Monoclonal antibodies that are cross-reactive against bacterial collagen binding proteins

#The present invention relates in general to monoclonal antibodies that have been generated against collagen binding proteins and peptides from bacteria such as Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium, as well as streptococcal bacteria, and in particular to monoclonal antibodies against certain peptide fragments from the collagen binding domains from these proteins such as ACE19 and ACE40 which evidence cross-reactivity across species, as well as their use in treating or preventing bacterial infections.

#The magnitude of gram-positive nosocomial infections has been documented extensively in both the scientific literature as well as in the lay press over the past two decades. Since staphylococci account for the single largest cause of nosocomial infections they have been the focus of most reports. Traditionally, the generic antibiotic vancomycin has been the drug of choice to treat gram-positive infections. However, the continued rise in the prevalence of methicillin resistant S. aureus (MRSA) and the emergence of vancomycin resistant isolates of S. aureus from intensive care units from around the world has served as a rallying point for the clinical community, biopharmaceutical companies, and governmental agencies to develop novel therapeutics. The continued overuse of vancomycin has not only led to the development of resistant S. aureus strains, but it has also resulted in the emergence of resistant strains of enterococci. In 1986, the first clinical isolates of Enterococcus faecium were reported in France. A decade later, vancomycin-resistant enterococci (VRE) have reported in 18 countries and 6 continents. The problem in the United States is extremely troublesome, were >20% of enterococcal isolates reported in the 1998 report of the National Nosocomial Infectious Surveillance System (NNIS) hospitals were vancomycin resistant. The resistant rate was >50% higher than that reported for the same hospitals from the period 1993-1997. Enterococci now account for 10% of all nosocomial bloodstream and 20% of cardiovascular infections in the U.S. Moreover, VRE tend to be concomitantly resistant to moderate to high levels of penicillins and aminoglycosides and therefore must be treated with unproven combinations of antibiotics. Even with the recent introductions of linezolid and quinupristin/dalfopristin for the treatment of certain types of VRE infections, a significant gap in the therapeutic armamentarium of the clinician exists. These data indicate that the development of novel therapies that can prevent infection in a prophylactic manner or enhance current treatment modalities or are warranted.

#In the U.S., VRE infections are typically seen in moderately to severely ill patients. Therefore, it makes sense that most data detailing the VRE infections come from acute-care hospitals, specifically ICUs, oncology wards, and transplantation units. Host factors attributed with VRE infections include advanced age, APACHE score, neutronpenia, hematological malignancy, and prior nosocomial infection. Prolonged antibiotic exposure to vancomycin has also been associated as a risk factor for VRE infection. The most significant risk factors are length of hospital stay, proximity to a patient colonized or infected with VRE, and severe underlying illness.

#FIG. 1 is a graphic representation of the binding properties of an anti-ACE40 monoclonal antibody to E. faecium.

#FIG. 2 is a graphic representation of the binding properties of an anti-ACE19 monoclonal antibody to E. faecium.

#FIG. 3 is a graphic representation of the binding properties of an anti-ACE40 monoclonal antibody to E. faecalis.

#In accordance with the present invention, there are provided cross-reactive monoclonal antibodies that have been generated from regions of the ACE protein, which is a 74 kDa protein which has a structural similarity to that of MSCRAMM® proteins from other Gram-positive bacteria. The ACE protein of the invention is an extracellular matrix-binding protein of enterococcal bacteria such as Enterococcus faecalis, which can bind with collagens such as collagen type I and type IV and with laminin, and has the sequence as disclosed in PCT publication, WO00/68242, incorporated herein by reference. The collagen-binding Ace protein from Enterococcus faecalis has the amino acid sequence set forth herein as SEQ ID No. 1, and the nucleic acid sequence encoding the Ace protein is set forth herein as SEQ ID No. 2.

#As discussed further herein, certain regions of the ACE protein have been identified, and these include regions known as the A domain (or ACE40) at amino acids 32-367 of the E. faecalis ACE protein, and fragment ACE19 located at amino acids 174-319 of the ACE protein. In accordance with the present invention, there are provided monoclonal antibodies which are generated from and which can recognize can bind to ACE A domain (ACE40) and ACE19 fragment, and these generated monoclonal antibodies have been isolated and purified by the present inventors and shown to have cross-reactive properties as set forth below. The monoclonal antibodies in accordance with the invention can be used to treat or protect against infections by enterococcal and staphylococcal bacteria as discussed further below. In addition, there are provided cross-reactive monoclonal antibodies that have been generated from regions of the CNA protein, another MSCRAMM® protein from the Gram-positive bacteria, Staphylococcus aureus. The collagen binding protein identified as CNA has been disclosed, e.g., in WO97/43314, incorporated herein by reference, and monoclonal cross-reactive antibodies of the invention are generated from fragments such as CNA19, amino acids 151-318 of the CNA protein. As set forth below, these cross-reactive monoclonal antibodies can be used to treat or prevent a wide variety of bacterial infections.

#In accordance with the invention, the cross-reactive monoclonal antibodies of the invention may be prepared in a number of suitable ways that would be well known in the art, such as the well-established Kohler and Milstein method described above which can be utilized to generate monoclonal antibodies. In one such suitable method, mice are injected intraperitoneally once a week for a prolonged period with a purified recombinant protein such as ACE40, ACE19 or CNA19 as described above, followed by a test of blood obtained from the immunized mice to determine reactivity to the purified protein or fragment. Following identification of mice reactive to the tested protein, lymphocytes isolated from mouse spleens are fused to mouse myeloma cells to produce hybridomas positive for the antibodies against these proteins which are then isolated and cultured, following by purification and isotyping.