/treatment strategy promulgated by the American Society for Infectious Diseases (IDSA) or the Chinese medical Society, preferably vancomycin, norvancomycin, teicoplanin, linezolid, daptomycin, cephapiprep, fusidic acid, ceftaroline.
The invention also provides a method of diagnosing an infection caused by alpha-hemolysin or a microorganism producing alpha-hemolysin, said method comprising contacting said antibody or fragment thereof, nucleic acid molecule, vector, host cell, conjugate, fusion protein and/or pharmaceutical composition thereof with a sample from a subject.
Compared with the prior art, the alpha-hemolysin (alpha-Toxin) of the staphylococcus aureus and the alpha-hemolysin mutant protein (alpha-Toxin H35L) of the staphylococcus aureus without virulence are obtained by prokaryotic expression of escherichia coli. The method comprises the steps of successfully immunizing a mouse by utilizing alpha-Toxin H35L, taking splenocytes, establishing an antibody library by utilizing a hybridoma technology, screening to obtain an alpha-hemolysin monoclonal antibody which has high affinity with alpha-hemolysin (alpha-Toxin) and biological activity, and further humanizing the alpha-hemolysin monoclonal antibody to obtain a lead antibody molecule (IgG1 subtype).
Specifically, the invention utilizes alpha-Toxin H35L to successfully immunize a mouse, then takes splenocytes, utilizes a hybridoma technology to establish an antibody library, and screens to obtain the alpha-hemolysin monoclonal antibody which has high affinity with alpha-hemolysin (alpha-Toxin) and biological activity. And 16 lead antibody molecules are obtained in total, and not only have high affinity with alpha-hemolysin, but also have the activity of blocking alpha-hemolysin. Particularly, on the aspects of antigen selection and antibody screening, the invention adopts the strategies of weak toxicity immunity and strong toxicity screening.
Based on the murine lead antibody molecules, the humanized antibody molecules with similar biological activity to the murine antibody are finally obtained by carrying out recombinant expression on the human murine affinity antibody and the humanized antibody.
In vitro pharmacodynamic studies show that the humanized antibody can block the hemolytic effect of alpha-hemolysin on rabbit erythrocytes and the damage effect of alpha-hemolysin on lung epithelial cells in a dose-dependent manner. In addition, the invention also utilizes a mouse alpha-hemolysin sepsis model, an MRSA bacteremia model and an MRSA pulmonary infection model to carry out pharmacodynamic evaluation on the lead antibody molecule in animals. Research results show that the humanized antibody has a remarkable protective effect on a mouse alpha-hemolysin sepsis model; the survival time of a mouse MRSA bacteremia model can be remarkably prolonged; can obviously reduce the tissue bacterium carrying amount of a mouse MRSA lung infection model. Moreover, the humanized antibody of the invention is combined with common antibacterial drugs of vancomycin, linezolid and the like to be applied, and shows remarkable synergistic effect in a mouse alpha-hemolysin sepsis model, an MRSA bacteremia model and an MRSA lung infection model.
Furthermore, single dose pharmacokinetic studies (N ═ 3) were performed on the antibodies of the invention in cynomolgus monkeys at a dose of 20mg/kg, and the results showed that the basic pharmacokinetic parameters met the criteria for druggability (Table 1), the in vivo elimination half-life (T1/2) was 137 ± 36.9h, and the plasma Clearance (CL) was 0.501 ± 0.222 ml/h/kg. In addition, the antibody acute toxicity test research (N is 10) is carried out in a mouse body, and the result shows that when 125mg/kg of the antibody is administered within 24 hours at most, the mouse does not die, no discomfort phenomenon of the animal is found after continuous observation for 14 days, and no abnormality is found when the main organs (heart, liver, spleen, lung, kidney and brain) of the killed animal are generally observed; when the cynomolgus monkey is administrated with the antibody of the invention at a dose of 10mg/kg once, no death of the animals occurs, and no discomfort of the animals occurs after continuous observation for 28 days. Acute toxicity test research shows that the antibody of the invention has good safety.
The antibody of the invention can effectively neutralize toxin, block the damage of the toxin to tissue cells of a patient and simultaneously improve the immunity of the patient, thereby relieving the tissue damage caused by clinical staphylococcus aureus infection, promoting the infectious bacteria in the body of the patient to be removed more quickly, and preventing or relieving sepsis. Under the action of the antibody, a patient can be quickly changed from intravenous infusion therapy to oral therapy, and the treatment course is shortened; meanwhile, the antibody of the invention has better clinical curative effect and tolerance, and is a favorable supplement for the existing antibiotic therapy.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the construction of recombinant expression plasmids of Staphylococcus aureus alpha-hemolysin (alpha-Toxin) protein fused to a His tag.
FIG. 2 shows the construction of recombinant expression plasmids of His-tag fused S.aureus mutant alpha-hemolysin (H35L alpha-Toxin) proteins.
FIG. 3 shows the 10% SDS-PAGE electrophoresis results of recombinant expressed Staphylococcus aureus mutant alpha-hemolysin and its mutant H35L alpha-Toxin, wherein FIG. 3A is alpha-Toxin, FIG. 3B is H35L alpha-Toxin, and the loading amount is 10 μ g.
FIG. 4 shows the hemolytic effect of Staphylococcus aureus alpha-hemolysin and its mutant (H35L alpha-Toxin) on sheep blood plates.
FIG. 5 shows the hemolytic effect of Staphylococcus aureus alpha-hemolysin and its mutant (H35L alpha-Toxin) on rabbit blood, where FIG. 5A is alpha-Toxin and FIG. 5B is H35L alpha-Toxin.
FIG. 6 shows the detection results of the hybridoma cell line screening process, wherein FIG. 6A is the ELISA detection results of the antibody in the supernatants of different cell lines binding to alpha-hemolysin, and FIG. 6B is the inhibition results of the antibody in the supernatants of different cell lines to alpha-hemolysin.
Fig. 7 shows the results of ELISA detection of the binding of the antibody of the present invention to α -hemolysin, wherein fig. 7A to 7D show the binding of the screened antibodies 78D4, 16H4, 78F4 and 98G9 to α -hemolysin, respectively.
Fig. 8 shows Octect binding and dissociation curves of the antibody α -hemolysin of the present invention, wherein fig. 8A to 8D show binding of the screened 78D4, 16H4, 78F4 and 98G9 humanized versions of the antibodies to α -hemolysin, respectively.
Fig. 9 shows the effect of the antibody of the present invention on the hemolytic activity of α -hemolysin, wherein fig. 9A to 9C show the results at different antibody amounts, respectively.
Figure 10 shows the therapeutic effect of the antibodies of the invention in an animal model of α -hemolysin-induced sepsis.
Figure 11 shows the therapeutic effect of the antibodies of the invention in an animal model of bacteremia caused by methicillin-resistant staphylococcus aureus.
Figure 12 shows the therapeutic effect of the antibodies of the invention in an animal model of pneumonia resistant to methicillin-resistant staphylococcus aureus.
Figure 13 shows the results of a pharmacokinetic study of the antibodies of the invention following a single administration in cynomolgus monkeys.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.
Known antibodies are used herein as controls, wherein:
fully human antibody R-301 from Aridis pharmaceuticalAbbreviated 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.
Humanized antibody MEDI4893, abbreviated as AZ (see US20140072577A1), from Astrazeneca pharmaceuticals, the heavy chain variable region is shown in SEQ ID NO:84 and the light chain variable region is shown in SEQ ID NO: 85.
The antibody provided by the invention has a heavy chain constant region shown as SEQ ID NO. 86 and a light chain constant region shown as SEQ ID NO. 87.
Example 1Recombinant expression of His-tag fused Staphylococcus aureus alpha-hemolysin (alpha-Toxin)
The alpha-hemolysin amino acid sequence of staphylococcus aureus is taken as a target sequence, a corresponding base sequence is artificially synthesized, and the corresponding base sequence is cloned into a Pet-21a plasmid containing a His tag by utilizing enzyme cutting sites NdeI and XhoI. Wherein the staphylococcus aureus alpha-hemolysin amino acid sequence is shown as SEQ ID NO. 78, the corresponding base sequence is shown as SEQ ID NO. 79, and the construction of the recombinant plasmid is shown as figure 1.
The obtained recombinant plasmid was transformed into competent cells BL21(DE3) pLysS, and a single colony was picked the next day and inoculated into LB liquid medium containing 100. mu.g/ml ampicillin, followed by shaking culture at 37 ℃ overnight. Inoculating overnight cultured bacterial liquid into LB liquid culture medium containing 100 μ g/ml ampicillin at a volume ratio of 1:100, and performing shaking culture at 200rpm at 37 deg.C to OD600About 0.6 to 0.8, IPTG was added to the bacterial solution to a final concentration of 0.1mM, and induction was carried out at 16 ℃ for 16 to 18 hours. Taking the induced bacterial liquid, centrifuging at 8,000rpm for 3min, collecting thallus, and storing at-80 deg.C.
Example 2Recombinant expression of His-tag fused Staphylococcus aureus alpha-hemolysin mutant (H35L alpha-Toxin)
Based on an amino acid sequence of alpha-hemolysin of staphylococcus aureus, 35-bit histidine (His) is mutated into leucine (Leu) to obtain a mutated amino acid sequence, a corresponding base sequence is artificially synthesized, and the mutated amino acid sequence is cloned into a Pet-21a plasmid containing a His label by utilizing enzyme cutting sites NdeI and XhoI. Wherein the amino acid sequence of the staphylococcus aureus mutant alpha-hemolysin (H35L alpha-Toxin) is shown as SEQ ID NO. 80, the corresponding base sequence is shown as SEQ ID NO. 81, and the construction of the recombinant plasmid is shown as figure 2.
The obtained recombinant plasmid was transformed into competent cell BL21(DE3) pLysS, and a single colony was picked the next day and inoculatedThe mixture was cultured overnight at 37 ℃ in LB liquid medium containing 100. mu.g/ml ampicillin with shaking. Inoculating overnight cultured bacterial liquid into LB liquid culture medium containing 100 μ g/ml ampicillin at a volume ratio of 1:100, and performing shaking culture at 200rpm at 37 deg.C to OD600About 0.6 to 0.8, IPTG was added to the bacterial solution to a final concentration of 0.25mM, and the mixture was induced at 25 ℃ for 4.5 hours. Taking the induced bacterial liquid, centrifuging at 8,000rpm for 3min, collecting thallus, and storing at-80 deg.C.
Example 3Purification of Staphylococcus aureus alpha-hemolysin (alpha-Toxin) and its mutant (H35L alpha-Toxin)
Respectively crushing escherichia coli for inducing expression of staphylococcus aureus alpha-hemolysin and mutants thereof by using an ultrasonic crusher, working at 180W for 3s, and pausing for 3s for 7-9 min; centrifugation was carried out at 13,000rpm for 30min, and the supernatant was collected and sterilized by filtration through a 0.22. mu.mL filter.
The Ni column was loaded into the packed column by mixing with the filtered supernatant on a rotary mixer for 1h at room temperature. And eluting the protein which is not specifically bound with the Ni column by using BD liquid (containing imidazole with the concentration of 30mM) with the volume of 5 times of the column bed until the color of the protein is not changed, and then eluting the target protein by using BB liquid (containing imidazole with the concentration of 300mM) with the volume of 5 times of the column bed. Then, the eluate containing the target protein was concentrated and displaced using a 10kD concentration tube in PBS. The electrophoresis results of the obtained protein are shown in FIG. 3.
Example 4Staphylococcus aureus alpha-hemolysin (alpha-Toxin) and its mutant (H35L alpha-Toxin) biological activity detection (in vitro hemolysis experiment and mouse in vivo toxicity effect)
Respectively dripping 10 mu l of staphylococcus aureus alpha-hemolysin with different concentrations (5 mu g/ml, 0.5 mu g/ml and 0.05 mu g/ml) and mutants thereof on the surface of a sheep blood plate (Shanghai Kema Jia microbial technology Co., Ltd.), placing the blood plate in an incubator at 37 ℃ for incubation for 24h, dripping obvious hemolysin which is visible around the alpha-hemolysin, wherein the diameter of the hemolysin is related to the concentration thereof; and no hemolytic ring was found around the alpha-hemolysin mutant. The results of the experiment are shown in FIG. 4.
To 96-well plates were added 75. mu.l of rabbit erythrocytes (Bio-channel Biotechnology) and Staphylococcus aureus alpha-hemolysin of varying mass per wellOr a mutant thereof, and adding PBS buffer to a final volume of 150. mu.l. Incubate at 37 ℃ for 1h and centrifuge 96-well plates at 3000rpm for 3 min. 100 μ l of the supernatant was assayed for absorbance (OD) at 405nm using a microplate reader405) The hemolytic activity was evaluated. The results show that alpha-hemolysin is dose-dependent on the hemolytic activity of rabbit erythrocytes, whereas alpha-hemolysin mutants have no hemolytic activity. The results of the experiment are shown in FIG. 5.
The lowest lethal dose of staphylococcus aureus alpha-hemolysin or mutants thereof, recombinantly expressed according to the methods described in example 1, example 2 and example 3, to C57 mice was found to be 3 μ g/mouse when administered to C57 mice at different doses by tail vein injection; the highest injection of alpha-hemolysin mutant was 200. mu.g/mouse, and no adverse reaction occurred in the mice.
Example 5Immunization of Balb/c mice with Staphylococcus aureus alpha-hemolysin mutant (H35L alpha-Toxin)
Alpha-hemolysin or alpha-hemolysin mutant was diluted to different concentrations with PBS and injected tail vein into C57 mice. The toxic effects of the two on C57 mice were compared to select an immunogen suitable for subsequent immunization of animals and the amount thereof. The results are shown in Table 1.
TABLE 1 toxic Effect of different doses of alpha-hemolysin and alpha-hemolysin mutants on mice
Example 6Immunization of Balb/c mice with Staphylococcus aureus alpha-hemolysin mutant (H35L alpha-Toxin)
Referring to Antibodies a Laboratory Manual, Second Edition (Edward A. Greenfield 2012), 8-week-old Balb/c mice were immunized in a total of 42 days at 14-day intervals.
The staphylococcus aureus alpha-hemolysin mutant is emulsified in complete or incomplete Freund's adjuvant and injected into subcutaneous tissues and peritoneal cavity of the neck, back, tail root and groin 3 of the mouse in a unilateral manner. On the 35 th day of immunization, tail vein blood was collected, and spleen cells of immunized mice were fused with myeloma cells after antibody titer detection by ELISA.
Example 7Screening and identification of hybridoma cell strain and determination of antibody sequence
Spleen cells of Balb/c mice immunized with staphylococcus aureus alpha-hemolysin mutant (H35L alpha-Toxin) were fused with myeloma cells P3X63Ag8.653 by PEG or electrofusion. The fused hybridoma cells were inoculated in 30 384-well plates, and 24 hours later, HAT-containing medium and HT-containing medium were added to select hybridoma cells. After culturing in 384-well plates for 10-14 days, cell supernatants were taken and subjected to ELISA assay with α -Toxin to screen hybridoma clones secreting antibodies that specifically bind to α -hemolysin (see FIG. 6A). Subsequently, 94 wells were picked per plate and transferred to a 96-well plate for culture (of which plate No. 30 was less positive for ELISA detection, and unselected wells were transferred to plates) in order of ELISA OD values from high to low, and 29 96-well plates were co-transferred.
Under physiological conditions, the presence of alpha-hemolysin leads to lysis of red blood cells and the release of the lysate leads to a change in the color of the supernatant of the solution. And the anti-alpha-hemolysin antibody in the cell secretion supernatant can inhibit the cracking of the alpha-hemolysin to red blood cells. By detecting the light absorption value of the supernatant, the cracking degree of alpha-hemolysin on cells and the inhibition of the alpha-hemolysin by the antibody can be detected.
The hemolysis experiment detection is carried out on the culture supernatant of the 96-well plate after the plate transfer, and the steps are as follows: diluting WT-alpha-toxin hemolysin to 5 mu g/mL mother liquor, mixing 25 mu L with equal volume of cell culture supernatant, adding into 96-well plate containing 5% rabbit red blood cells (diluted to 75 mu L by PBS), and placing in an incubator at 37 ℃ for incubation for 1 h. Centrifuge the 96-well plate centrifuge at 3000rpm for 3 min. Add 75. mu.l of the supernatant to a new 96-well plate and measure the OD405 and OD450 absorbance values using a microplate reader. Partial results are shown in FIG. 6B.
Adding the screened hybridoma parent clones secreting the anti-alpha-hemolysin antibody into a 96-well plate paved with feeder cells by a limiting dilution method, observing and marking monoclonal cells under a microscope after 2-3 days, and screening the monoclonal hybridoma cells capable of secreting the anti-alpha-hemolysin monoclonal antibody by an ELISA (enzyme-linked immuno sorbent assay) experiment after 7 days.
After the monoclonal hybridoma secreting the anti-alpha-hemolysin monoclonal antibody is subjected to expanded culture, extracting total RNA of the cell according to the steps of an RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); reverse transcribing hybridoma cell total RNA to cDNA using 5 XPrimeScript RT Master Mix (Takara); amplification of antibody light chain variable region IgVL (kappa) and heavy chain variable region V using degenerate primers (Anke Krebber.1997) and Extaq PCR reagents (Takara)HAnd (4) sequencing. Purifying the PCR amplification product by using a PCR clean-up Gel extraction kit (Macherey-Nagel Co.); connecting the amplified PCR product to a T Vector according to the specification of a pClone007Simple Vector Kit (Scopheraceae Biotechnology limited), converting escherichia coli competent cells, amplifying the strain, extracting plasmids, and performing DNA sequencing to obtain the variable region sequence of the monoclonal antibody.
Murine antibody variable region sequences were obtained and analyzed as follows:
>98G9 murine antibody
Heavy chain variable region:
DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISGGSSTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCASGYPYGLDYWGQGTSVTVSS(SEQ ID NO:44)
light chain variable region:
DIDMTQSPSSMYASLGERVTITCKASQDINWYLSWFQQKPGKSPKTLIYRGNRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPFTFGSGTKLEIK(SEQ ID NO:46)
the heavy and light chain CDRs of the murine 98G9 antibody were defined according to various methods, see table 2.
TABLE 2.98G 9 CDR sequences of murine antibodies
>78F4 murine antibody
Heavy chain variable region:
QVQLQQPGAELVRPGASVKLSCKASGYSFTSYWMNWVKQRPGQGLEWIGMIHPSDSETRLSQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRFDWDRAMDYWGQGTSVTVSS(SEQ ID NO:52)
light chain variable region:
DIQMTQSPASLSASVGETVTITCRASENIFSYLAWYQQKQGKSPQLLVYNTRSLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHYGTPWTFGGGTKLEIK(SEQ ID NO:54)
the heavy and light chain CDRs of the murine 78F4 antibody were defined according to various methods, see table 3.
TABLE 3.78F 4 CDR sequences of murine antibodies
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 of
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 of
SEQ ID NO:19
SEQ ID NO:20
SEQ ID NO:21
>78D4 murine antibody
Heavy chain variable region:
EVHLQQSGPELMKPGASVKISCKTSGYTFSEYTMHWVKQSHGKSLEWIGSINPNNGGTTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYNCARTRDYDNDGGLFAYWGQGTLVTVSA(SEQ ID NO:60)
light chain variable region:
DVQITQSPSYLAASPGETITINCRASKNISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGNRSGTDFTLTISSLEPEDFAMYYCQQHYEYPFTFGGGTKLEIK(SEQ ID NO:62)
the heavy and light chain CDRs of the 78D4 murine antibody were defined according to various methods, see table 4.
TABLE 4.78 CDR sequences of murine 4 antibodies
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 of
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 of
SEQ ID NO:30
SEQ ID NO:32
SEQ ID NO:33
>16H4 murine antibody
Heavy chain variable region:
EVQLQQSGAELVKPGASVTLSCTVSGFNIKDTYMHWVKQRPEQGLEWIGKIDPASGNTKYDPQFQGKATITADTSSNTAYLHLSSLTSEDSAVYFCASPYGNDYAMNYWGQGTSVTVSS(SEQ ID NO:68)
light chain variable region:
DIQMTQSPASLSASVGETVTITCRASEKIYSFLAWYQQKQEKSPQLLVYNAETLAEGVPSRFSGTGSGIQFSLKIISLQPEDFGIYYCQHHYGTPYTFGGGTKLEIK(SEQ ID NO:70)
the heavy and light chain CDRs of the murine 16H4 antibody were defined according to various methods, see table 5.
TABLE 5.16H 4 CDR sequences of murine antibodies
Method
Heavy chain CDR1
Heavy chain CDR2
Heavy chain CDR3
Chothia
SEQ ID NO:34
SEQ ID NO:37
SEQ ID NO:40
AbM
SEQ ID NO:35
SEQ ID NO:38
SEQ ID NO:40
Kabat
SEQ ID NO:36
SEQ ID NO:39
SEQ ID NO:40
Combination of
SEQ ID NO:35
SEQ ID NO:39
SEQ ID NO:40
Method
Light chain CDR1
Light chain CDR2
Light chain CDR3
Chothia
SEQ ID NO:41
SEQ ID NO:42
SEQ ID NO:43
AbM
SEQ ID NO:41
SEQ ID NO:42
SEQ ID NO:43
Kabat
SEQ ID NO:41
SEQ ID NO:42
SEQ ID NO:43
Combination of
SEQ ID NO:41
SEQ ID NO:42
SEQ ID NO:43
Example 8Binding of the antibodies of the invention to alpha-hemolysin (alpha-Toxin)
Alpha-hemolysin was diluted to 1. mu.g/ml with PBS buffer and incubated overnight at 4 ℃ in 96-well plates (Microwell 96F 167008, Thermo) coated with 100. mu.l per well; the next day the 96-well plate was removed and washed with PBST (containing 0.5% PBS) and the residual water was spun off thoroughly after 1min of each soak. Adding 200 μ l PBST containing 5% BSA into sample wells, and sealing at 37 deg.C for 1 h; the plates were then washed with PBST and the wells were spin dried.
100. mu.l of recombinantly expressed anti- α -hemolysin monoclonal antibody (antibody concentration see FIG. 7 abscissa) was added in each 96-well plate at a dilution of each in two-fold and incubated overnight at 4 ℃. After the 96-well plate was taken out, the plate was washed with PBST, and then 100. mu.l of an anti-mouse IgG secondary antibody (IH-0031, Biotechnology of Ming Changsheng, Beijing) was added to each well, and incubated at 37 ℃ for 1 hour. Washing with PBST for 5 times, adding 100 μ l of Substrate Solution (Invitrogen) per well, and incubating at 37 deg.C for 10 min; after the reaction was terminated by adding 50. mu.l of 2N sulfuric acid to each well, absorbance was measured at a wavelength of 450nm using a microplate reader (Multiskcin FC, Thermo).
The results are shown in FIG. 7.
Example 9Obtaining of chimeric antibody and humanized antibody of the present invention
First, the complete light and heavy chain variable regions of the murine antibody are combined with the human light and heavy chain constant regions to obtain a chimeric antibody form as a control. The resulting chimeric antibody was named "murine antibody abbreviated as-xi".
After comprehensive analysis of the heavy chain sequence of the murine antibody, the antigen Complementarity Determining (CDR) region of the antibody binding to the antigen and the framework region (framework) supporting the conserved three-dimensional conformation of the antibody are determined, the sequences of the known humanized antibody are searched by analysis, the heavy chain sequence of the humanized antibody which is most similar to the murine antibody, such as IGHV1-3, is selected, and the sequence of the framework region of the antibody is used as a template. The murine heavy chain CDRs were embedded into the human framework regions to generate humanized antibody heavy chain sequences (heavy chain version 0). Subsequently, individual amino acid positions of the murine framework regions that may be involved in antigen-antibody binding are reverted to generate humanized antibody heavy chain sequences (version 1, 2, 3 … …). The same procedure generates humanized antibody light chain sequences (version 0, 1, 2 … …). The humanized antibody designed to be synthesized is co-transfected into 293 cells with light and heavy chains and the humanized antibody is expressed recombinantly (version designations are e.g.H 0L0, which is co-expressed as H0L0, and may be further abbreviated as version 00). Experiments prove that the purified humanized antibody and the murine parent antibody show consistent activity of specifically binding the alpha-hemolysin protein.
The finally obtained humanized antibody version and chimeric antibody xi version were compared for antigen binding using an Octect instrument, and FIG. 8 shows the results of the partial antibody assay. From the curve of the binding and dissociation phases of the antibody binding to the antigen, a particular humanized antibody version shows similar or better properties in the binding, dissociation phase of the antigen antibody than the control antibody (including the chimeric antibody of the invention or the AZ, AR antibody).
The humanized antibody having the following structure was obtained by screening.
Humanized antibody 98G9-02(98G9-H0L2)
Heavy chain variable region (H0):
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWVSYISGGSSTIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASGYPYGLDYWGQGTLVTVSS(SEQ ID NO:48)
light chain variable region (L2):
DIQMTQSPSSLSASVGDRVTITCKASQDINWYLSWFQQKPGKAPKTLIYRGNRLVDGVPSRFSGSGSGQDYTFTISSLQPEDMATYYCLQYDEFPFTFGQGTKVEIK(SEQ ID NO:76)
humanized antibody 98G9-03(98G9-H0L3)
Heavy chain variable region (H0):
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWVSYISGGSSTIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASGYPYGLDYWGQGTLVTVSS(SEQ ID NO:48)
light chain variable region (L3):
DIQMTQSPSSLSASVGDRVTITCKASQDINWYLSWFQQKPGKAPKTLIYRGNRLVEGVPSRFSGSGSGQDYTFTISSLQPEDMATYYCLQYDEFPFTFGQGTKVEIK(SEQ ID NO:50)
humanized antibody 78F4-00(78F4-H0L0)
Heavy chain variable region (H0):
QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMNWVRQAPGQGLEWMGMIHPSDSETRLSQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARFDWDRAMDYWGQGTLVTVSS(SEQ ID NO:56)
light chain variable region (L0):
DIQMTQSPSSLSASVGDRVTITCRASENIFSYLAWYQQKPGKAPKLLIYNTRSLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPWTFGQGTKLEIK(SEQ ID NO:58)
humanized antibody 78D4-33(78D4-H3L3)
Heavy chain variable region (H3):
QVQLVQSGAEVKKPGASVKVSCKTSGYTFSEYTMHWVRQAPGQRLEWMGSINPNQGGTTYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCARTRDYDNDGGLFAYWGQGTLVTVSS(SEQ ID NO:64)
light chain variable region (L3):
DVQITQSPSFLSASVGDRVTITCRASKNILKYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSRSGTEFTLTISSLQPEDFATYYCQQHYEYPFTFGQGTKLEIK(SEQ ID NO:66)
humanized antibody 16H4-11(16H4-H1L1)
Heavy chain variable region (H1):
EVQLVQSGAEVKKPGATVKISCKVSGFNIKDTYMHWVQQAPGKGLEWMGKIDPASGNTKYDPQFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCATPYGNDYAMNYWGQGTLVTVSS(SEQ ID NO:72)
light chain variable region (L1):
DIQMTQSPSSLSASVGDRVTITCRASEKIYSFLAWYQQKPGKAPKLLLYNAETLAEGVPSRFSGSGSGIDFTLTISSLQPEDFATYYCQHHYGTPYTFGGGTKVEIK(SEQ ID NO:74)
the heavy and light chain CDRs of the humanized antibodies are shown in table 6.
TABLE 6 CDR sequences of humanized antibodies
Example 10Detection of hemolytic Activity of the antibody of the present invention against alpha-hemolysin (alpha-Toxin)
To a 96 well plate, 75 μ l of 5% rabbit red blood cells (Bio-channel Biotechnology), 12.5ng Staphylococcus aureus α -hemolysin and varying masses of anti- α -hemolysin antibodies (12.5ng, 25ng, 50ng) were added per well and PBS buffer was added to a final volume of 150 μ l. Incubate at 37 ℃ for 1h and centrifuge 96-well plates at 3000rpm for 3 min. 100 μ l of the supernatant was assayed for absorbance (OD) at 405nm using a microplate reader405) The hemolytic activity was evaluated.
The experimental result shows that the chimeric antibody and the humanized antibody have obvious alpha-hemolysin resisting activity and dose dependence, and the activity of partial antibodies is equivalent to that of control antibodies AR and AZ. The results are shown in FIG. 9.
Example 11Binding kinetics (K) of the antibodies of the invention to alpha-hemolysin (alpha-Toxin)on，Koff) And affinity constant KDDetection of (2)
Antibody-antigen interaction was measured using a GE BIAcore instrument S200.
Referring to the operation of commercial kit (product No. BR-1008-39, Lot10261753) of GE company Human antigen capture kit, firstly, the maximum amount of anti-Human Fc antibody is saturated and coupled in an analysis channel and a control sample channel of a sensor chip CM5, then 7.5ug/ml of the antibody to be detected flows through the analysis channel, the antibody is uniformly distributed, finally, the antigen sample with gradient dilution is flowed through the analysis channel and the sample channel together (the initial concentration is 20nM, 8 concentration points are diluted by 1:3, and the concentration points are set to be 0.741nM repeatedly), and the photoreaction value occurring after the antibody and the antigen are combined is measured. Subsequently, the binding constant Kon and dissociation constant Koff, as well as the affinity constant KD of the antibody were finally obtained by instrumental software fitting (1:1) analysis.
The results are shown in Table 7.
TABLE 7 binding kinetics and affinity constants of the antibodies of the invention to antigens
Antibodies
ka(1/Ms)
kd(1/s)
KD(M)
Rmax(RU)
Chi2(RU2)
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
Example 12Animal model replication of sepsis caused by alpha-hemolysin (alpha-Toxin) and detection of therapeutic effect of antibody of the present invention
The C57BL/6J mice were randomly divided into a model control group and a monoclonal antibody drug-treated group according to body weight. 30min before the experiment, the tail vein of the treatment group is injected with anti-alpha-hemolysin monoclonal antibody (6 mug/mouse), the control group of mice is injected with PBS with the same dose, then all mice are injected with alpha-hemolysin (3 mug/mouse) in the tail vein to establish a sepsis infection mouse model, the survival time of the experimental animals is observed and recorded, and the result is shown in figure 10.
Example 13Methicillin-resistant strainsStaphylococcus aureus-induced bacteremia animal model replication and detection of therapeutic effect of the antibodies of the invention
Methicillin-resistant Staphylococcus aureus USA300 was activated for 2 generations on TSB solid medium plate, inoculated into TSB liquid medium overnight for culture, centrifuged at 12,000rpm to collect the thallus, and resuspended in normal saline for use.
Tail vein infection of C57BL/6J mice USA 3006X 107CFU/mouse, and randomly divided into a model Control group (Control) and different anti-alpha-hemolysin monoclonal antibody drug treatment groups according to the body weight. After 2h infection, the mice were observed and recorded for survival time by tail vein injection of 15mg/kg corresponding antibody for each group of monoclonal antibody treatment groups and PBS for the control group at the same dose, and the results are shown in FIG. 11.
Example 14Animal model replication of methicillin-resistant staphylococcus aureus in pneumonia and detection of therapeutic effect of antibodies of the invention
Methicillin-resistant Staphylococcus aureus USA300 was activated for 2 generations on TSB solid medium plate, inoculated into TSB liquid medium overnight for culture, centrifuged at 12,000rpm to collect the thallus, and resuspended in normal saline for use.
C57BL/6J mice were infected transtracheally with USA 3001.8X 108CFU/mouse, and randomly divided into a model control group, a monoclonal antibody drug treatment group, a vancomycin treatment group and a vancomycin + monoclonal antibody treatment group according to the body weight. After 2h of infection, the injection is carried out through tail vein injection, each group of animals are treated by corresponding medicines, the dosage of the monoclonal antibody is 15mg/kg, the dosage of the vancomycin is 1.25mg/kg, and a control group is injected with PBS with the same dosage. The animals are sacrificed 24h after infection, lung tissues are taken to be homogenized, weighed and homogenized, and coated on a TSB solid culture medium to detect the tissue bacterial load, and the experimental result shows that the anti-alpha-hemolysin antibody can enhance the pharmacodynamic action of vancomycin in treating the pneumonia infection of methicillin-resistant staphylococcus aureus.
The results are shown in FIG. 12.
Example 15Acute toxicity study of the antibodies of the invention
Acute toxicity test studies of the 78D 4H 3L3 antibody molecules of the invention were performed in mice and cynomolgus monkeys (mouse experiment N ═ 10, cynomolgus monkey experiment N ═ 3).
C57BL/6 mice (18-20g), hermaphrodite, were injected with 125mg/kg of the 78D 4H 3L3 antibody molecule per mouse over a 24 hour period via the tail vein. The result shows that when the 125mg/kg dose is given within 24 hours at most, the mice do not die, the animals do not have any discomfort after continuous observation for 14 days, and the main organs (heart, liver, spleen, lung, kidney and brain) of the killed animals are generally observed without abnormality.
The antibody molecule 78D 4H 3L3 was administered to each male cynomolgus monkey at 10mg/kg by intravenous drip to the extremities in a single dose. The result shows that no animal death occurs when the antibody molecule of 78D 4H 3L3 is dosed at 10mg/kg, and no discomfort occurs when the animals are continuously observed for 28 days.
Acute toxicity test research shows that the 78D 4H 3L3 antibody molecule has good safety.
Example 16Pharmacokinetic study of the antibodies of the invention
The 78D 4H 3L3 antibody molecule was subjected to a single dose pharmacokinetic study in cynomolgus monkeys at a dose of 10mg/kg (N ═ 3).
The antibody molecule 78D 4H 3L3 was administered by intravenous drip of limbs to cynomolgus monkeys, males, and each animal at 10mg/kg in a single dose. Blood was collected at 0h (pre-dose, 0h) before administration, 0.25h (15min), 0.5h (needle withdrawal point), 4h, 24h (D2), 48h (D3), 96h (D5), 168h (D8), 336h (D15), 504h (D22), and 672h (D29) after the start of administration. The blood sampling part is used for sampling blood from peripheral veins (non-administration limbs) or inguinal veins of four limbs of the animals. The amount of blood collected was about 1mL whole blood/mouse/time point. Measuring the antibody concentration in the cynomolgus monkey serum by adopting an ELISA method; pharmacokinetic parameters such as AUClast, CL and T1/2 were calculated by WinNonlin Phoenix (v6.4, Pharsight) software using a method other than compartmental model analysis.
The ELISA method is adopted to determine the concentration of the 78D 4H 3L3 antibody in the cynomolgus monkey serum, the individual graph of the serum drug concentration is shown in figure 13, the pharmacokinetic parameters are summarized in table 8, and the drug exposure can be seen after the administration of each animal.
TABLE 8 pharmacokinetic results for antibody 78D 4H 3L3
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
Sequence listing
<110> Mewey (Shanghai) Biotech Co., Ltd
Shanghai Puming Biotechnology Co.,Ltd.
<120> anti-alpha-hemolysin antibody and use thereof
<130> LC19110042
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Gly Phe Thr Phe Ser Thr Phe
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Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Ser Gln Lys Phe Lys
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<210> 18
<211> 9
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<221> PEPTIDE
<222> ()..()
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<210> 19
<211> 11
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain, CDR1
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<210> 20
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<222> ()..()
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Asn Thr Arg Ser Leu Ala Glu
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Gln His His Tyr Gly Thr Pro Trp Thr
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<212> PRT
<213> Artificial (artificial)
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<221> PEPTIDE
<222> ()..()
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Gly Tyr Thr Phe Ser Glu Tyr
1 5
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<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
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Gly Tyr Thr Phe Ser Glu Tyr Thr Met His
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<213> Artificial (artificial)
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Glu Tyr Thr Met His
1 5
<210> 25
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Asn Pro Asn Asn Gly Gly
1 5
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<210> 27
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<213> Artificial (artificial)
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<210> 28
<211> 17
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Gly
<210> 29
<211> 13
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<210> 30
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<210> 31
<211> 11
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Gly Phe Asn Ile Lys Asp Thr
1 5
<210> 35
<211> 10
<212> PRT
<213> Artificial (artificial)
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<221> PEPTIDE
<222> ()..()
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Gly Phe Asn Ile Lys Asp Thr Tyr Met His
1 5 10
<210> 36
<211> 5
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
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Asp Thr Tyr Met His
1 5
<210> 37
<211> 6
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
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Asp Pro Ala Ser Gly Asn
1 5
<210> 38
<211> 10
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
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<400> 38
Lys Ile Asp Pro Ala Ser Gly Asn Thr Lys
1 5 10
<210> 39
<211> 17
<212> PRT
<213> Artificial (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 (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 (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 (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 (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 (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
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<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
ccctatggtt tggactactg gggtcaagga acctcagtca ccgtctcctc a 351
<210> 46
<211> 107
<212> PRT
<213> Artificial (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 (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 (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 (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 (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 (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
ggcaaagccc caaaaaccct catctaccgg ggaaacagac tggtggaagg ggtgcccagc 180
agatttagcg gaagcggcag cggccaggac tacaccttca ccatatcaag cctgcagccc 240
gaagacatgg ccacctacta ctgcctgcag tacgacgaat ttccatttac cttcggacag 300
ggaaccaaag tcgagatcaa a 321
<210> 52
<211> 118
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<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 (artificial)
<220>
<221> gene
<222> ()..()
<223> heavy chain variable region
<400> 53
caggtccaac 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 (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 (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 (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
<210> 57
<211> 354
<212> DNA
<213> Artificial (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 (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 (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 (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 (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 (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
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 63
<211> 321
<212> DNA
<213> Artificial (artificial)
<220>
<221> gene
<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 (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 (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 (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 (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 (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 (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 (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 (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
gaagattttg ggatttatta ctgtcaacat cattatggta ctccgtacac gttcggaggg 300
gggaccaagt tggaaataaa a 321
<210> 72
<211> 119
<212> PRT
<213> Artificial (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 (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 (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 (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
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 76
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 Asp 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> 77
<211> 321
<212> DNA
<213> Artificial (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 (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 (Staphylococcus aureus)
<400> 79
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 g 891
<210> 80
<211> 294
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> mutant alpha-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 (artificial)
<220>
<221> gene
<222> ()..()
<223> mutant alpha-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 g 891
<210> 82
<211> 129
<212> PRT
<213> Artificial (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 (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 (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
<212> PRT
<213> Artificial (artificial)
<220>
<221> PEPTIDE
<222> ()..()
<223> light chain variable region
<400> 85
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 (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 (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