WO2008089476A1 - Monoclonal antibodies recognizing the enterococcus faecium acm protein - Google Patents

Abstract

Monoclonal antibodies which can bind to the ACM protein of Enterococcus faecium are provided which can be useful in the treatment and protection against infection from enterococcal bacteria such as E. faecium and other bacteria from the genus Enterococcus. The monoclonal antibodies of the invention can be prepared from the ACM protein or from its ligand binding A domain, and these antibodies can also be useful in diagnosing infections caused by E. faecium. Suitable pharmaceutical compositions based on the monoclonal antibodies of the invention, as well as methods for their use, are also provided.

Description

MONOCLONAL ANTIBODIES RECOGNIZING THE ENTEROCOCCUS FAECIUM ACM PROTEIN

Cross Reference to Related Applications The present application claims the benefit of U.S. provisional applications Ser.

No. 60/886,125, filed January 23, 2007, and Ser. No. 60/885,693, filed January 19, 2007, both of said applications incorporated herein by reference.

Field of the Invention The present invention relates to the fields of microbiology, molecular biology, and immunology and more particularly relates to newly identified monoclonal antibodies, the use of monoclonal antibodies, as well as the production of such monoclonal antibodies and recombinant host cells transformed with the DNA encoding monoclonal antibodies to prevent, treat, or diagnose Enterococcus faecium infections in man and animals. The invention includes murine, chimeric, humanized, and human monoclonal antibodies, as well as fragments, regions and derivatives thereof. The antibodies detailed in this invention specifically a recognize Enterococci MSC RAM M^® protein, ACM expressed on the surface of E. faecium strains. In addition, the invention comprises monoclonal antibodies that bind to epitopes recognized by the manufactured monoclonal antibodies as set forth herein.

Background of the Invention

The Enterococci are members of the normal intestinal flora of man and animals have long been recognized as a common cause of endocarditis, and are now well recognized as important opportunistic pathogens, ranked third among the organisms isolated from nosocomial infections (Murray, 1990; Tannock and Cook, 2002). Among the Enterococcal species described, Enterococcus faecalis and Enterococcus faecium represent over 90% of clinical isolates and cause a wide spectrum of diseases, including intra-abdominal and surgical wound infections, urinary tract infections, catheter-related bacteremia and central nervous system infections, among others. Many of these infections, as well as endocarditis, are probably dependent upon the ability of the infecting bacterium to adhere to the layer of extracellular matrix (ECM) proteins exposed after tissue damage. Recently, E. faecium infections have become a life threatening challenge to clinicians because of the accumulation of multiple antibiotic resistances, including ampicillin and vancomycin (Murray, 2000). Enterococcus faecium, a normal inhabitant of the human gut, can cause infection when the organism spreads beyond its original niche into the bloodstream. Thus, adherence to colonic epithelium, to urinary epithelium or heart valves by Enterococci is likely to be a key to subsequent infections, which may involve some kind of adhesin via ECM proteins.

Colonization of host tissue is considered a vital step in the bacterial infection process (Patti et al, 1994a; Foster and Hook, 1998). Most pathogenic bacteria have been shown to recognize and adhere to various components of the ECM, thus leading to colonization of the host tissue. ECM is a stable macromolecular structure consisting of a complex mixture of glycoproteins and proteoglycans including collagens, laminin and fibronectin, which provides structural support to epithelial and endothelial cells in addition to other roles (Westerlund and Korhonen, 1993). Any trauma that damages host tissue integrity exposes underlying ECM proteins, thus making them accessible for bacterial adhesion. The major structural protein of the ECM is collagen, the single most abundant protein in vertebrates. There are at least 20 different collagens that are characterized by the formation of triple helices (consisting of repeats of the amino acid sequence GIy-X-Y).

A subfamily of bacterial surface adhesins, collectively known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), which specifically bind to host ECM, has been identified in a number of Gram-positive bacteria (Patti and Hook, 1994). MSCRAMMs that adhere to host collagen, fibronectin, fibrinogen and/or laminin have been isolated and the corresponding genes well characterized (Jonsson et al., 1991 ; Patti et al., 1992; Cheung et al., 1995; Rich et al., 1999; Holmes et al., 2001 ; Terao et al., 2002). In some cases, the primary binding sites in these MSCRAMMs have been localized to specific domains of the respective adhesins (Patti et al., 1993; Boden and Flock, 1994; Courtney et al., 1996; Hartford et al., 1999; Rich et al., 1999). Similar to other Gram-positive pathogens, opportunistic E. faecium possess genes such as ACM that code for a distinct surface protein that may play a role in bacterial colonization, multiplication within the host, assisting in evasion of host defenses which all lead to clinical infection of the host.

However, the role of such surface proteins has not been completely determined, and thus in many cases it is not known whether any particular surface proteins will be useful in methods of fighting or diagnosing infections. It is further not known whether monoclonal antibodies can be generated from such surface proteins, and if so, which of those monoclonals will be effective tools against infection. With regard to the ACM surface protein from E. faecium, it has not heretofore been known to generate monoclonal antibodies from said surface protein, and it thus remains a challenge and a desirable object to obtain effective methods of treating and/or preventing enterococcal infections utilizing these surface proteins, and to obtain monoclonal antibodies which recognize such surface proteins so as to be useful in treating and/or preventing infections from these bacteria.

Summary of the Invention

Accordingly, it is an object of the present invention to provide monoclonal antibodies that can bind to the ACM surface protein from Enterococcus faecium with high affinity and which can thus be useful in methods to treat, prevent or diagnose enterococcal infections.

It is also an object of the present invention to provide monoclonal antibodies which are able to bind ACM and which are generated from the ACM or its binding domain or A domain, and which can be utilized in methods of treating or protecting against enterococcal infections. It is also an object of the present invention to provide monoclonal antibodies which recognize the ACM protein, and which can also recognize subdomains of ACM, including the A domain covering the N1 , N2 and N3 subregions, as well as the N1 , N2, truncated N2, N3, N1 N2, truncated N2N3, and N2N3 subdomains

It is also an object of the present invention to provide monoclonal antibodies to the ACM protein which can be useful in inhibiting the binding of ACM to collagen.

It is a further object of the present invention to provide antibodies and antisera which recognize the binding domain of the ACM protein and which can be useful in methods of treating, preventing, identifying or diagnosing enterococcal infections.

These and other objects are provided by virtue of the present invention which comprises the generation and use of monoclonal antibodies which can recognize the

ACM protein from Enterococcus faecium, or its ligand binding A domain, for the treatment or prevention of Enterococcal infections. In accordance with the invention, suitable compositions and passive vaccines based on the monoclonal antibodies of the invention, as well as methods for their use, are also provided as set forth in the detailed description hereinbelow.

Brief Description of the Drawing Figures

Figure 1 is a graphic representation showing the inhibition of ACM-collagen binding in the present of anti-ACM Mabs. Detailed Description of the Preferred Embodiments

In accordance with the present invention, there are provided monoclonal antibodies which can recognize and bind to the extracellular MSC RAM M® protein ACM, a surface localized protein from E. faecium, and subregions included therein including the N1 , N2 and N3 regions, which together form the ligand binding A domain of ACM, and other subdomains and combinations of these regions. In the preferred method of generating these monoclonal antibodies, they are raised against an E. coli expressed and purified A domain (N1 N2N3) of the ACM protein used to generate a panel of murine monoclonal antibodies. However, monoclonal antibodies recognizing ACM or its subregions can be raised from other subregions or larger parts of the protein as long as they are immunogenic and will be able to generate antibodies that recognize ACM and/or its subregions.

The ACM protein from Enterococcus faecium is well known and has been disclosed in the literature, including Nallapareddy et al., "Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family", Molecular Microbiology 47 (6), 1733-1747 (2003), said article incorporated herein by reference. Additional information regarding the ACM protein is set forth in Nallapareddy et al., "Inhibition of Enterococcus faecium Adherence to Collagen by Antibodies against High-Affinity Binding Subdomains of Acm, Infect Immun. 75(6): 3192-3196 (2007), said article incorporated herein by reference. As indicated in these references, ACM is a well characterized protein having binding region subdomains of the type observed with other MSCRAMMΘs, including in particular the CNA protein of Staphylococcus aureus, and has at least those binding subdomains as set forth in the Nallapareddy 2007 article cited above. The ACM protein is coded by a nucleotide sequence (SEQ ID NO:1 ) as set forth below (or degenerates thereof):

TCTAAATAAA GAAACAGATG TTTCTTTATA TTGTATTTTA TCCTTTAATT AACACAAAAA

CAGCTAGTAT AAACTAATAA TGTTAATTAA AAGTGTATTT TATGAAACTA TCGTTATTTA

TTAATGTTGC ATTTTTGTAA GTTAAAAATT GGAAATATAG ATTGAAAAAA TGAAAGCAAT AAGTAAAATA AAATGAATAT ATGCGAAAAA ATAATTTACA TTAGGAAAAA CCTTGTAAAA

ACAGTTTTTT GGCATATTTA TTTTTTATAA ATATTTGTAA AATAACTATC AATTTTTTAG

TTAAAAGATT GGAAATGAGT GGGGGAGGAA TTGAAGAAAG GATTTTTGAG AGATGATATA

GTAGGTTTTA TTTTTAGTTA AAATAGGGGA GATAGAAAAA ATGAAAAAGT GTAGAAATAT

TATTTTATCA ATGTTATTTA TTATTACTAA TATAACATCA CTGATTCCTG TTCATGTCTA TGCGGATGCA GGCAGAGATA TCAGCAGTAA TGTCACTTCG TTGACCGTGG ACCCAACGAA

TATTACAGAT GGAGGAAACA TAAAAGTAAA ATTCTCGTTT GACGAAAAAA AACAAAATAT

CCAACCAGGT GATTATCTTT GGATCAATTG GCCAAGTGAA GGAAATATTC GCGGTGAAGG

ATTCCAAAAA GAAATCCCAT TAATGATTGA AAATAAAAAT GTTGGAACTT TAACGGTCAG

AAAAGATTCT GCGCAGGTTG TGTTTAATGA AAATATTAAA AACCTAGACT CAGTAGAAGG TTGGGGGCAA TTTGAGATAC AGGCCAGAAA CGTAACCGAT ACCGGTGAAG AAAATACGGG

ACCATTCACC GTGACGAGCG GTGATAAAAC AGCTACTGTA AATGTGACAA AGCCAGCTTC

TGGTTCTTCT TCTAGCGTTT TTTATTATAA AACAGGAGAC ATGCTACCTG AAGATACTAA GCACATTCGA TGGTTTTTGA ATATTAACAA TAACGGCACT TATGTTGAAC AACCAGTTAA

AATCAGTGAT GAAATCCAAA GTGGACAAAG ATTAGATCCA AGTACGTTTG AAATTAATCA

AATACACTTA GGTGAACAGA AGGTTTATAG AGGAGAAGAG GGAATCCAAC AATTTTTACA

AGATTTCCCC AGCGCTACAT TTAATTTTTC AGTAACTGAT AATTACATTG AAATAACTAT ACCAAAAAAC TTTGTCAATC TTAGGAAAAT AATGGTGAGT TATAAGACGA TCATAGAAAA

CCCAGAACAA ATAAATTTTG AGAATCATTC GGAAGCTTGG TTTAAAGAAT TTAATAAACC

TGCTGTTGAT GGAGAAAGTT TCAATCATAC AGTTAAAAAT ATTTCAGCTT CTGGTGGAGT

CAACGGTACA GTTAGAGGCG AATTGAAGAT TTTCAAATAT ATCAATGACA CAGAGATTGG

TATCCCAAAT GTGACTTTCG AATTAAGACG AGCAGATGAA CAGCCTATTC AAGGACAGTC TAGTATCCTG TTGACTTCGA ATGAACAGGG AGAAGCTAGT ATCAAAGGCC TGCAAGTAGG

AGACTATGTT GTAAAAGAAA AAGAAGCACC CAATTGGATT GATTTTGATC CACTAAGTTC

AAATGAACTC AAATTCTCCA TAAACGAAAA TGATACAGAG GGAGTCAGTT TACCTATTTA

TAACAAGAAA AAGGTTACGA ACATCACAGC AACTAAAATT TGGAACGGTG GAACCACTCC

AAGACCGAGT ATTTATTTTA AATTATTCAG AGCAAGTACA AACAATTGGG AACCAGTTCC TGATGCAGAA ACAAAACGAT TAGACAATGG AATAACTTCA GTGACTTGGG AAGATATCCA

ACAGTATGAT GATAGTGGAA ACGAATATAC GTTTAAAGTG CAAGAAGTAG ATCAAAACGG

AAATGATTAT GTCCCTTCAG GTTATCAAAA AATTGAAAAC GGGCTAGTCG TTACAAATGA

GAATAAAGAA GTCATTTCAG TCTCAGGACA AAAAACATGG GAAGACAATG AGAACCAAGA

CGGCAAACGA CCGACGACGA TCACAGTAAA TCTATTAGCC GACGGTCAAC CGATCCAACA CAAAGAAGTA AGTGAAAAAG ATGATTGGCG CTATCGTTTC ACAAATCTGC CAAAATACAG

AGACGGGCAA GAAATCATCT ATACTGTAAC GGAAGACAAC GTGCCCGAAT ACAGTACGAC

GATTGACGGG TATAACATCA AAAACAGTTA TACACCGGAA AAAACAAATA TATCTATACC

AGGTAGCCAT ATAACACCGT GGAAAAATTT TCCAGAGAAG ACAGAGAGAG AGAAAATTAG

AAATCTATTC TCTCAAAAAT TGCAAATATT TGCTCATACA GGAGAATCCA GCGAACAAAG AAGCAATTAT ATTAGTAAGA GAGCTGTACC TAAACAGATA ACAAATAAAT CAAAAAGCAT

TCTTCCTAAA ACAAGTAGTA AAAAATCTAG TTTTGCTGTA ATCATTGGTT TATTGATAGT

AACTATCTGT GCTGGGATAT TCTTACAAAA ATATAAAAAA GTTTAGTTAG AATATAATAT

TTTTTGTTAA ATATGACTGA AATCAAAAGA ATAAAATTCC GATTTATAAT CGAAAAAAGA

AGCATTTTTA TATATGCTTC TTTTTTCGTA GAATCAATAA ATATGGATTT GGCTAACTAT

The full ACM protein itself has the amino acid sequence (SEQ ID NO:2) as set forth below:

MKKCRNIILSMLFIITNITSLIPVHVYADAGRDISSNVTSLTVD

PTNITDGGNIKVKFSFDEKKQNIQPGDYLWINWPSEGNIRGEGFQKEIPLMIENKNVG

TLTVRKDSAQWFNENIKNLDSVEGWGQFEIQARNVTDTGEENTGPFTVTSGDKTATV

NVTKPASGSSSSVFYYKTGDMLPEDTKHIRWFLNINNNGTYVEQPVKISDEIQSGQRL

DPSTFEINQIHLGEQKVYRGEEGIQQFLQDFPSATFNFSVTDNYIEITIPKNFVNLRK IMVSYKTIIENPEQINFENHSEAWFKEFNKPAVDGESFNHTVKNISASGGVNGTVRGE

LKIFKYINDTEIGIPNVTFELRRADEQPIQGQSSILLTSNEQGEASIKGLQVGDYVVK

EKEAPNWIDFDPLSSNELKFSINENDTEGVSLPIYNKKKVTNITATKIWNGGTTPRPS

IYFKLFRASTNNWEPVPDAETKRLDNGITSVTWEDIQQYDDSGNEYTFKVQEVDQNGN DYVPSGYQKIENGLVVTNENKEVISVSGQKTWEDNENQDGKRPTTITVNLLADGQPIQ HKEVSEKDDWRYRFTNLPKYRDGQEIIYTVTEDNVPEYSTTIDGYNIKNSYTPEKTNI SIPGSHITPWKNFPEKTEREKIRNLFSQKLQIFAHTGESSEQRSNYISKRAVPKQITN KSKSILPKTSSKKSSFAVIIGLLIVTICAGIFLQKYKKV As indicated in the Nallapareddy 2007 article referred to above, the ACM protein has several well-defined subdomains, including regions N1 (aa 29-150), N2 (aa 151 - 346), and N3 (347-529), which collectively form the A domain, and other subregions can be constructed including one identified as the truncated N2 region (aa 151 -320) and other constructs that can be prepared based on combinations of subdomains including N1 N2 (29-346), N1 N2 truncate (aa 29-320) and N2N3 (151 -529).

The ACM A domain is coded by a nucleotide sequence (SEQ ID NO:3) as set forth below (or degenerates thereof) including an initial sequence (bolded and underlined) coding for a 6-His tag used for purification :

AAAATTGAAAACGGGCTAGTCGTTACAAATGAGAATAAATAG

The ACM A domain has the amino acid sequence (SEQ ID NO:4) as set forth below which includes the 6-His tag (bolded and underlined) for the purification step as described below: MRGSHHHHHHGSDAGRDISSNVTSLTVDPTNITDGGNIKVKFSFDEKKQNIQPGDYLWINWPSEG NIRGEGFQKEIPLMIENKNVGTLTVRKDSAQWFNENIKNLDSVEGWGQFEIQARNVTDTGEENTGPFTV TSGDKTATVNVTKPASGSSSSVFYYKTGDMLPEDTKHIRWFLNINNNGTYVEQPVKISDEIQSGQRLDPS TFEINQIHLGEQKVYRGEEGIQQFLQDFPSATFNFSVTDNYIEITIPKNFVNLRKIMVSYKTIIENPEQI NFENHSEAWFKEFNKPAVDGESFNHTVKNISASGGVNGTVRGELKIFKYINDTEIGIPNVTFELRRADEQ PIQGQSSILLTSNEQGEASIKGLQVGDYWKEKEAPNWIDFDPLSSNELKFSINENDTEGVSLPIYNKKK VTNITATKIWNGGTTPRPSIYFKLFRASTNNWEPVPDAETKRLDNGITSVTWEDIQQYDDSGNEYTFKVQ EVDQNGNDYVPSGYQKIENGLWTNENK

Once the His tag is removed, the ACM A domain has the amino acid sequence (SEQ ID NO:6) as set forth below: DAGRDISSNVTSLTVDPTNITDGGNIKVKFSFDEKKQNIQPGDYLWINWPSEGNIRGEGFQKEIP LMIENKNVGTLTVRKDSAQWFNENIKNLDSVEGWGQFEIQARNVTDTGEENTGPFTVTSGDKTATVNVT KPASGSSSSVFYYKTGDMLPEDTKHIRWFLNINNNGTYVEQPVKISDEIQSGQRLDPSTFEINQIHLGEQ KVYRGEEGIQQFLQDFPSATFNFSVTDNYIEITIPKNFVNLRKIMVSYKTIIENPEQINFENHSEAWFKE FNKPAVDGESFNHTVKNISASGGVNGTVRGELKIFKYINDTEIGIPNVTFELRRADEQPIQGQSSILLTS NEQGEASIKGLQVGDYWKEKEAPNWIDFDPLSSNELKFSINENDTEGVSLPIYNKKKVTNITATKIWNG GTTPRPSIYFKLFRASTNNWEPVPDAETKRLDNGITSVTWEDIQQYDDSGNEYTFKVQEVDQNGNDYVPS GYQKIENGLWTNENK

This amino acid sequence can thus be coded by the nucleotide sequence included below (or degenerates thereof) which is identified as SEQ ID NO:5:

AAATAG

Accordingly, in one aspect, the present invention provides monoclonal antibodies which recognize the ACM protein from E. faecium and/or which can be raised against or which can recognize the other subdomains and subregions of the ACM protein including the A domain (N1 N2N3) and the other subregions, constructs and combinations as set forth above. In addition, the invention relates to monoclonal antibodies which can be useful in inhibiting the ACM binding to collagen and in inhibiting the binding of E. faecium to host cells, and which can thus be useful in methods of treating, preventing or diagnosing enterococcal infections. Even further, the present invention contemplates other monoclonals that will bind to epitopes recognized by the specific monoclonals described hereinbelow. In the preferred method of making monoclonal antibodies in accordance with the invention, these antibodies may be obtained in conventional ways including steps of introducing the ACM and/or its binding subdomains such as the A domain into a host animal, followed by isolation of sera and formation of a suitable hybridoma. In one suitable method, the proteins of the invention were produced and purified using standard PCR techniques in which the A domain of ACM was amplified from E. faecium TX2555 genomic DNA (from sequences described above) and subcloned into a suitable E. coli expression vector such as PQE-30 (QIAGEN®), which allows for the expression of a recombinant fusion protein containing six histidine residues. After expression either by shake flasks or in bioreactors, the cells were harvested by centrifugation and the cell paste frozen such at around -80° C. Cells can be lysed in 1 X PBS (1 OmL of buffer/1 g of cell paste) using multiple passes through a microfluidizer at 10,000 psi. Lysed cells can then be spun down at 17,000rpm for 30 minutes to remove cell debris. Supernatant could then be passed over a 5-mL HiTrap Chelating (Pharmacia) column charged with 0.1 M NiCI₂. After loading, the column can be washed with 5 column volumes of 1 OmM Tris, pH 8.0, 10OmM NaCI (Buffer A). Protein can be eluted using a 0-100% gradient of 1 OmM Tris, pH 8.0, 10OmM NaCI, 50OmM imidazole (Buffer B). Fractions containing ACM can then be dialyzed such as in 1 x PBS. In such a process, it is also preferred that the resulting ACM protein be put through an endotoxin removal protocol. Buffers that can be used during this protocol include those which can be made endotoxin free by passing over a 5-mL Mono-Q SEPHAROSE® (Pharmacia) column. Protein can be divided evenly between 4x 15mL tubes, the volume of which may be brought to 9mL with Buffer A. 1 mL of 10% TRITON® X-1 14 can be added to each tube and incubated with rotation for 1 hour at 4° C. Tubes can be placed in a 37° C water bath to sparate phases and can be spun down at 2,000rpm for 10 minutes to allow the upper aqueous phase from each tube to be collected and the detergent extraction repeated. Aqueous phases from the 2nd extraction can be combined and passed over a 5-mL IDA chelating (SIGMA®) column, charged with 0.1 M NiCI₂ to remove remaining detergent. The column can be washed with 9 column volumes of Buffer A before protein elution which can be carried out with 3 column volumes of Buffer B. The eluant can be passed over a 5-mL Detoxigel (Sigma) column and the flow-through collected and reapplied to the column. The flow-through from the second pass can be collected and dialyzed in 1 x PBS. The purified product can then be isolated and should be analyzed for concentration, purity and endotoxin level before administration into mice for polyclonal or monoclonal antibody production.

In accordance with the present invention, the next step would be to prepare monoclonal antibodies against the purified ACM protein, preferably using techniques by which mAbs could be generated that are of high affinity, that are able to interrupt or restrict the binding of extracellular matrix proteins (ECM), and which demonstrate therapeutic or diagnostic efficacy. In one such method, E. coli expressed and purified ACM protein was used to generate a panel of murine monoclonal antibodies. Briefly, a group of Balb/C or SJL mice received a series of subcutaneous immunizations of 1 -10 mg of protein in solution or mixed with a suitable adjuvant in a series of injections. Seven days after a boost, serum was collected and titered in ELISA assays against in immunizing ACM protein. Three days after the final boost, the spleen was removed, teased into a single cell suspension and the lymphocytes harvested. The lymphocytes were then fused to a P3X63Ag8.653 myeloma cell line (ATCC #CRL-1580). Cell fusion, subsequent plating and feeding were performed according to the Production of Monoclonal Antibodies protocol from Current Protocols in Immunology (Chapter 2, Unit 2.), said reference work incorporated herein by reference. Any hybridoma clones generated from the ACM fusions were screened for specific anti-ACM antibody production using a standard ELISA assay. Positive clones were expanded and tested further for activity in a whole bacterial cell binding assay by flow cytometry and ACM binding by Biacore analysis. In this analysis, lmmulon 2-HB high-binding 96-well microtiter plates (Dynex) were coated with 1 μg/well of ACM in 1 X PBS, pH 7.4 and incubated for 2 hours at room temperature. All washing steps in ELISAs were performed three times with 1 X PBS, 0.05% Tween-20 wash buffer. Plates were washed and blocked with a 1 % BSA solution at room temperature for 1 hour before hybridoma supernatant samples were added to wells. Plates were incubated with samples and relevant controls such as media alone for one hour at room temperature, washed, and goat anti-mouse IgG-AP (Sigma) diluted 1 :5000 in 1 X PBS, 0.05 % Tween-20, 0.1% BSA was used as a secondary reagent. Plates were developed by addition of 1 mg/ml solution of 4-nitrophenyl phosphate (pNPP) (Sigma), followed by incubation at 37° C for 30 minutes. Absorbance was read at 405 nm using a SpectraMax 190 Plate Reader (Molecular Devices Corp.). Antibody supernatants that had an OD₄₀S > 3 times above background (media alone, ~ 0.1 OD) were considered positive. Throughout the analysis, the flow rate remained constant at 10 ml/min. Prior to the ACM injection, test antibody was adsorbed to the chip via RAM-Fc binding. At time 0, ACM at a concentration of 30 μg/ml was injected over the chip for 3 min followed by 2 minutes of dissociation. This phase of the analysis measured the relative association and disassociation kinetics of the mAb / ACM interaction.

Bacterial samples were collected, washed and incubated with mAb or PBS alone (control) at a concentration of 2 μg/ml after blocking with rabbit IgG (50 mg/ml). Following incubation with antibody, bacterial cells were incubated with Goat-F_(ab'₎₂-Anti- Mouse-F₍ab'₎₂-FITC which served as the detection antibody. After antibody labeling, bacterial cells were aspirated through the FACScaliber flow cytometer to analyze fluorescence emission (excitation: 488, emission: 570). For each bacterial strain, 10,000 events were collected and measured. Tests from this analysis showed that ACM positive hybridomas were generated in a frequency of 0.7 - 2.1 % of the growth positive wells. Interestingly, most of the ACM Biacore positive hybridomas were also positive by on whole cells. Positive examples (by ELISA, Biacore and Flow) were also shown. To determine that the anti-ACM mAbs generated and selected with recombinant

ACM cross-reacted with native ACM expressed on Enterococcus strains of bacteria, flow cytometric analysis was used. Anti-ACM mAbs varied in reactivity to E. faecium strains. Single cell cloned mAbs with high affinity activity against ACM as suggested by the Biacore analysis, demonstrated remarkable activity in the inhibition of ACM - Collagen binding as well as binding to whole Enterococcus bacteria. In such tests, lmmulon 2-HB high-binding 96-well plates were coated with 2.5 μg/ml ACM in PBS and incubated 2 hours at room temperature. Plates were washed and blocked with 1% BSA solution for 1 hour, then washed and incubated with monoclonal antibody (either hybridoma supernatant or purified antibody) for 1 hour at room temperature. Following incubation with antibody, plates were either washed or left untreated, and 2μg/ml solution of human type I collagen was added. Plates were incubated 1 hour at 37 ⁰C and washed. Detection was performed with 1 :2,000 diluted biotinylated rabbit anti- collagen Ab (Rockland) and 1 :8,000 diluted HRP conjugated neutralite Avidin (Southern Biotech). Following incubation with conjugate, plates were washed and ABTS substrate was added. Plates then incubated 10 minutes at room temperature, the reaction was stopped with addition of 10% SDS, and absorbance was read at 405 nm. All data was analyzed using SOFTmax Pro v.3.1 .2. software (Molecular Devices Corp., Sunnyvale, California, USA). The data reflected that anti-ACM mAbs were able to inhibit ACM-collagen binding. In addition, tests were conducted to determine if the mAbs to ACM in accordance with the invention could bind to whole bacteria. In such tests, bacterial samples were collected, washed and incubated with mAb or PBS alone (control) at a concentration of 2 μg/ml. Following incubation with antibody, bacterial cells were incubated with Goat-F_(ab'₎₂-Anti-Mouse-F_(ab'₎₂-FITC which served as the detection antibody. After antibody labeling, bacterial cells were aspirated through the FACScaliber flow cytometer to analyze fluorescence emission (excitation: 488, emission: 570). For each bacterial strain, 10,000 events were collected and measured. An affinity determination was made using BIACORE® wherein Surface plasmon resonance (SPR) was used to test ELISA-positive clones for the ability to bind rACM protein. Kinetic analysis was performed on a BIACORE® 3000 (Biacore, Piscataway, NJ) using the ligand capture method included in the software. The anti-ACM mAbs were passed over a Goat anti-mouse-F(ab')₂ chip, allowing binding and capture via the Fc portion. Varying concentrations of the rACM protein were then passed over the chip, and data collected. Using the Biacore provided Evaluation software (Version 3.1 ), ko_n and k_Off were measured and K_A and K₀ were calculated. As a result of these tests, it was determined that the ACM mAbs of the present invention could bind to whole bacteria and thus be used in the inhibition of or interference with the binding of that bacteria to host cell. Accordingly, the present invention provides monoclonal antibodies which recognize the ACM protein and which can bind to E. faecium so as to be useful in methods of treating, preventing or diagnosing enterococcal infections. In addition, the invention provides monoclonals that can recognize subdomains of ACM, including the A domain, and the other subdomains, constructs and subregions as described herein such as N1 N2, N2N3, etc. Accordingly, the present invention contemplates these monoclonal antibodies, and other monoclonals recognizing the same epitopes of the specific monoclonals described herein.

Accordingly, the present invention relates to an isolated and/or purified monoclonal antibody which can bind to the ACM protein and/or their binding subdomains, and which thus can be useful in methods of inhibiting adherence of E. faecium to host cells and thus treat or prevent a enterococcal infection when used in amounts effective to prevent or treat such infections. In addition to the methods described above, these monoclonal antibodies may be produced using any of a variety of conventional methods, e.g., the method of Kohler and Milstein, Nature 256:495-497 (1975), or other suitable ways known in the field. In addition, it will be recognized that these monoclonals can be prepared in a number of forms, including chimeric, humanized, or human in addition to murine in ways that would be well known in this field. Still further, monoclonal antibodies may be prepared from a single chain, such as the light or heavy chains, and in addition may be prepared from active fragments of an antibody which retain the binding characteristics (e.g., specificity and/or affinity) of the whole antibody. By active fragments is meant an antibody fragment which has the same binding specificity as a complete antibody which binds to extracellular matrix binding proteins, and the term "antibody" as used herein is meant to include said fragments. Additionally, antisera prepared using monoclonal or polyclonal antibodies in accordance with the invention are also contemplated and may be prepared in a number of suitable ways as would be recognized by one skilled in the art.

Although production of antibodies as indicated above is preferably carried out using synthetic or recombinantly produced forms of the ACM protein or antigenic subregions therefrom, antibodies may also be generated from natural isolated and purified ACM proteins or subregions, or active fragments thereof. Still other conventional ways are available to generate the ACM antibodies of the present invention using recombinant or natural purified ACM proteins or their active regions, as would be recognized by one skilled in the art.

As would be recognized by one skilled in the art, the antibodies of the present invention may also be formed into suitable pharmaceutical compositions for administration to a human or animal patient in order to treat or prevent an infection caused by enterococcal bacteria. Pharmaceutical compositions containing the antibodies of the present invention, or effective fragments thereof, may be formulated in combination with any suitable pharmaceutical vehicle, excipient or carrier that would commonly be used in this art, including such conventional materials for this purpose, e.g., saline, dextrose, water, glycerol, ethanol, other therapeutic compounds, and combinations thereof. As one skilled in this art would recognize, the particular vehicle, excipient or carrier used will vary depending on the patient and the patient's condition, and a variety of modes of administration would be suitable for the compositions of the invention, as would be recognized by one of ordinary skill in this art. Suitable methods of administration of any pharmaceutical composition disclosed in this application include, but are not limited to, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal administration.

If topical administration is desired, the composition may be formulated as needed in a suitable form, e.g., an ointment, cream, gel, lotion, drops (such as eye drops and ear drops), or solution (such as mouthwash). Wound or surgical dressings, sutures and aerosols may be impregnated with the composition. The composition may contain conventional additives, such as preservatives, solvents to promote penetration, and emollients. Topical formulations may also contain conventional carriers such as cream or ointment bases, ethanol, or oleyl alcohol.

Additional forms of antibody compositions, and other information concerning compositions, methods and applications with regard to the MSC RAM M® ACM of the present invention can also be found from other patent references concerning other MSCRAMMΘs which will generally be applicable to the present invention as well, and these patents include U.S. Pat. Nos. 7,045,131 ; 6,994,855; 6,979,446; 6,841 ,154; 6,703,025; 6,692,739; 6,685,943; 6,680,195; 6,635,473; 6,288,214; 6,177,084; and 6,008,341 , all of said patents incorporated herein by reference.

The antibody compositions of the present invention which are generated against the N1 N2N3 regions from the ACM protein from E. faecium may also be administered with a suitable adjuvant in an amount effective to enhance the immunogenic response against the conjugate. For example, suitable adjuvants may include alum (aluminum phosphate or aluminum hydroxide), which is used widely in humans, and other adjuvants such as saponin and its purified component Quil A, Freund's complete adjuvant, RIBBI adjuvant, and other adjuvants used in research and veterinary applications. Still other chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman-Snitkoff et al. J. Immunol. 147:410-415 (1991 ) and incorporated by reference herein, encapsulation of the conjugate within a proteoliposome as described by Miller et al., J. Exp. Med. 176:1739-1744 (1992) and incorporated by reference herein, and encapsulation of the protein in lipid vesicles such as NOVASOME® lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH) may also be useful.

The antibody compositions of the present invention will thus be useful for interfering with, modulating, or inhibiting binding interactions between the ACM protein on enterococcal bacteria and its ligand on host cells and tissues, and will thus have particular applicability in developing compositions and methods of preventing or treating enterococcal infection, and in inhibiting binding of enterococcal bacteria to host tissue and/or cells.

In accordance with the present invention, methods are provided for preventing or treating a enterococcal infection which comprise administering an effective amount of the monoclonal antibody of the present invention as described above in amounts effective to treat or prevent the infection. In addition, these monoclonal antibodies have been shown to have high affinity in binding of enterococcal bacteria, and can bind whole bacteria, and thus should be effective in treating or preventing infection from bacteria such as E. faecium. Further, these monoclonals will be useful in inhibiting E. faecium binding to the extracellular matrix of the host, and in reducing or eliminating the adherence of E. faecium on host cells or on other surfaces, e.g., medical equipment, implants or prosthetics. The mAbs of the invention can thus be used to inhibit or interfere with the binding of the ACM protein to collagen, and thus to also inhibit or interfere with the binding of E. faecium to host cells. Accordingly, in accordance with the invention, administration of the antibodies of the present invention in any of the conventional ways described above (e.g., topical, parenteral, intramuscular, etc.), and will thus provide an extremely useful method of treating or preventing enterococcal infections in human or animal patients. By effective amount is meant that level of use, such as of an antibody titer, that will be sufficient to either prevent adherence of the bacteria, to inhibit binding of bacteria to host cells and thus be useful in the treatment or prevention of infection. As would be recognized by one of ordinary skill in this art, the level of antibody titer needed to be effective in treating or preventing enterococcal infection will vary depending on the nature and condition of the patient, and/or the severity of the pre-existing enterococcal infection. In addition to the use of antibodies of the present invention to treat or prevent E. faecium infection as described above, the present invention contemplates the use of these antibodies in a variety of ways, including the detection of the presence of E. faecium to diagnose infection, whether in a patient or on medical equipment, implants or prosthetics which may also become infected. In accordance with the invention, a preferred method of detecting the presence of infections involves the steps of obtaining a sample suspected of being infected by one or more enterococcal bacteria species or strains, such as a sample taken from an individual, for example, from one's blood, saliva, tissues, bone, muscle, cartilage, or skin. The cells can then be lysed, and the ACM A domain or fragments (N1 , N2, N3) can be extracted, precipitated or amplified. Following isolation of the sample, diagnostic assays utilizing the antibodies of the present invention may be carried out to detect the presence of E. faecium, and such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoasssay, Western blot analysis and ELISA assays. In general, in accordance with the invention, a method of diagnosing an E. faecium infection is contemplated wherein a sample suspected of being infected with E. faecium infection has added to it the monoclonal antibody in accordance with the present invention, and E. faecium is indicated by antibody binding to the ACM proteins in the sample.

Accordingly, antibodies in accordance with the invention may be used for the specific detection or diagnosis of enterococcal proteins, for the prevention of infection from bacteria, for the treatment of an ongoing infection, or for use as research tools. The term "antibodies" as used herein includes monoclonal, polyclonal, chimeric, single chain, bispecific, simianized, and humanized or primatized antibodies as well as Fab fragments, such as those fragments which maintain the binding specificity of the antibodies to the ACM proteins, including the products of an Fab immunoglobulin expression library. Accordingly, the invention contemplates the use of single chains such as the variable heavy and light chains of the antibodies as will be set forth below. Generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art. In the present case, monoclonal antibodies to ACM proteins have been generated against its ligand binding domain A (made up of subregions N1 , N2 and N3) and have been isolated and shown to have high affinity to E. faecium. Moreover, the monoclonals of the present invention have been shown to recognize a high number of strains, on an equivalent level to that recognize by polyclonal antibodies to ACM, and thus can be used effectively in methods to protect against enterococcal infection or treat same.

When so desired for medical or research purposes, any of the above described antibodies may be labeled directly with a detectable label for identification and quantification of bacteria. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).

Alternatively, the antibody may be labeled indirectly by reaction with labeled substances that have an affinity for immunoglobulin. The antibody may be conjugated with a second substance and detected with a labeled third substance having an affinity for the second substance conjugated to the antibody. For example, the antibody may be conjugated to biotin and the antibody-biotin conjugate detected using labeled avidin or streptavidin. Similarly, the antibody may be conjugated to a hapten and the antibody-hapten conjugate detected using labeled anti-hapten antibody. These and other methods of labeling antibodies and assay conjugates are well known to those skilled in the art.

Antibodies to ACM as described above may also be used in production facilities or laboratories to isolate additional quantities of the proteins, such as by affinity chromatography. For example, the antibodies of the invention may also be utilized to isolate additional amounts of the ACM proteins or their active fragments.

The isolated antibodies of the present invention, or active fragments thereof, may also be utilized in the development of vaccines for passive immunization against infections. Further, when administered as pharmaceutical composition to a wound or used to coat medical devices or polymeric biomaterials in vitro and in vivo, the antibodies of the present invention, may be useful in those cases where there is a previous infection because of the ability of this antibody to further restrict and inhibit E. faecium binding to fibronectin and thus limit the extent and spread of the infection. In addition, the antibody may be modified as necessary so that, in certain instances, it is less immunogenic in the patient to whom it is administered. For example, if the patient is a human, the antibody may be "humanized" by transplanting the complimentarity determining regions (CDR's) of the hybridoma-derived antibody into a human monoclonal antibody as described, e.g., by Jones et al., Nature 321 :522-525 (1986) or Tempest et al. Biotechnology 9:266-273 (1991 ) or "veneered" by changing the surface exposed murine framework residues in the immunoglobulin variable regions to mimic a homologous human framework counterpart as described, e.g., by Padlan, Molecular Imm. 28:489-498 (1991 ) and U.S. Pat. No. 6,797,492, all of these references incorporated herein by reference. Even further, when so desired, the monoclonal antibodies of the present invention may be administered in conjunction with a suitable antibiotic to further enhance the ability of the present compositions to fight bacterial infections.

As indicated above, enterococcal infections are not only a problem with patients but also may affect medical devices, implants and prosthetics, and thus the present invention can be utilized to protect these devices from enterococcal infection as well, e.g., by coating these devices with the compositions of the present invention. Medical devices or polymeric biomaterials to be coated with the antibody compositions described herein include, but are not limited to, staples, sutures, replacement heart valves, cardiac assist devices, hard and soft contact lenses, intraocular lens implants (anterior chamber or posterior chamber), other implants such as corneal inlays, kerato- prostheses, vascular stents, epikeratophalia devices, glaucoma shunts, retinal staples, scleral buckles, dental prostheses, thy ro plastic devices, laryngoplastic devices, vascular grafts, soft and hard tissue prostheses including, but not limited to, pumps, electrical devices including stimulators and recorders, auditory prostheses, pacemakers, artificial larynx, dental implants, mammary implants, other implants, cranio/facial tendons, artificial joints, tendons, ligaments, menisci, and disks, artificial bones, artificial organs including artificial pancreas, artificial hearts, artificial limbs, and heart valves; stents, wires, guide wires, intravenous and central venous catheters, laser and balloon angioplasty devices, vascular and heart devices (tubes, catheters, balloons), ventricular assists, blood dialysis components, blood oxygenators, urethral/ureteral/urinary devices (Foley catheters, stents, tubes and balloons), airway catheters (endotracheal and tracheostomy tubes and cuffs), enteral feeding tubes (including nasogastric, intragastric and jejunal tubes), wound drainage tubes, tubes used to drain the body cavities such as the pleural, peritoneal, cranial, and pericardial cavities, blood bags, test tubes, blood collection tubes, vacutainers, syringes, needles, pipettes, pipette tips, and blood tubing.

It will be understood by those skilled in the art that the term "coated" or "coating", as used herein, means to apply the antibody or pharmaceutical composition derived therefrom, to a surface of the device, preferably an outer surface that would be exposed to streptococcal bacterial infection. The surface of the device need not be entirely covered by the protein, antibody or active fragment.

In a preferred embodiment, the antibodies may also be used as a passive vaccine which will be useful in providing suitable antibodies to treat or prevent a enterococcal infection. As would be recognized by one skilled in this art, a vaccine may be packaged for administration in a number of suitable ways, such as by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. One such mode is where the vaccine is injected intramuscularly, e.g., into the deltoid muscle, however, the particular mode of administration will depend on the nature of the bacterial infection to be dealt with and the condition of the patient. The vaccine is preferably combined with a pharmaceutically acceptable vehicle, carrier or excipient to facilitate administration, and such a suitable carrier or other moiety may be water or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution.

The preferred dose for administration of an antibody composition in accordance with the present invention is that amount will be effective in preventing of treating a enterococcal infection, and one would readily recognize that this amount will vary greatly depending on the nature of the infection and the condition of a patient. As indicated above, an "effective amount" of antibody or pharmaceutical agent to be used in accordance with the invention is intended to mean a nontoxic but sufficient amount of the agent, such that the desired prophylactic or therapeutic effect is produced. As will be pointed out below, the exact amount of the antibody or a particular agent that is required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular carrier or adjuvant being used and its mode of administration, and the like. Accordingly, the "effective amount" of any particular antibody composition will vary based on the particular circumstances, and an appropriate effective amount may be determined in each case of application by one of ordinary skill in the art using only routine experimentation. The dose should be adjusted to suit the individual to whom the composition is administered and will vary with age, weight and metabolism of the individual. The compositions may additionally contain stabilizers or pharmaceutically acceptable preservatives, such as thimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Company, St. Louis, MO).

When used with suitable labels or other appropriate detectable biomolecule or chemicals, the monoclonal antibodies described herein are useful for purposes such as in vivo and in vitro diagnosis of enterococcal infections or detection of enterococcal bacteria. Laboratory research may also be facilitated through use of such antibodies. Various types of labels and methods of conjugating the labels to the antibodies of the invention are well known to those skilled in the art, such as the ones set forth below.

For example, the antibody can be conjugated (directly or via chelation) to a radiolabel such as, but not restricted to, ³²P, ³H, ¹⁴C, ³⁵S, ¹²⁵I, or ¹³¹ I. Detection of a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography. Bioluminescent labels, such as derivatives of firefly luciferin, are also useful. The bioluminescent substance is covalently bound to the protein by conventional methods, and the labeled protein is detected when an enzyme, such as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light. Fluorogens may also be used to label proteins. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, and Texas Red. The fluorogens are generally detected by a fluorescence detector. The location of a ligand in cells can be determined by labeling an antibody as described above and detecting the label in accordance with methods well known to those skilled in the art, such as immunofluorescence microscopy using procedures such as those described by Warren et al. (MoI. Cell. Biol., 7: 1326-1337, 1987). As indicated above, the monoclonal antibodies of the present invention, or active portions or fragments thereof, are particularly useful for interfering with the initial physical interaction between a enterococcal pathogen responsible for infection and a mammalian host, such as the adhesion of the bacteria to mammalian extracellular matrix proteins, and this interference with physical interaction may be useful both in treating patients and in preventing or reducing bacteria infection on in-dwelling medical devices to make them safer for use.

In another embodiment of the present invention, a kit which may be useful in isolating and identifying enterococcal bacteria and infection is provided which comprises the antibodies of the present invention in a suitable form, such as lyophilized in a single vessel which then becomes active by addition of an aqueous sample suspected of containing the enterococcal bacteria. Such a kit will typically include a suitable container for housing the antibodies in a suitable form along with a suitable immunodetection reagent which will allow identification of complexes binding to the ACM antibodies of the invention. For example, the immunodetection reagent may comprise a suitable detectable signal or label, such as a biotin or enzyme that produces a detectable color, etc., which normally may be linked to the antibody or which can be utilized in other suitable ways so as to provide a detectable result when the antibody binds to the antigen.

In short, the antibodies of the present invention which bind to the ACM protein or active fragments or subregions thereof are thus extremely useful in treating or preventing enterococcal infections in human and animal patients and in medical or other in-dwelling devices. Accordingly, the present invention relates to methods of identifying and isolating antibodies which can bind to ACM and which can be used in methods of treatment of infections which involve opsonophagocytic killing of the bacteria. Antibodies which are identified and/or isolated using the present method, such as the antibodies which can bind to the ACM protein or its subregions and which can prevent or treat infection from E. faecium, and antibodies recognizing the same epitopes as those recognized by the monoclonals described herein, are thus a part of the present invention EXAMPLES

The following examples are provided which exemplify aspects of the preferred embodiments of the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1. Expression and Purification of ACM

To characterize the utility of this invention, the A domain of ACM from E. faecium was cloned, expressed recombinantly and purified. Bolded and underlined sequence represents the addition of the 6-His tag used for purification.

ACM:

Nucleotide Sequence

AAAATTGAAAACGGGCTAGTCGTTACAAATGAGAATAAATAG

Amino Acid Sequence

MRGSHHHHHHGSDAGRDISSNVTSLTVDPTNITDGGNIKVKFSFDEKKQNIQPGDYLWINWPSEGNIRGE GFQKEIPLMIENKNVGTLTVRKDSAQWFNENIKNLDSVEGWGQFEIQARNVTDTGEENTGPFTVTSGDK TATVNVTKPASGSSSSVFYYKTGDMLPEDTKHIRWFLNINNNGTYVEQPVKISDEIQSGQRLDPSTFEIN QIHLGEQKVYRGEEGIQQFLQDFPSATFNFSVTDNYIEITIPKNFVNLRKIMVSYKTIIENPEQINFENH SEAWFKEFNKPAVDGESFNHTVKNISASGGVNGTVRGELKIFKYINDTEIGIPNVTFELRRADEQPIQGQ SSILLTSNEQGEASIKGLQVGDYWKEKEAPNWIDFDPLSSNELKFSINENDTEGVSLPIYNKKKVTNIT ATKIWNGGTTPRPSIYFKLFRASTNNWEPVPDAETKRLDNGITSVTWEDIQQYDDSGNEYTFKVQEVDQN GNDYVPSGYQKIENGLWTNENK Protein Production and Purification

Using PCR, the A domain of ACM was amplified from E. faecium TX2555 genomic DNA (from sequences described above) and subcloned into the E. coli expression vector PQE-30 (Qiagen), which allows for the expression of a recombinant fusion protein containing six histidine residues. After expression either by shake flasks or in bioreactors, the cells were harvested by centrifugation and the cell paste frozen at -80° C. Cells were lysed in 1 X PBS (1 OmL of buffer/1 g of cell paste) using 2 passes through a microfluidizer at 10,000 psi. Lysed cells were spun down at 17,000rpm for 30 minutes to remove cell debris. Supernatant was passed over a 5-mL HiTrap Chelating (Pharmacia) column charged with 0.1 M NiC^- After loading, the column was washed with 5 column volumes of 1 OmM Tris, pH 8.0, 10OmM NaCI (Buffer A). Protein was eluted using a 0-100% gradient of 1 OmM Tris, pH 8.0, 10OmM NaCI, 50OmM imidazole (Buffer B). Fractions containing ACM were dialyzed in 1 x PBS. The ACM protein was then put through an endotoxin removal protocol. Buffers used during this protocol were made endotoxin free by passing over a 5-mL Mono-Q sepharose (Pharmacia) column. Protein was divided evenly between 4x 15mL tubes. The volume of each tube was brought to 9mL with Buffer A. 1 mL of 10% Triton X-1 14 was added to each tube and incubated with rotation for 1 hour at 4° C. Tubes were placed in a 37° C water bath to separate phases. Tibes were spun down at 2,000rpm for 10 minutes and the upper aqueous phase from each tube was collected and the detergent extraction repeated. Aqueous phases from the 2nd extraction were combined and passed over a 5-mL IDA chelating (Sigma) column, charged with 0.1 M NiCI₂ to remove remaining detergent. The column was washed with 9 column volumes of Buffer A before the protein was eluted with 3 column volumes of Buffer B. The eluant was passed over a 5-mL Detoxigel (Sigma) column and the flow-through collected and reapplied to the column. The flow-through from the second pass was collected and dialyzed in 1 x PBS. The purified product was analyzed for concentration, purity and endotoxin level before administration into mice for polyclonal or monoclonal antibody production.

Example 2. Immunization Strategies for Monoclonal Antibody Production

With the goal of generating and characterizing monoclonal antibodies (mAbs), strategies were formulated to generate mAbs against ACM that were of high affinity, able to interrupt or restrict the binding of extracellular matrix proteins (ECM) and demonstrate therapeutic or diagnostic efficacy.

E. coli expressed and purified ACM protein was used to generate a panel of murine monoclonal antibodies. Briefly, a group of Balb/C or SJL mice received a series of subcutaneous immunizations of 1 -10 mg of protein in solution or mixed with adjuvant as described below in Table 1 :

Table 1. Immunization Scheme

Conventional

Injection Da^ Amount (uq) Route Adjuvant

Primary 0 5 Subcutaneous FCA

Boost #1 14 1 Intraperitoneal RIBI

Boost #2 28 1 Intraperitoneal RIBI

Boost #3 4? 1 Intraperitoneal RIBI

Seven days after a boost, serum was collected and titered in ELISA assays against in immunizing ACM protein. Three days after the final boost, the spleen was removed, teased into a single cell suspension and the lymphocytes harvested. The lymphocytes were then fused to a P3X63Ag8.653 myeloma cell line (ATCC #CRL- 1580). Cell fusion, subsequent plating and feeding were performed according to the Production of Monoclonal Antibodies protocol from Current Protocols in Immunology (Chapter 2, Unit 2.).

Example 3. Screening and Selection of Anti-ACM Monoclonal Antibodies

Any hybridoma clones generated from the ACM fusions were screened for specific anti-ACM antibody production using a standard ELISA assay. Positive clones were expanded and tested further for activity in a whole bacterial cell binding assay by flow cytometry and ACM binding by Biacore analysis.

ELISA Analysis lmmulon 2-HB high-binding 96-well microtiter plates (Dynex) were coated with 1 μg/well of ACM in 1 X PBS, pH 7.4 and incubated for 2 hours at room temperature. All washing steps in ELISAs were performed three times with 1 X PBS, 0.05% Tween-20 wash buffer. Plates were washed and blocked with a 1 % BSA solution at room temperature for 1 hour before hybridoma supernatant samples were added to wells.

Plates were incubated with samples and relevant controls such as media alone for one hour at room temperature, washed, and goat anti-mouse IgG-AP (Sigma) diluted 1 :5000 in 1 X PBS, 0.05 % Tween-20, 0.1 % BSA was used as a secondary reagent. Plates were developed by addition of 1 mg/ml solution of 4-nitrophenyl phosphate (pNPP) (Sigma), followed by incubation at 37° C for 30 minutes. Absorbance was read at 405 nm using a SpectraMax 190 Plate Reader (Molecular Devices Corp.). Antibody supernatants that had an OD₄₀S > 3 times above background (media alone, ~ 0.1 OD) were considered positive.

Biacore Analysis

Throughout the analysis, the flow rate remained constant at 10 ml/min. Prior to the ACM injection, test antibody was adsorbed to the chip via RAM-Fc binding. At time

0, ACM at a concentration of 30 μg/ml was injected over the chip for 3 min followed by

2 minutes of dissociation. This phase of the analysis measured the relative association and disassociation kinetics of the mAb / ACM interaction.

Binding to Whole Bacteria

Bacterial samples were collected, washed and incubated with mAb or PBS alone (control) at a concentration of 2 μg/ml after blocking with rabbit IgG (50 mg/ml). Following incubation with antibody, bacterial cells were incubated with Goat-F_(ab'₎₂-Anti- Mouse-F₍ab'₎₂-FITC which served as the detection antibody. After antibody labeling, bacterial cells were aspirated through the FACScaliber flow cytometer to analyze fluorescence emission (excitation: 488, emission: 570). For each bacterial strain, 10,000 events were collected and measured.

Table 2. ACM Screening Summary

From the above analysis, ACM positive hybridomas were generated in a frequency of 0.7 - 2.1 % of the growth positive wells. Interestingly, most of the ACM Biacore positive hybridomas were also positive by on whole cells. Positive examples (by ELISA, Biacore and Flow) are shown in Table 3. Table 3. Representative Examples of Hybridoma Supernatants From Fusions in Table 3

Example 4. Anti-ACM mAbs Bind E. faecium Bacteria and Inhibit ACM - Collagen Binding

To determine that the anti-ACM mAbs generated and selected with recombinant ACM cross-reacted with native ACM expressed on Enterococcus strains of bacteria, flow cytometric analysis was used. Anti-ACM mAbs varied in reactivity to E. faecium strains. Single cell cloned mAbs with high affinity activity against ACM as suggested by the Biacore analysis presented in Table 3, demonstrated remarkable activity in the inhibition of ACM - Collagen binding as well as binding to whole Enterococcus bacteria.

ELISA Based ACM - Collagen Binding Inhibition lmmulon 2-HB high-binding 96-well plates were coated with 2.5 μg/ml ACM in PBS and incubated 2 hours at room temperature. Plates were washed and blocked with 1 % BSA solution for 1 hour, then washed and incubated with monoclonal antibody (either hybridoma supernatant or purified antibody) for 1 hour at room temperature. Following incubation with antibody, plates were either washed or left untreated, and 2μg/ml solution of human type I collagen was added. Plates were incubated 1 hour at 37 ⁰C and washed. Detection was performed with 1 :2,000 diluted biotinylated rabbit anti-collagen Ab (Rockland) and 1 :8,000 diluted HRP conjugated neutralite Avidin (Southern Biotech). Following incubation with conjugate, plates were washed and ABTS substrate was added. Plates then incubated 10 minutes at room temperature, the reaction was stopped with addition of 10% SDS, and absorbance was read at 405 nm. All data was analyzed using SOFTmax Pro v.3.1 .2. software (Molecular Devices Corp., Sunnyvale, California, USA). The data reflected that anti-ACM mAbs were able to inhibit ACM-collagen binding (see Fig. 1 ).

Binding to Whole Bacteria

Bacterial samples (see Table 4) were collected, washed and incubated with mAb or PBS alone (control) at a concentration of 2 μg/ml. Following incubation with antibody, bacterial cells were incubated with Goat-F_(ab'₎₂-Anti-Mouse-F₍ab'₎₂-FITC which served as the detection antibody. After antibody labeling, bacterial cells were aspirated through the FACScaliber flow cytometer to analyze fluorescence emission (excitation: 488, emission: 570). For each bacterial strain, 10,000 events were collected and measured.

Affinity Determination by Biacore

Surface plasmon resonance (SPR) was used to test ELISA-positive clones for the ability to bind rACM protein. Kinetic analysis was performed on a Biacore 3000 (Biacore, Piscataway, NJ) using the ligand capture method included in the software. The anti-ACM mAbs were passed over a Goat anti-mouse-F(ab')₂ chip, allowing binding and capture via the Fc portion. Varying concentrations of the rACM protein were then passed over the chip, and data collected. Using the Biacore provided Evaluation software (Version 3.1 ), k_on and k_Off were measured and K_A and K₀ were calculated.

Table 4. Flow Cytometric Straining of Whole Enterococcus Bacteria

Table 5. Affinity Measurements by Biacore

This data demonstrates that it is possible, although an extremely rare event, to select monoclonal antibodies immunized against the ACM antigen in mice and selected for high affinity binding, the ability to inhibit ACM - collagen binding as well as cell surface binding on E. faecium strains.

The following references cited above are incorporated herein as if set forth in their entirety:

Boden, M. K., and Flock, J.I. (1994) Cloning and characterization of a gene for a 19 kDa fibrinogen-binding protein from Staphylococcus aureus. MoI Microbiol 12: 599-606.

Cheung, A.I., Projan, SJ. , Edelstein, R.E., and Fischetti, V.A. (1995) Cloning, expression, and nucleotide sequence of a Staphylococcus aureus gene (fbpA) encoding a fibrinogen binding protein. Infect I 'mm un 63: 1914-1920.

Courtney, H. S., Dale, J. B., and Hasty, D.I. (1996) Differential effects of the streptococcal fibronectin-binding protein, FBP54, on adhesion of group A streptococci to human buccal cells and HEp-2 tissue culture cells. Infect lmmun 64: 2415-2419.

Foster, TJ. , and Hook, M. (1998) Surface protein adhesins of Staphylococcus aureus. Trends Microbiol 6: 484-488.

Hartford, O., McDevitt, D., and Foster, TJ. (1999) Matrix binding proteins of Staphylococcus aureus: functional analysis of mutant and hybrid molecules. Microbiology 145: 2497-2505.

Holmes, A. R., McNab, R., Millsap, K.W., Rohde, M., Hammerschmidt, S., Mawdsley, J. L., and Jenkinson, H. F. (2001 ) The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence. MoI Microbiol 41 : 1395-1408.

Jonsson, K., Signas, C, Muller, HP., and Lindberg, M. (1991 ) Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene. Eur J Biochem 202: 1041 -1048.

Murray, B. E. (1990) The life and times of the Enterococcus. Clin Microbiol Rev 3: 46- 65.

Murray, B. E. (2000) Vancomycin-resistant Enterococcal infections. N Engl J Med 342: 710-721 . Patti, J. M., Jonsson, H., Guss, B., Switalski, L.M., Wiberg, K., Lindberg, M., and Hook, M. (1992) Molecular characterization and expression of a gene encoding a Staphylococcus aureus collagen adhesin. J Biol Chem 267: 4766-4772.

Patti, J. M., Boles, J.O., and Hook, M. (1993) Identification and biochemical characterization of the ligand binding domain of the collagen adhesin from Staphylococcus aureus. Biochemistry 32: 1 1428-1 1435.

Patti, J. M., Allen, B.L., McGavin, MJ. , and Hook, M. (1994a) MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol 48: 585-617.

Patti, J. M., and Hook, M. (1994) Microbial adhesins recognizing extracellular matrix macromolecules. Curr Opin Cell Biol 6: 752-758.

Rich, R. L., Kreikemeyer, B., Owens, R.T., LaBrenz, S., Narayana, S.V.L., Weinstock, G. M., et al. (1999) Ace is a collagen-binding MSCRAMM from Enterococcus faecalis. J Biol Chem 274: 26939-26945.

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Claims

What Is Claimed Is:

1 . A monoclonal antibody which binds to the ACM protein from Enterococcus faecium.

2. The monoclonal antibody according to Claim 1 wherein the antibody is raised against the A domain of the ACM protein from Enterococcus faecium.

3. The monoclonal according to Claim 1 , wherein said antibody treats or prevents E. faecium infection in a human or animal.

5. The monoclonal antibody according to Claim 1 , wherein said antibody inhibits binding of the ACM protein to collagen.

6. The monoclonal antibody according to Claim 1 , wherein said antibody is suitable for parenteral, oral, intranasal, subcutaneous, aerosolized or intravenous administration in a human or animal.

7. The monoclonal antibody according to Claim 1 wherein the monoclonal antibody is of a type selected from the group consisting of murine, chimeric, humanized and human monoclonal antibodies.

8. The monoclonal antibody according to Claim 1 wherein the antibody is a single chain monoclonal antibody.

9. The monoclonal antibody according to Claim 1 which comprises an antibody fragment having the same binding specificity of an antibody which binds to the ACM protein from Enterococcus faecium.

10. The monoclonal antibody according to Claim 1 that is raised against a peptide having the amino acid sequence of SEQ ID NO:2.

1 1 . The monoclonal antibody according to Claim 1 wherein the monoclonal antibody recognizes a protein or peptide selected from the group consisting of the ACM protein of E. faecium and the A domain of the ACM protein of E. faecium.

12. The monoclonal antibody according to Claim 1 wherein the monoclonal antibody recognizes a peptide selected from the group consisting of the N1 , N2, N3, N1 N2, truncated N2, N1 N2 truncate, and N2N3 constructs from the ACM protein of E. faecium.

13. The monoclonal antibody according to Claim 1 that recognizes a protein or peptide having the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.

14. The monoclonal antibody according to Claim 1 that recognizes a protein or peptide encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:5 and degenerates thereof.

15. A monoclonal antibody that binds to an epitope recognized by a monoclonal antibody selected from the group consisting monoclonal antibodies 103-21 , 103-24, 103- 32, 103-55, 106-5 and 107-37.

16. Isolated antisera containing an antibody according to Claim 1 .

17. A diagnostic kit comprising the antibody according to Claim 1 and means for detecting binding by that antibody.

18. The diagnostic kit according to Claim 17 wherein said means for detecting binding comprises a detectable label that is linked to said antibody.

19. A method of diagnosing an infection of E. faecium comprising adding the antibody according to Claim 1 to a sample suspected of being infected with E. faecium, and determining if antibodies have bound to the sample.

20. A pharmaceutical composition comprising the antibody of Claim 1 and a pharmaceutically acceptable vehicle, carrier or excipient.

21. The pharmaceutical composition according to Claim 20 further comprising a physiologically acceptable antibiotic.

22. A method of treating or preventing an infection of E. faecium comprising administering to a human or animal patient an effective amount of the antibody according to Claim 1 .

23. A method of making a monoclonal antibody that recognizes the ACM protein from E. faecium comprising administering to a host animal an immunogenic amount of the ACM protein from E. faecium, forming a hybridoma from antibodies generated by said peptide, and isolating a monoclonal antibody from said hybridoma.

24. A method of making a monoclonal antibody that recognizes the ACM protein from E. faecium comprising administering to a host animal an immunogenic amount of the ACM ligand binding A domain, forming a hybridoma from antibodies generated by said peptide, and isolating a monoclonal antibody from said hybridoma.

25. A passive vaccine comprising the antibody of claim 1 in an amount effective to inhibit binding of the ACM protein to collagen and a physiologically acceptable vehicle, carrier, or excipient.

26. A method of inhibiting binding of E. faecium to host cells comprising administering to a human or animal patient an effective amount of the antibody according to Claim 1 .

27. A method of inhibiting binding of the ACM protein of E. faecium to collagen comprising administering to a suitable host or device an effective amount of the antibody according to Claim 1 .

28. A monoclonal antibody that binds to an epitope recognized by monoclonal antibody 103-21.10.