WO2020098701A1 - Anticorps dirigé contre α-hémolysine de staphylococcus aureus et utilisations associées - Google Patents

Anticorps dirigé contre α-hémolysine de staphylococcus aureus et utilisations associées Download PDF

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WO2020098701A1
WO2020098701A1 PCT/CN2019/117997 CN2019117997W WO2020098701A1 WO 2020098701 A1 WO2020098701 A1 WO 2020098701A1 CN 2019117997 W CN2019117997 W CN 2019117997W WO 2020098701 A1 WO2020098701 A1 WO 2020098701A1
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antibody
seq
amino acid
acid sequence
sequence shown
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PCT/CN2019/117997
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English (en)
Chinese (zh)
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廖化新
郑伟宏
王孝丽
袁晓辉
王月明
李成明
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珠海泰诺麦博生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

  • the present invention relates to the field of medicine and immunology, and in particular to an antibody against staphylococcus aureus alpha-hemolysin.
  • the source of human pathogens is mostly Gram-positive bacteria, and the important member is Staphylococcus aureus, about 20% of long-term carriers of Staphylococcus aureus in humans.
  • the infections caused by it are characterized by acute and purulent, which can lead to skin, mucous membrane, deep tissue infections and endocarditis, pneumonia, sepsis, osteomyelitis, meningitis, scalded skin syndrome, toxic shock syndrome, etc.
  • the mortality of infections of various diseases, serious infections and complications is as high as 20%.
  • Staphylococcus aureus is widely distributed in nature and has strong resistance. It is an important pathogen causing bacterial food poisoning.
  • the present invention provides an antibody or antibody fragment thereof against S. aureus ⁇ -hemolysin, the antibody or antibody fragment thereof comprising a light chain variable region and a heavy chain variable region:
  • the functionally active CDR variant includes an amino acid sequence in which at least one amino acid is modified in the parental CDR sequence, and includes at least 60% sequence identity with the parental CDR sequence, preferably at least 70%, at least 80%, at least 90 Amino acid sequence with% sequence identity, or consists of an amino acid sequence with at least 60% sequence identity with the parental CDR sequence, preferably with at least 70%, at least 80%, at least 90% sequence identity.
  • the modification may be a chemical change or a partial modification of the amino acid sequence, the modification allows the variant to retain the biological properties of the unmodified sequence, and the partial modification may be the deletion or replacement of one to several amino acids, for example, 1, 2, 3 , 4 or 5 amino acids, or adding or inserting one to several amino acids, for example 1, 2, 3, 4 or 5 amino acids, or chemically derivatizing one to several amino acids, for example, 1, 2, 3, 4 or 5 amino acids, or a combination of them.
  • the substitution of amino acid residues may be a conservative substitution, for example, replacing one hydrophobic amino acid with one hydrophobic amino acid.
  • the CDR regions of the light chain are as follows:
  • the monoclonal antibody or antibody fragment thereof is not higher than 1 * 10 -5 M, for example, 1 * 10 -6 M, 1 * 10 -7 M, 1 * 10 -8 M , 1 * 10 -9 M or 1 * 10 -10 M or less KD dissociates from Staphylococcus aureus ⁇ -hemolysin.
  • KD refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, and indicates the degree of dissociation of the antibody and antigen when in equilibrium. The smaller the KD, the smaller the dissociation, which means the stronger the affinity between antibody and antigen.
  • the product is a drug. In some embodiments, the product is a vaccine.
  • the medicament of the present invention must be sterile and stable under the conditions of manufacture and storage.
  • the preferred methods of preparation are vacuum drying and lyophilization, which produce activity from a previously sterile filtered solution of the active ingredient and other desired ingredients Powder of ingredients and other desired ingredients.
  • the medicament of the present invention may be in a solution, and an appropriate pharmaceutically acceptable excipient may be added and / or mixed before or during delivery to provide an injectable unit dosage form.
  • the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentration, can maintain proper fluidity, and can delay absorption if necessary.
  • Example 5 is a graph of WB results of Hla denatured rubber in Example 8 of the present invention.
  • Example 7 is a graph showing the results of TRN1016 neutralizing wHla lysis of red blood cells in Example 10 of the present invention.
  • Figure 8 is a graph showing the results of TRN1016 neutralizing wHla lysing A549 cells of the present invention.
  • the signs of inflammation may not be obvious early in the infection. If the bacteria are not deep, signs of inflammation such as redness and swelling or burning skin show up quickly. The skin color may progress to purple, and blisters may form, with subsequent necrosis (eg, death) of subcutaneous tissue. Patients with necrotizing fasciitis typically have fever and appear to be very ill. If not treated, that is, without proper medical assistance, the rate of infection progressing rapidly and leading to death is as high as 73%.
  • Staphylococcus aureus may be "scalded skin syndrome” (also known as “staphylococcal scalded skin syndrome”), "toxic epidermal necrosis”, “limited bullous pustulosis”, “Li The main pathogenic factors of "Ritter's disease” and “Lyell's disease”.
  • Scald-like skin syndrome frequently occurs in older children, typically from S. aureus strains that produce epidermolysis exotoxins (eg, epidermal exfoliating toxins A and B, sometimes referred to as scalded skin syndrome toxins) In an outbreak caused by flowering, these epidermises release exotoxins and cause separation within the epidermal layer.
  • the bacteria can initially infect only a smaller lesion, however, the toxin disrupts the intercellular connection, spreads the epidermal layer, and allows the infection to penetrate the outer layer of the skin, producing desquamation that is a disease.
  • the shedding of the outer layer of skin usually exposes the underlying normal skin, but without proper treatment, fluid loss in the process can cause serious damage in young children.
  • Toxic shock syndrome is caused by a strain of Staphylococcus aureus that produces a so-called "toxic shock syndrome toxin".
  • the disease can be caused by infection of Staphylococcus aureus in any part, but it is often mistakenly regarded as a disease exclusively for women using sanitary napkins. The disease involves toxemia and sepsis and can be fatal.
  • TSS due to the infection of the bacterium Staphylococcus aureus is typically additionally manifested in otherwise healthy individuals with high fever, accompanied by low blood pressure, discomfort and confusion, which can rapidly progress to stupor, coma, and many Organ failure.
  • the characteristic rash often seen in the early course of the disease is similar to sunburn and can affect any area of the body, including lips, mouth, eyes, palms, and soles of the feet. In patients who survived the initial onslaught of infection, the rash was desquamated or exfoliated after 10-14 days.
  • Genomic DNA from S. aureus strain was amplified by PCR to the wHla gene. Then use QuickChange II XL site-directed mutagenesis kit to generate H35L variants by site-directed mutagenesis of wild-type genes. After DNA sequencing confirmed, it was expressed in E. coli, cultured in LB medium containing ampicillin at 37 ° C overnight, and cells were harvested by centrifugation. Ni-NTA purification was used to obtain wHla protein and recombinant Hla protein.
  • Flow cytometry sorting of single plasma cells Determine the antibody titer of the sample according to serological experiments (ELISA detection with HLA protective antigen protein (HPLC purity> 95%) as antigen), select the sample with high antibody titer, pass Flow cytometry sorts individual plasma cells, and sorts by CD3 / CD14 / CD16 / CD235a-CD19 + CD20 +/- CD38hi CD27hi gate sorting. The plasma cell populations at different time points were separated.
  • the first strand of cDNA was synthesized using SuperscriptV reverse transcriptase (Invitrogen, Carlsbad, CA) and random primers.
  • Human Ig V H and V K / L were amplified by PCR using the Ig primer set (constant region primer sequence of heavy chain: GCGGCCCTGGGCTGCCTGGTCAAG; constant region primer sequence of light chain: AGGAGAGTGTCACAGAGCAGGACAG).
  • the PCR amplified VH and VK / L products were cloned into TOPO TA vector and sequenced. Then, the above amplification product was re-amplified by PCR, and the product was identified by 1.2% agarose gel electrophoresis.
  • Antibody gene sequence determination and bioinformatics analysis the PCR product of the antibody gene identified by gel electrophoresis as positive, and the heavy chain and light chain can be matched in pairs is purified with Qiagen PCR product purification kit, and from the forward and reverse respectively
  • the IMGT online server http://imgt.cines.fr/ was used to analyze antibody gene families, mutation rates, subtypes and CDR regions. The new antibody genes are added to the antibody gene library.
  • the PCR product of the antibody variable region gene identified by gel electrophoresis as positive, and the heavy chain and light chain can be paired is connected to the pcDNA3.3 vector by TA cloning method to construct an expression vector of fully human anti-Hla antibody
  • the expression vector was transformed into DH5 ⁇ competent bacteria, and cultured overnight on a plate containing ampicillin at 37 ° C. Ten single colonies were picked for PCR with specific primers.
  • the reaction conditions were: 94 ° C pre-denaturation for 3min; 94 ° C denaturation for 30s , Annealing at 55 ° C for 30s, extension at 72 ° C for 100s, 28 cycles; extension at 72 ° C for 5min. Take 5 ⁇ L of PCR product and detect by 1% agarose gel electrophoresis.
  • CM5 chip surface plasmon resonance measurement was used to measure the KD value (BIACORE3000), and the isolated TRN1016 monoclonal antibody was coupled to the CM5 chip using an amine coupling kit.
  • CM5 carboxymethylated dextran biosensor chip
  • EDC N-ethyl-N ′-(3-dimethylaminopropyl) -carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un anticorps dirigé contre α-hémolysine de staphylococcus aureus et des utilisations associées. La CDR de la région variable de chaîne lourde et de la région variable de chaîne légère dudit anticorps comprend une séquence d'acides aminés (ou un variant de CDR actif fonctionnel ayant la même fonction) telle que représentée dans SEQ ID NO. 1 à 3 ou une séquence d'acides aminés (ou un variant de CDR actif fonctionnel ayant la même fonction) telle que représentée dans SEQ ID NO. 4 à 6. L'invention concerne en outre une molécule d'acide nucléique codant pour l'anticorps et des utilisations de l'anticorps dans la préparation de produits α-hémolysine de staphylococcus aureus ayant une liaison spécifique, de vaccins contre staphylococcus aureus et autres. De plus, des produits qui peuvent être utilisés en tant que traitement ou traitement adjuvant pour des infections par staphylococcus aureus peuvent être obtenus sur la base de l'anticorps La découverte de nouveaux médicaments anti-MRSA hautement efficaces et faiblement toxiques est devenue une direction importante ces dernières années dans le domaine de la recherche de traitement pour MRES.
PCT/CN2019/117997 2018-11-14 2019-11-13 Anticorps dirigé contre α-hémolysine de staphylococcus aureus et utilisations associées WO2020098701A1 (fr)

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CN201811349827.8 2018-11-14
CN201811349827.8A CN109400704B (zh) 2018-11-14 2018-11-14 一种抗金黄色葡萄球菌α-溶血素的抗体及其应用

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CN109400704B (zh) * 2018-11-14 2020-07-21 珠海泰诺麦博生物技术有限公司 一种抗金黄色葡萄球菌α-溶血素的抗体及其应用
CN112538112B (zh) * 2019-09-20 2023-10-27 迈威(上海)生物科技股份有限公司 抗α-溶血素的抗体及其应用
CN110498854B (zh) * 2019-09-28 2021-01-29 中国人民解放军陆军军医大学 一种抗金黄色葡萄球菌肠毒素b的抗体及其应用
CN113444172A (zh) * 2020-03-25 2021-09-28 兴盟生物医药(苏州)有限公司 金黄色葡萄球菌α-毒素特异性抗体及其应用
CN113214368A (zh) * 2021-04-14 2021-08-06 中国人民解放军陆军军医大学 用于体外检测血清中金黄色葡萄球菌溶血素的溶血中和活性的溶血素抗原表位肽及其应用
WO2023208123A1 (fr) * 2022-04-28 2023-11-02 珠海泰诺麦博制药股份有限公司 Anticorps monoclonal entièrement humain se liant spécifiquement à la toxine hla de staphylococcus aureus
CN116789813B (zh) * 2023-06-27 2024-04-26 重庆原伦生物科技有限公司 一种抗金黄色葡萄球菌α-溶血素的单克隆抗体及其应用
CN116789814B (zh) * 2023-06-27 2024-03-29 重庆原伦生物科技有限公司 一种特异性结合金黄色葡萄球菌α-溶血素的抗体及其应用
CN117771379A (zh) * 2023-10-25 2024-03-29 珠海泰诺麦博制药股份有限公司 特异性结合金黄色葡萄球菌Hla毒素的全人源抗体与抗生素的组合

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