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Multifunctional monoclonal antibodies directed to peptidoglycan of gram-positive bacteria

a gram-positive bacteria, multi-functional technology, applied in the field of immunology and infectious diseases, can solve the problems of host invasion, infection and occasionally death, and the purity of cell wall preparations cannot be verified, so as to enhance phagocytosis and kill gram-positive bacteria, block colonization, and enhance phagocytosis

Inactive Publication Date: 2008-01-17
BIOSYNEXUS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent is about a new invention that provides therapeutic compositions containing protective monoclonal antibodies (MAbs) to peptidoglycan (PepG) that can enhance phagocytosis, block colonization, and inhibit PepG-induced toxicity. These MAbs can be used to prevent and treat infections caused by Gram-positive bacteria, particularly staphylococci, which are often associated with nasal colonization and systemic infections. The MAbs can be administered alone or in combination to enhance phagocytosis, inhibit bacterial infection, and reduce the spread of Gram-positive bacteria. The invention also includes methods of using MAbs to elicit opsonic antibodies to Gram-positive bacteria and peptides that mimic PepG epitopes as vaccines to enhance immunity against these bacteria.

Problems solved by technology

The systems for opsonization and phagocytosis are significant because defective phagocytosis and killing of staphylococci (and other Gram-positive bacteria) leads to host invasion, infection and occasionally death.
Thus, the composite function of the antibodies in polyclonal serum may account for the serum's functional activity: However, such polyclonal IgG is clearly not always protective, as evidenced by the continued presence of infections due to such bacteria.
However, many bacterial cell extracts that are used for immunization are not pure for one epitope or antigen, so the activity of the resulting antibodies may represent activities against several different cell wall components.
This is particularly problematic if the cell wall is the antibody target, and the purity of the cell wall preparation cannot be verified.
Moreover, although perhaps protective in some individuals, polyclonal serum cannot be used to elucidate the functional role of an antibody to a single epitope because, by definition, a polyclonal serum contains many different antibodies, which bind to multiple antigens and epitopes.
Moreover, until recently, determining the role of peptidoglycan or of antibodies to peptidoglycan was complicated by the impurity of peptidoglycan preparations.
These teichoic acid moieties can easily contaminate peptidoglycan preparations, which are prepared from cell wall extracts.
Furthermore, since peptidoglycan is ubiquitous in the bacterial world, highly specific opsonic or protective antibodies to peptidoglycan seem unlikely.
One explanation is that the smaller fragments could not support binding of a sufficient number of different antibodies, and that antibodies to a single epitope on peptidoglycan are not opsonic.
Consequently, while peptidoglycan can activate the alternative pathway, which promotes opsonization and phagocytosis of S. aureus by complement alone, the role of antibodies and the classical pathway in opsonization and phagocytosis remained difficult to understand.

Method used

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  • Multifunctional monoclonal antibodies directed to peptidoglycan of gram-positive bacteria
  • Multifunctional monoclonal antibodies directed to peptidoglycan of gram-positive bacteria
  • Multifunctional monoclonal antibodies directed to peptidoglycan of gram-positive bacteria

Examples

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Effect test

example 1

The Production of Hybridomas and Monoclonal Antibodies to S. aureus PepG Immunization of Mice

[0112] To produce monoclonal antibodies directed against S. aureus PepG, immunizations were carried out using 5-6 week old female BALB / c mice, obtained from Harlan Sprague Dawley (Indianapolis, Ind.). The immunogen for the primary immunization was S. aureus PepG (gift from Roman Dziarski; PepG can also prepared as described in Example 2). Five microliters of PepG (7 mg / ml suspension) was mixed with 345 μl of PBS and 350 μl of RIBI adjuvant (RIBI Immunochemicals, Hamilton, N.H.). The resulting suspension contained 50 μg / ml of PepG. Each mouse was immunized with a subcutaneous (sc) dose of 0.1 ml (5 μg per mouse).

[0113] Approximately four weeks following the initial immunization, a booster immunization was given. PBS (873 μl) was mixed with 7.1 μl of PepG (7 mg / ml suspension) and 120 μl of Alhydrogel (Accurate Chemical and Scientific, Co., Westbury, N.Y.). Each mouse received an sc dose of 0...

example 2

Production of Hybridomas and Monoclonal Antibodies to B. subtilis PepG Purification of Peptidoglycan

[0122]Bacillus subtilis HR (gift of Howard Roger, University of Kent, UK) vegetative cell walls were made as previously described under stringent conditions, using lipopolysaccharide-free materials (6, 38), which are herein incorporated for any purpose). Proteins were removed from the peptidoglycan by treatment with pronase, and teichoic acid and other attached polymers were removed by treatment with HF (48% v / v) for 24 h at 4° C. The insoluble peptidoglycan was pelleted by centrifugation (13,000 g, 5 min, 4° C.) and resuspended in distilled water to 2 mg / ml PepG. This step was repeated once. The peptidoglycan was then pelleted and resuspended in 50 mM Tris HCl pH7.5 to 2 mg / ml PepG, and this step was repeated once. Finally, the peptidoglycan was pelleted and resuspended in distilled water to 2 mg / ml PepG three more times. The peptidoglycan was resuspended at about 10 mg / ml in distil...

example 3

Substrate Affinities of PepG Antibodies

Cellosyl Digestion of PepG

[0132] PepG from Bacillus subtilis, Staphylococcus aureus, Streptococcus mutans, Bacillus megaterium, Enterococcus faecalis, Staphylococcus epidermidis, and Listeria monocytogenes was purified as described in Example 2. One milliliter of each PepG (10 mg / ml) in 25 mM sodium phosphate buffer pH5.6 was digested with 250 μg / ml cellosyl (Hoechst AG) for 15 hours at 37° C. The samples were boiled for 3 minutes to stop the reaction and insoluble material was removed by centrifugation (14,000×g for 8 minutes at room temperature). The soluble cellosyl-digested PepG was stored at −20° C.

[0133] Staphylococcal PepG has a unique pentaglycine crossbridge, which can be cleaved by lysostaphin, a glycine-glycine endopeptidase. Lysostaphin (25 μg / ml; Sigma Cat No. L0761) was added to the cellosyl digestion of S. aureus and S. epidermidis PepG to cleave this peptide cross bridge. S. aureus was also digested without lysostaphin.

ELI...

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Abstract

The present invention encompasses protective monoclonal antibodies that bind to peptidoglycan of Gram-positive bacteria. The antibodies also bind to whole bacteria and enhance phagocytosis and killing of the bacteria in vitro and block nasal colonization by Gram-positive bacteria in vivo. The invention also provides human, humanized and chimeric antibodies. The invention also sets forth the heavy chain and light chain variable regions of an antibody within the invention.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation application of U.S. application Ser. No. 10 / 323,903, filed Dec. 20, 2002; which is a non-provisional application of U.S. Provisional Application No. 60 / 343,444, filed Dec. 21, 2001. [0002] This application is related to U.S. patent application Ser. No. 09 / 097,055, filed Jun. 15, 1998, and to U.S. Patent Application No. 60 / 341,806, and the application entitled, Methods for Blocking or Alleviating Staphylococcal Nasal Colonization by Intranasal Application of Monoclonal Antibodies filed on Dec. 20, 2001, and to U.S. Pat. Nos. 5,571,511 and 5,955,074. The entire contents of each of the above mentioned applications and patents are hereby incorporated by reference in their entirety.DESCRIPTION OF THE INVENTION [0003] 1. Field of the Invention [0004] This invention in the fields of immunology and infectious diseases relates to protective antibodies that are specific for Gram-positive bacteria, particularly t...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/40A61K39/02A61K39/07C07K16/12A61K39/085A61K39/09A61K39/00
CPCA61K39/00A61K2039/505C07K16/1271C07K2319/30C07K16/1278C07K2317/24C07K2317/77C07K16/1275
Inventor SCHUMAN, RICHARD F.KOKAI-KUN, JOHN FITZGERALDFOSTER, SIMON J.STINSON, JEFFREY R.FISCHER, GERALD WALTER
Owner BIOSYNEXUS INC
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