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Human binding molecules having killing activity against enterococci and staphylococcus aureus and uses thereof

A staphylococcus and enterococcus technology, applied in the fields of application, bacterial antigen components, bacteria, etc., can solve the problems of unpublished human antibodies, unpublished sequences, etc.

Active Publication Date: 2012-10-17
JANSSEN VACCINES & PREVENTION BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although WO99 / 18996 mentions human antibodies as the desired molecules, the antibodies actually disclosed and used therein are of rabbit origin, and the document does not actually disclose any human antibodies, nor their sequences

Method used

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  • Human binding molecules having killing activity against enterococci and staphylococcus aureus and uses thereof
  • Human binding molecules having killing activity against enterococci and staphylococcus aureus and uses thereof
  • Human binding molecules having killing activity against enterococci and staphylococcus aureus and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Construction of scFv Phage Display Libraries Using RNA Extracted from Donors for Screening for Opsonization Activity

[0135]Blood samples were drawn from donors aged 25-50 years who reported recent Gram-positive bacterial infection as well as healthy adults. Peripheral blood leukocytes were separated by centrifugation, and serum was stored and frozen at -80°C. Donor sera were screened for killing activity using the opsonophagocytic killing assay (Huebner et al., 1999) and compared to normal rabbit sera. Sera from donors with higher than normal phagocytic activity were selected for the generation of phage display libraries. Total RNA was prepared from peripheral blood leukocytes of these donors using organic phase separation followed by ethanol precipitation. The resulting RNA was dissolved in RNase-free water and its concentration determined by OD260nm detection. After that, the RNA was dissolved to a concentration of 100 ng / µl. Next, 1 μg of RNA was converted into...

Embodiment 2

[0139] Construction of scFv phage display libraries using RNA extracted from memory B cells

[0140] Peripheral blood was collected from normal healthy donors, recovering donors, or vaccinated donors by venipuncture using EDTA anticoagulated tubes. Blood samples (45ml) were diluted twice with PBS and 30ml aliquots placed under 10ml Ficoll-Hypaque (Pharmacia) and centrifuged at 900 xg for 20 minutes at room temperature without pause. The supernatant was carefully removed to just above the fraction containing lymphocytes and platelets. Next, carefully remove this layer (about 10 ml) and transfer to a new 50 ml tube, wash 3 times with 40 ml PBS, and centrifuge at 400 xg for 10 minutes at room temperature to remove platelets. The resulting lymphocyte-containing pellet was resuspended in RPMI medium containing 2% PBS, and the cell number was determined by cell counting. Use CD24, CD27, and surface IgM as markers to separate switched and IgM memory B cells for approximately 1X10 ...

Embodiment 3

[0142]Selection of phages carrying single-chain Fv fragments that specifically bind enterococci

[0143] Using antibody phage display libraries, common phage display techniques, and Techniques for selection of antibody fragments are primarily described in US Patent No. 6,265,150 and in WO 98 / 15833 (both incorporated herein by reference). The antibody phage libraries used were the screened donor library prepared as described in Example 1 and the IgM memory library prepared as described in Example 2. The methods and helper phages described in WO 02 / 103012 (incorporated herein by reference) are used in the present invention. To identify phage antibodies that recognize enterococci, phage selection experiments were performed using live bacteria in suspension or immobilized bacteria in immunotubes. The strains used are described in Table 8. All phage antibodies were isolated from selections in which, in at least one step, E. faecalis 12030 in suspension was used. Antibodies des...

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PUM

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Abstract

Disclosed is a human monoclonal antibody, wherein said antibody comprises: a heavy chain CDR1 region comprising amino acid sequence SEQ ID NO: 296; a heavy chain CDR2 region comprising amino acid sequence SEQ ID NO: 297; a heavy chain CDR3 region comprising amino acid sequence SEQ ID NO: 298; a light chain CDR1 region comprising amino acid sequence SEQ ID NO: 299; a light chain CDR2 region comprising amino acid sequence SEQ ID NO: 300; a light chain CDR3 region comprising amino acid sequence SEQ ID NO: 301; characterized in that said antibody has opsonic phagocytic killing activity against at least one strain of each of at least two different Enterococcus species and against at least one strain of Staphylococcus aureus.

Description

[0001] This application is a divisional application of an invention patent application with an application date of June 5, 2007, an application number of 200780020712.8, and an invention title of "Human binding molecule with killing activity against Enterococcus and Staphylococcus aureus and its application". field of invention [0002] The present invention relates to medicines. In particular, the invention relates to the diagnosis, prevention and / or treatment of enterococcal infections. Background of the invention [0003] Enterococci are Gram-positive, facultative anaerobic bacteria of the family Enterococcaceae. They were previously classified as group D strep. Enterococci are found in the intestines of most humans and are usually isolated from toilets, urine, and sites of intra-abdominal and lower extremity infections. Bacteria of the Enterococcus genus are generally considered harmless commensals of the gastrointestinal tract, but within the past 10 years they have b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/63C12N1/21C12N1/19C12N5/10C12P21/08A61K39/40A61P31/04G01N33/577
CPCY02A50/30A61K39/116A61K39/395C07K16/00C07K16/12C07K16/1228
Inventor M·思罗斯比R·A·克拉梅C·A·德克吕夫
Owner JANSSEN VACCINES & PREVENTION BV
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