WO2007016556A1

WO2007016556A1 - Multifunctional polymers for promotion of opsonization of target cells and organisms

Info

Publication number: WO2007016556A1
Application number: PCT/US2006/029913
Authority: WO
Inventors: Vijay M. Krishnamurthy; Lee J. Quinton; Lara A. Estroff; Steven J. Metallo; Joseph P. Mizgerd; George M. Whitesides
Original assignee: President And Fellows Of Harvard College
Priority date: 2005-08-02
Filing date: 2006-08-01
Publication date: 2007-02-08

Abstract

A multifunctional polymer is provided that presents groups capable of binding to and modifying the surfaces of bacteria, cancer cells, or other target cells or organisms. The polymer can be used to associate hapten moieties with the surface of the target cell or organism to promote opsonization and subsequent phagocytosis of the opsonized target cell or organism by macrophages.

Description

MULTIFUNCTIONAL POLYMERS FOR PROMOTION OF OPSONIZATION OF TARGET CELLS AND ORGANISMS

RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Provisional Application No. 60/704,715, filed August 2, 2005 and U.S. Provisional Application No. 60/758,675, filed January 13, 2006, the disclosures of which are hereby expressly incorporated by reference in their entirety.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The immunological system is able to recognize foreign substances (antigens) which stimulate the system to produce antibody-mediated immunity and/or cell-mediated immunity, or both. An antigen is a substance, usually macromolecular, that induces an immunological response. Because of its complex macromolecular structure, a single microorganism consists of multiple antigens (e.g., surface structures such as cell wall components, fimbriae, flagella, etc., or extracellular proteins, such as toxins or enzymes produced by the microorganism, or other functional groups). The coat proteins and some of the envelope proteins of animal viruses are also usually antigenic. The host is able to respond specifically to each and every antigen that comes into contact with the components of the immunological system.

Antibody-mediated immunity is a type of immunity that is mediated by soluble host proteins called antibodies or immunoglobulins. Antibodies are proteins produced by lymphocytes that can specifically bind a wide variety of protein and polysaccharide antigens and elicit a response that is significant in antimicrobial defense. In conjunction with the complement system, antibodies are the mediators of humoral immunity, and their presence on mucosal surfaces provides resistance to many infectious agents. Antibodies are essential for the prevention and/or cure of many types of bacterial and viral infections. There are several classes or types of antibodies, but all members of the classes of antibodies that are produced in response to a specific antigen react stereochemically with that antigen and not with other antigens. The host has the genetic capacity to produce specific antibodies to thousands of different antigens, but does not do so until there is an appropriate antigenic stimulus. Due to clonal selection, the host produces only the homologous antibodies that will react with that antigen. These antibodies are found in the blood plasma and lymph and in many extravascular tissues.

Induction of a primary immune response begins when an antigen penetrates epithelial surfaces. It eventually comes into contact with macrophages or certain other classes of antigen presenting cells, which include B cells, monocytes, dendritic cells, Langerhans cells and endothelial cells. Antigens, such as bacterial cells, are internalized by endocytosis and processed by the antigen presenting cells, then presented to immunocompetent lymphocytes to initiate the early steps of the immunological response. Antibodies enhance phagocytic engulfment of microbial antigens, a process referred to as opsonization. IgG and IgM antibodies have a combining site for the antigen and a site for cytophilic association with phagocytes. Bacteria and viral particles are thus ingested with increased efficiency by, e.g., macrophages, a process referred to as phagocytosis.

Gram-positive bacteria are all important to public health, and all can cause bacterial infections, which remain an important cause of morbidity and mortality. Immunocompromised patients are particularly susceptible to such infections (Graham, N. M. The epidemiology of acute respiratory infections in children and adults: a global perspective. Epidemiol. Rev. 12, 149-178 (1990)). The effective design of vaccines against these infections has proven challenging because bacterial surfaces are often poorly antigenic (Picard, C, Puel, A., Bustamante, J., Ku, C. L. & Casanova, J. L. Primary immunodeficiencies associated with pneumococcal disease. Curr. Opin. Allergy Clin. Immunol. 3, 451-459 (2003); Verbrugh, H. A., Peterson, P. K., Nguyen, B. Y., Sisson, S. P. & Kim, Y. Opsonization of encapsulated Staphylococcus aureus: the role of specific antibody and complement. J. Immunol 129, 1681-1687 (1982); Casal, J. & Tarrago, D. Immunity to Streptococcus pneumoniae: Factors affecting production and efficacy. Curr. Opin. Infect. Dis. 16, 219-224 (2003); Bogaert, D., Hermans, P. W. H., Adrian, P. V., Rumke, H. C. & de Groot, R. Pneumococcal vaccines: an update on current strategies. Vaccine 22, 2209-2220 (2004)).

The rise of drug resistant strains of bacteria has highlighted the need for antibacterial agents with new mechanisms of action (Hubbard, B. K. & Walsh, C. T. Vancomycin assembly: Nature's way. Angew. Chem., Int. Ed. Eng. 42, 730-765 (2003)). Several strategies have been employed to attach monomelic, bifunctional molecules noncovalently to the surface of pathogens (or to soluble truncations of proteins on the surface of pathogens), and to recruit components of the immune system (e.g., antibodies, complement, or macrophages) to the bound species (Byrn, R. A. et al. Biological properties of a CD4 immunoadhesin. Nature 344, 667-670 (1990); Shokat, K. M. & Schultz, P. G. Redirecting the Immune Response: Ligand-Mediated Immunogenicity. J. Am. Chem. Soc. 113, 1861-1862 (1991); Bertozzi, C. R. & Bednarski, M. D. Antibody Targeting to Bacterial-Cells Using Receptor- Specific Ligands. J. Am. Chem. Soc. 114, 2242-2245 (1992); Bertozzi, C. R. & Bednarski, M. D. A Receptor-Mediated Immune-Response Using Synthetic Glycoconjugates. J. Am. Chem. Soc. 114, 5543-5546 (1992)).

One approach uses fusion proteins of a surface recognition protein (the soluble region of CD4 (sCD4)) and the Fc region of the heavy chain of IgG to induce phagocytic cell- mediated killing of HIV-I infected T-lymphoblastoid cells (which display gρl20, the target of CD4, at their surface) (Byrn, R. A. et al. Biological properties of a CD4 immunoadhesin. Nature 344, 667-670 (1990)).

Another approach uses bifunctional molecules to couple antibodies (added in a second step) to a soluble version of the HIV surface protein or to the surface of bacteria. A dinitrophenyl (DNP)-CD4 conjugate has been used to direct anti-DNP antibodies to purified gpl20; the bound antibodies were able to recruit the initial component of the complement cascade, CIq, to the complex in vitro (Shokat, K. M. & Schultz, P. G. Redirecting the Immune Response: Ligand-Mediated Immunogenicity. J. Am. Chem. Soc. 113, 1861-1862 (1991).

This bifunctional, antibody-targeting approach has also been applied to the Gram- negative bacterium E. coli, exploiting the binding of mannose by FimH, the adhesion protein on the tips of type 1 pili on the bacterial surface, using small molecule conjugates (Bertozzi, C. R. & Bednarski, M. D. Antibody Targeting to Bacterial-Cells Using Receptor-Specific Ligands. J. Am. Chem. Soc. 114, 2242-2245 (1992); Bertozzi, C. R. & Bednarski, M. D. A Receptor-Mediated Immune-Response Using Synthetic Glycoconjugates. J. Am. Chem. Soc. 114, 5543-5546 (1992)) or polymers (Li, J. et al. Bacteria targeted by human natural antibodies using alpha-Gal conjugated receptor-specific glycopolymers. Bioorg. Med. Chem. 7, 1549-1558 (1999)).

A mannose-biotin conjugate was synthesized to which was bound avidin and an anti- avidin antibody to generate a complex that displayed both mannose and antibody molecules (Bertozzi, C. R. & Bednarski, M. D. Antibody Targeting to Bacterial-Cells Using Receptor- Specific Ligands. J. Am. Chem. Soc. 114, 2242-2245 (1992); Bertozzi, C. R. & Bednarski, M. D. A Receptor-Mediated Immune-Response Using Synthetic Glycoconjugates. J. Am. Chem. Soc. 114, 5543-5546 (1992)). Binding of the mannose-avidin-IgG complex to the surface of E. coli and subsequent recognition of the IgG portion of the complex to activate complement and enable recognition and killing of the bacteria by macrophages was demonstrated (Bertozzi, C. R. & Bednarski, M. D. A Receptor-Mediated Immune-Response Using Synthetic Glycoconjugates. J. Am. Chem. Soc. 114, 5543-5546 (1992)).

A polymer has also been developed that presents both mannoside units (to bind to the bacterial surface) and α-Gal units (with the potential to recruit innate anti-α-Gal antibodies to the polymer adsorbed on bacteria). Targeting of antibodies to a bacterial surface was not demonstrated, however (Li, J. et al. Bacteria targeted by human natural antibodies using alpha-Gal conjugated receptor-specific glycopolymers. Bioorg. Med. Chem. 7, 1549-1558 (1999)).

SUMMARY OF THE INVENTION

It is desirable to provide a functionalized polymer capable of carrying out three distinct functions based on polyvalent molecular recognition: i) recognition of the surface of a target cell, ii) modification of the target cell surface to generate specific binding sites recognized by an antibody, and iii) promotion of phagocytosis of the opsonized target cell. The preferred embodiments provide polymers capable of performing these functions and provide methods of treating Gram-positive bacterial infections, attacking breast tumor cells, .and destroying other target cells through opsonization and subsequent phagocytosis.

Li a first aspect, a pharmaceutical composition for facilitating opsonization of a target cell or a target organism in a host is provided, the composition comprising a polymeric substance, the polymeric substance comprising a plurality of first functional groups and a plurality of second functional groups, wherein the first functional group is capable of specific binding to the target cell or the target organism, and wherein the second functional group comprises a hapten capable of causing an antibody to associate with the target cell or the target organism; and a pharmaceutically acceptable excipient. hi an embodiment of the first aspect, the hapten is capable of causing an antibody naturally occurring in the host to associate with the target cell or the target organism. hi an embodiment of the first aspect, the hapten is capable of causing only a synthetic antibody or an antibody not naturally occurring in the host to associate with the target cell or the target organism. hi an embodiment of the first aspect, the polymeric substance comprises a functionalized polyacrylamide. hi an embodiment of the first aspect, the first functional group comprises a sialic acid moiety capable of specific binding to a hemagglutinin moiety on the target cell or the target organism. hi an embodiment of the first aspect, the first functional group comprises a mannose moiety capable of specific binding to a FimH adhesin moiety on the target cell or the target organism. hi an embodiment of the first aspect, the first functional group comprises a Glc-Nac moiety capable of specific binding to a pneumococcal surface adhesin A moiety or a pilus adhesin moiety on the target cell or the target organism. hi an embodiment of the first aspect, the first functional group comprises a Neu-Ac moiety or a lacto-N-neotetraose moiety capable of specific binding to a choline binding protein A moiety on the target cell or the target organism. In an embodiment of the first aspect, the first functional group comprises a Neu-Ac moiety capable of specific binding to a pilus adhesin moiety on the target cell or the target organism.

In an embodiment of the first aspect, the first functional group comprises a plasmin moiety or a plasminogen moiety capable of specific binding to an α-enolase moiety on the target cell or the target organism.

In an embodiment of the first aspect, the first functional group comprises a collagen moiety capable of specific binding to a collagen adhesin moiety on the target cell or the target organism.

In an embodiment of the first aspect, the first functional group comprises a lactose moiety capable of specific binding to a pilus adhesin moiety on the target cell or the target organism.

In an embodiment of the first aspect, the first functional group comprises a vancomycin moiety capable of specific binding to a D-AIa-D-AIa moiety on the target cell or the target organism.

In an embodiment of the first aspect, the first functional group comprises an anti-Her2 peptide moiety capable of specific binding to a Her2 receptor moiety on the target cell or the target organism.

In an embodiment of the first aspect, the second functional group comprises a polysaccharide capable of specific binding to an anti-alpha(Gal) antibody.

In an embodiment of the first aspect, the pharmaceutical composition is in unit dosage form.

In an embodiment of the first aspect, the target cell is a cancer cell.

In an embodiment of the first aspect, the target organism is a pathogenic bacterium.

In an embodiment of the first aspect, the pharmaceutical composition is for use in treating an infection by a pathogenic bacterium.

In an embodiment of the first aspect, the pharmaceutical composition is for use in treating breast cancer or prostate cancer.

In an embodiment of the first aspect, the phaπnaceutical composition is for use in killing the target cell, e.g., a cancer cell or a B cell, or the target organism, e.g., enteroaggregative E. coli, fϊmbrilated E. colt, S. pneumoniae, P. aeruginosa, S. aureus, S. epidermidis, or E.faecalis.

In an embodiment of the first aspect, the pharmaceutical composition is for use in facilitating opsonization and subsequent phagocytosis of the target cell or the target organism.

In a second aspect, a method of opsonizing a target cell or a target organism in a host is provided, the method comprising the step of administering a polymeric substance to the host, the polymeric substance comprising a plurality of first functional groups and a plurality of second functional groups, wherein the first functional group is capable of specific binding to the target cell or target organism, and wherein the second functional group comprises a hapten capable of causing an antibody to associate with the target cell or target organism, whereby the target cell or the target organism is opsonized.

In an embodiment of the second aspect, the antibody is naturally occurring in the host.

In an embodiment of the second aspect, the antibody is a synthetic antibody or an antibody not naturally occurring in the host, and the method further comprises the step of administering the antibody to the host.

In an embodiment of the second aspect, the first functional group comprises a moiety capable of specific binding to a target organism comprising a pathogenic bacterium.

In an embodiment of the second aspect, the first functional group comprises a moiety capable of specific binding to a target organism comprising a Gram-positive bacterium.

In an embodiment of the second aspect, the target organism is selected from the group consisting of enteroaggregative E. coli, fϊmbrilated E. coli, S. pneumoniae, P. aeruginosa, S. aureus, S. epidermidis, and E. faecalis.

In an embodiment of the second aspect, the target cell is a cancer cell.

In an embodiment of the second aspect, the target cell is a breast cancer cell.

In an embodiment of the second aspect, the target cell is a prostate cancer cell.

In an embodiment of the second aspect, the target cell is a B cell.

In an embodiment of the second aspect, the step of administering further comprises phagocytosis of the opsonized target cell or the opsonized target organism. In a third aspect, a pharmaceutical kit is provided, the kit comprising a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a polymeric substance, the polymeric substance comprising a plurality of first functional groups and a plurality of second functional groups, wherein the first functional group is capable of specific binding to a target cell or a target organism, and wherein the second functional group comprises a hapten capable of causing an antibody to associate with the target cell or the target organism; and directions for administering the pharmaceutical composition to a patient in need thereof, whereby the target cell or the target organism is opsonized in the patient.

In an embodiment of the third aspect, the kit further comprises an antibody, wherein the antibody is caused to associate with the target cell or the target organism by the hapten; and directions for administering the antibody to a patient in need thereof. hi an embodiment of the third aspect, the antibody is a synthetic antibody or an antibody not naturally occurring in the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 provides the structure of a polymer of a preferred embodiment. The subscripts x, y, and z denote the average number of side-chains of vancomycin, unsubstituted amide, and fluorescein, respectively, per polymer strand.

Figure 2 provides a schematic representation of a process that results in phagocytosis of a Gram-positive bacterium directed by a bifunctional polymer (pA-V-F) that binds to the cell wall via vancomycin and displays fluorescein as an antigen, (a) The cell wall of a Gram- positive bacterium is composed predominantly of peptidoglycan consisting of repeating disaccharide units with a peptide moiety (terminating in D-AIa-D-AIa; represented as filled triangles) acting as cross-linking agent, (b) The bifunctional polymer binds to the bacterial surface mediated by the vancomycin side chains (represented as "Y") interacting with D-AIa- D-AIa residues of the peptidoglycan. (c) The bacterial surface is subsequently recognized by anti-fluorescein IgG (from mouse) specific for the second group (fluorescein, represented as open circles) that the polymer presents, (d) Macrophages (culture J774 cells from mouse) bind to the opsonized bacteria mediated by interactions with the Fc region of the bound IgGs and (e) internalize the bacteria-polymer-antibody aggregate into a phagosome. Figure 3 provides fluorescence micrographs demonstrating binding of polyacrylamide with pendant vancomycin and fluorescein groups (pA-V-F) to Gram-positive bacteria and not to Gram-negative bacteria.

Figure 4 provides fluorescence micrographs demonstrating binding of pA-V-F to S. epidermidis and subsequent decoration with R-phycoerythrin-labeled anti-fluorescein IgG (IgG(anti-fluor)phyoo) .

Figure 5 provides a schematic representation for the preparation of samples for opsonization and phagocytosis experiments. The three groups of bacteria used in phagocytosis studies are untreated bacteria (no polymer or antibody) (1), pA-V-F / IgG(control) (3a), and pA-V-F / IgG(anti-fluor) (4a). The four groups of bacteria used in opsonization studies are untreated bacteria (no polymer or antibody) (1), pA-F / IgG(anti- fluor) / IgG(anti-mouse)phyco (2b), pA-VF / IgG(control) / IgG(anti-mouse)phyco (3b), and pA- V-F / IgG(anti-fluor) / IgG(anti-mouse)phyco (4b). The amount of primary antibody (IgG(anti- fluor) and IgG(control)) bound to the bacterial surface is only qualitatively represented, and does not quantitatively reflect the relative amount of each antibody bound.

Figure 6 provides representative flow cytometry histograms demonstrating specific binding of antifluorescein IgG to pA-V-F labeled bacteria. The dotted line indicates the maximum phycoerythrin fluorescence intensity observed for the untreated bacteria (threshold).

Figure 7 provides flow cytometry data demonstrating greater phagocytosis of pA- V-F- labeled S. aureus when treated with anti-fluorescein IgG (IgG(anti-fluor)) than when treated with isotype-control IgG (IgG(control)). (a) Representative histograms (with one hour incubation of bacteria with macrophages) showing the fluorescence intensity of 5000 events of the macrophage population. The dotted line indicates the fluorescence threshold that was set on the basis of the maximum fluorescence intensity from untreated control bacteria. The fluorescein fluorescence intensity shifts to higher values for the macrophage population when incubated with IgG(anti-fluor) than when incubated with IgG(control). Summarized data from four independent experiments are expressed as (b) the percentage of macrophages that are fluorescent (based on the threshold set in (a)), and (c) mean fluorescence intensity of all events. The error bars in (b) and (c) represent standard errors of the mean from the independent measurements. The legend in (b) applies to both (b) and (c). Comparisons were made among groups within each time point and the differences between all three groups were found to be statistically significant (p < 0.05) using one-way analysis of variance (ANOVA) followed by a Bonferroni post-hoc test.

Figure 8 depicts S. aureus bacteria labeled with pA-V-F and anti-fluorescein IgG (IgG(anti-fluor)) are ingested by J774 macrophages, (a) Unfixed macrophages were prepared for visualization by optical microscopy by centrifugation and by staining with the Diff-Quik® stain set (Dade Behring). In this image of a representative macrophage, the S. aureus appear as small (0.8 μm diameter), darkly-stained spheres (referred to as cocci). Cocci are evident both associated with the external cell membrane of the macrophage (thick black arrow) and internalized in intracellular phagosomes of the macrophage (thin black arrow). The large, dark object in the center of the macrophage (white arrow) is its nucleus, (b) Unstained macrophages were also imaged using fluorescence microscopy. This image is a fluorescence micrograph (fluorescence from fluorescein) merged with a phase contrast micrograph of the same field. The fluorescent signals (white) arising from the pA-V-F-labeled S. aureus appear to originate from within the macrophage. This intensity must arise from a large cluster of cocci; single cocci are not resolved using this technique. The scale bar applies to both images.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following description and examples illustrate a preferred embodiment of the present invention in detail. Those of skill in the art will recognize that there are numerous variations and modifications of this invention that are encompassed by its scope. Accordingly, the description of a preferred embodiment should not be deemed to limit the scope of the present invention. Certain embodiments are described in relation to multifunctional polymers comprising polyacrylamide functionalized with vancomycin and fluorescein groups, and opsonization and subsequent phagocytosis of Gram-positive bacteria. It is to be understood that such embodiments also encompass other multifunctional polymer systems, and other target cells, as described in detail herein.

A multifunctional polymer is provided that presents a recognition group or ligand capable of binding to and modifying the surface of a target cell, and that also presents a hapten capable of binding to an antibody. Such polymers include, for example, a bifunctional polymer presenting vancomycin recognition groups and hapten fluorescein groups capable of binding to and modifying the surfaces of Gram-positive bacteria (e.g., S. aureus, S. epidermidis, S. pneumoniae, and E. faecalis). The vancomycin groups allow the specific recognition of a structural component of the bacterial cell wall, namely, peptides terminated in D-AIa-D-AIa. The fluorescein groups are low molecular weight haptens recognized by antifluorescein antibodies. The bound antibodies then promote phagocytosis of the bacteria by macrophages. Specifically, polymer-labeled S. aureus and S. pneumoniae can be efficiently opsonized by anti-fluorescein antibodies, and macrophages more readily ingest S. aureus decorated with the polymer-antibody complexes.

The multifunctional polymers of the preferred embodiments can carry out three distinct functions based on polyvalent molecular recognition: i) recognition of the surface of target cells, ii) modification of the cell surface to generate specific binding sites recognized by an antibody, and iii) promotion of phagocytosis of the opsonized cell. For example, a bifunctional polyacrylamide presenting both vancomycin and fluorescein groups as side chains (p A-V-F) (Figure 1) binds to the surfaces of Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis) (Figure 2). Gram-positive bacterial infections can be treated by converting the surfaces of Gram-positive bacteria into surfaces presenting controllable recognition elements by treatment with a bifunctional polymer (Figure 2b). After being labeled by the polymer, the bacteria are opsonized by antibodies (Figure 2c) directed against the haptens that are introduced by the polymer. The antibody-polymer-bacteria complexes are subsequently ingested by macrophages (Figures 2d and 2e). Likewise, other cells can be targeted, e.g., other types of bacteria, viruses, autoimmune disease causing B cells, and malignant cells, such as the cells associated with breast cancer or prostate cancer, by selecting a suitable recognition element (moiety or ligand).

The processes of the preferred embodiments provide new methods of promoting interaction between the components of the immune system (e.g., antibodies, macrophages) and cell surfaces (e.g., bacteria, viruses, cancer cells) not normally recognized by the immune system. The polymers and processes of preferred embodiments are typically directed toward specific types of cell markers, specific proteins, or other structures on the surface of a particular pathogen or target cell. The ligands that are targeted include, e.g., D-AIa-D-AIa residues at the carboxy terminus of some of the peptides in the peptidoglycan cell wall of bacteria (Figure 2a) (see, e.g., Rogers, H. J., Perkins, H. R. & Ward, J. B. Microbial Cell Walls and Membranes (Chapman and Hall, New York, 1980)). These residues provide recognition sites, dispersed throughout the cell wall, for the antibiotic vancomycin, which binds to them with good affinity (Kd- μM) (Nieto, M. & Perkins, H. R. Modifications of the acyl-D-alanyl-D-alanine terminus affecting complex-formation with vancomycin. Biochem. J. 123, 789-803 (1971); Nieto, M. & Perkins, H. R. Physicochemical properties of vancomycin and iodovancomycin and their complexes with diacetyl-L-lysyl-D-alanyl-D- alanine. Biochem. J. 123, 773-787 (1971)).

Vancomycin serves as an antibiotic by inhibiting cross-linking of the D-AIa-D-AIa- terminated peptides during formation and remodeling of the cell wall (i.e., cell wall biosynthesis) of Gram-positive bacteria by binding to these peptides at the membrane surface, and thus inhibiting the action of transpeptidases (Barna, J. C. & Williams, D. H. The structure and mode of action of glycopeptide antibiotics of the vancomycin group. Annu. Rev. Microbiol. 38, 339-357 (1984)). Vancomycin-resistant Gram-positive bacteria evade the cytotoxic properties of vancomycin by expressing three essential enzymes in response to the binding of vancomycin to VanS, a transmembrane sensor protein kinase (Walsh, C. T. Vancomycin Resistance: Decoding the Molecular Logic. Science 261, 308-309 (1993)). The net effect of the action of these enzymes is to replace D-AIa-D-AIa in the bacterial cell wall with D-Ala-D-Lac; vancomycin binds to D-Ala-D-Lac with 10³ -fold lower affinity than to D- Ala-D-Ala (Kd ~ mM), attenuating the potency of the antibiotic. Gram-negative bacteria (e.g., E. colϊ) are not susceptible to the antibiotic activity of vancomycin because their cell wall is protected by an outer membrane that is impermeable to vancomycin.

Vancomycin groups are preferably included in the bifunctional polymer pA-V-F to promote specific binding of the polymer to the surfaces of Gram-positive bacteria (Figure 2b), and to enhance avidity due to polyvalency so as to form a stable complex between pA-V- F and D-Ala-D-Lac-terminated peptides on the surface of a vancomycin-resistant bacterium. This approach exploits polyvalency by using a polyvalent scaffold, which can generate a high-avidity binder for a given surface from low-affinity recognition units even if not all of the recognition units are involved directly in binding to their cognate surface receptors (Mammen, M. et al. Optically controlled collisions of biological objects to evaluate potent polyvalent inhibitors of virus-cell adhesion. Chem. Biol. 3, 757-763 (1996); Mammen, M., Dahmann, G. & Whitesides, G. M. Effective Inhibitors of Hemagglutination by Influenza- Virus Synthesized from Polymers Having Active Ester Groups - Insight into Mechanism of Inhibition. J. Med. Chem. 38, 4179-4190 (1995); Lees, W. J., Spaltenstein, A., Kingerywood, J. E. & Whitesides, G. M. Polyacrylamides Bearing Pendant α-Sialoside Groups Strongly Inhibit Agglutination of Erythrocytes by Influenza A Virus: Multivalency and Steric Stabilization of Particulate Biological Systems. J. Med. Chem. 37, 3419-3433 (1994); Sigal, G. B., Mammen, M., Dahmann, G. & Whitesides, G. M. Polyacrylamides bearing pendant alpha-sialoside groups strongly inhibit agglutination of erythrocytes by influenza virus: The strong inhibition reflects enhanced binding through cooperative polyvalent interactions. J. Am. Chem. Soc. 118, 3789-3800 (1996); Mourez, M. et al. Designing a polyvalent inhibitor of anthrax toxin. Nat. Biotechnol. 19, 958-961 (2001); Sanders, W. J. et al. Inhibition of L-selectin-mediated leukocyte rolling by synthetic glycoprotein mimics. J. Biol. Chem. 274, 5271-5278 (1999); Yang, Z. Q., Puffer, E. B., Pontrello, J. K. & Kiessling, L. L. Synthesis of a multivalent display of a CD22-binding trisaccharide. Carbohydr. Res. 337, 1605-1613 (2002); Gestwicki, J. E., Strong, L. E. & Kiessling, L. L. Tuning chemotactic responses with synthetic multivalent ligands. Chem. Biol. 7, 583-591 (2000); Gestwicki, J. E. et al. Designed potent multivalent chemoattractants for Escherichia coll Bioorg. Med. Chem. 9, 2387-2393 (2001)).

The polyvalent binding of pA-V-F to a self-assembled monolayer (SAM) that presents D-AIa-D-AIa groups has been investigated using surface plasmon resonance spectroscopy (SPR) (Metallo, S. J., Kane, R. S., Holmlin, R. E. & Whitesides, G. M. Using bifunctional polymers presenting vancomycin and fluorescein groups to direct anti- fluorescein antibodies to self-assembled monolayers presenting D-alanine-D-alanine groups. J. Am. Chem. Soc. 125, 4534-4540 (2003)). These studies demonstrated that pA-V-F formed kinetically stable (koiτ~ 2 x 10^"6 s-i) complexes with high avidity at the surface presenting D- Ala-D-Ala groups. These studies also indicated that a thin (6.5 nm) layer of p A- V-F bound to the surface recruited anti-fluorescein antibodies to that surface almost as well as did a surface directly presenting fluorescein at a high surface density.

In preferred embodiments, molecular recognition of D-Ala-D-Ala by vancomycin (as opposed to the antibiotic properties of this molecule) to bind a polymer with pendant vancomycin groups to the surface of Gram-positive bacteria is employed. Polymers that present vancomycin in a polyvalent manner are provided which demonstrate vancomycin- dependent binding of the polymer to the cell wall of bacteria. This approach is different from that of previous uses of monofunctional polymers of vancomycin as potential antibiotics (Arimoto, H., Nishimura, K., Kinumi, T., Hayakawa, I. & Uemura, D. Multi-valent polymer of vancomycin: enhanced antibacterial activity against VRE. Cheni. Commun. 15, 1361-1362 (1999)).

The effective grafting of the polymer to the bacterial surface gives the ability to modify the molecules displayed at the surface of the bacteria by incorporating additional groups (i.e., in addition to vancomycin) of arbitrary structure into the polymer, e.g., fluorescein. Such a polymer (pAV-F) allowed bacteria that had their surface modified by adsorption of polymer to be visualized by fluorescence, and also promoted subsequent binding of anti-fluorescein antibodies to the modified bacteria (Figures 2b and 2c). These opsonized bacteria were then recognized and ingested by macrophages (Figures 2d and 2e). This provides new methods and polymers for the coupling of antibodies and macrophages to the surfaces of pathogens using a multifunctional polymer. Multifunctional Polymer m the methods of preferred embodiments, a plurality of hapten moieties attached to a polymer backbone are associated with a Gram-positive bacterium or other target cell via a plurality of recognition groups also attached to the polymer backbone. Any suitable polymer backbone can be employed. Suitable polymer backbones are preferably water soluble, or capable of being formulated or functionalized so as to be deliverable to the target cell.

Ligand-functionalized monomers can be polymerized directly using ring-opening metathesis polymerization (ROMP) (Trnka, T. M.; Grubbs, R. H., The development of L2X2Ru = CHR olefin metathesis catalysts: An organometallic success story. Ace. Chem. Res. 2001, 34, 18-29; Novak, B. M.; Risse, W., Grubbs, R. H., The Development of WeIl- Defined Catalysts for Ring-Opening Olefin Metathesis Polymerizations (ROMP). Advances in Polymer Science 1992, 102, Al -12). This process yields fully functionalized polymers of controllable valencies and lengths (Kanai, M.; Mortell, K. H.; Kiessling, L. L., Varying the size of multivalent ligands: The dependence of concanavalin a binding on neoglycopolymer length. J. Am. Chem. Soc. 1997, 119, 9931-9932; Strong, L. E.; Kiessling, L. L., A general synthetic route to defined, biologically active multivalent arrays. J. Am. Chem. Soc. 1999, 121, 6193-6196; Mortell, K. H.; Weatherman, R. V.; Kiessling, L. L., Recognition specificity of neoglycopolymers prepared by ring-opening metathesis polymerization. J. Am. Chem. Soc. 1996, 118, 2297-2298; Mortell, K. H.; Gingras, M.; Kiessling, L. L., Synthesis of Cell Agglutination Inhibitors by Aqueous Ring-Opening Metathesis Polymerization. J. Am. Chem. Soc. 1994, 116, 12053-12054; Gestwicki, J. E.; Kiessling, L. L., Inter-receptor communication through arrays of bacterial chemoreceptors. Nature 2002, 415, 81-84; Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L., Influencing receptor-ligand binding mechanisms with multivalent ligand architecture. J. Am. Chem. Soc. 2002, 124, 14922-14933). Polymers with less than quantitative loading of ligand can be synthesized by co-polymerizing ligand-functionalized and unfunctionalized monomers. (Spaltenstein, A.; Whitesides, G. M., Polyacrylamides Bearing Pendant α-Sialoside Groups Strongly Inhibit Agglutination of Erythrocytes by influenza Virus. J. Am. Chem. Soc. 1991, 113, 686-687; Lees, W. J.; Spaltenstein, A.; Kingerywood, J. E.; Whitesides, G. M., Polyacrylamides Bearing Pendant α-Sialoside Groups Strongly Inhibit Agglutination of Erythrocytes by Influenza A Virus: Multivalency and Steric Stabilization of Particulate Biological Systems. J. Med. Chem. 1994, 37, 3419-3433).

The degree of loading of the polymer with ligand depends on the ratio of ligand- functionalized to unfunctionalized monomer and their relative reactivities (Odian, G., Principles of Polymerization, ed.; John Wiley & Sons, Inc.: New York, 1991). This approach of polymerizing ligand-functionalized monomers offers the following advantages: i) monomers can be fully characterized before polymerization, and ii) controllable valencies are accessible using ROMP. The polymerization technique is selected so as to be compatible with the functional groups on the ligand. Preferably, the methods used are such that ligand- functionalized monomers can be readily synthesized with a predictable loading density of ligands (when ligand-functionalized and unfunctionalized monomers are co-polymerized, and have a difference in reactivity towards polymerization), and a predictable distribution of ligands (when ligand-functionalized and unfunctionalized monomers are co-polymerized) along the polymer backbone (block co-polymers or random co-polymers often cannot be readily distinguished).

It is generally preferred to maximize the number of recognition groups and haptens associated with the target cell. Optimal association is generally associated with a linear polymer backbone. However, in certain embodiments it can be acceptable, or even desirable, to employ a backbone with some degree of branching. A minor degree of branching is generally acceptable. A high degree of crosslinking or branching, e.g., as in a dendritic polymer, can reduce the number of recognition groups and haptens exposed to the target cell (on a weight basis of polymer), but may confer other advantages in certain embodiments.

It is generally preferred that each polymer strand include a plurality of hapten moieties and a plurality of recognition moieties. The number of hapten and recognition moieties that can be incorporated on the polymer strand depends upon the number of reactive groups present on the polymer strand (in the case of a polymer backbone that is functionalized), or the number and kind of monomers with reactive groups that are polymerized to yield the polymer chain, hi certain embodiments, it is preferred to maximize the number of hapten and/or recognition moieties per weight unit of polymer. Thus, in such embodiments, a shorter polymer strand is generally preferred over a longer polymer strand having the same number of hapten and recognition moieties, hi certain embodiments it can also be preferred to minimize the size of functionalized monomers, and to minimize the number of monomers not bearing hapten or recognition moieties when a mixture of functionalized and non- functionalized monomers is reacted to yield the polymer, so as to maximize the number of hapten and recognition moieties on a polymer weight basis, hi certain embodiments, however, a lower degree of loading of hapten and/or recognition moieties can be desirable, for example, to minimize aggregation or insolubility associated with a higher degree of loading. Polymers having more than about 20 repeating units are generally preferred. In certain embodiments, oligomers (e.g., having 10-20 repeating units or less) can be employed, provided that the oligomer backbone has one or more (preferably a plurality) recognition moieties and one or more (preferably a plurality) of hapten moieties. A polymer of any suitable number of repeating units can be employed as the backbone, however, lower molecular weights can be preferred as having an increased solubility when compared to higher molecular weight polymers. As discussed above, the optimal number of repeating units depends upon the nature of the repeating units and the resulting polymer.

A polymer backbone or reactive monomers of any suitable chemistry having groups capable of functionalization to add recognition groups and hapten groups can be employed. Polyacrylamide is particularly preferred, in that it presents both carboxyl and imide groups that can be functionalized. Carbohydrates are also suitable for use, in that they present a plurality of hydroxy! groups that can be functionalized. Suitable carbohydrates include hydroxypropylmethylcellulose and carboxymethylcellulose. Acrylic acid polymers which present carboxyl groups for reaction include polycarbophil, carbomer (acrylic acid polymer), poly(methylmethacrylate) acrylic acid/butyl acrylate copolymers, and the like.

A single functionalized polymer can be employed, or combinations of two or more different polymers (e.g., different recognition and/or hapten moieties, different molecular weights, different degrees of functionalization) can be employed. It is generally preferred to employ a polymer having a single peak molecular weight distribution; however, in certain embodiments it can be preferred to employ a polymer with a bimodal or multimodal molecular weight distribution, with varying amounts of polymer chains having different molecular weights.

If the polymer is a copolymer, it can be in any configuration, e.g., block, tapered, random, hi certain embodiments, the polymer can include a segment or segments having recognition moieties (same or different), a segment or segments having hapten moieties (same or different), a segment or segments having both recognition moieties (same or different) and hapten moieties (same or different), and/or a segment or segments imparting at least one other property to the polymer (e.g., increased solubility in aqueous environment, spacing between segments or hapten and recognition moieties, flexibility or rigidity, and the like). Any combination of segments or a single segment can be employed.

As stated above, in certain embodiments it can be preferred to include the maximum number of hapten and recognition groups on the polymer as is possible (about 100% reaction of groups capable of functionalization). However, in certain embodiments, a lesser degree of functionalization can be acceptable or even desirable, e.g., less than about 10, 20, 30, 40, 50, 60, 70 80, or 90 % of the available groups capable of functionalization actually undergoing functionalization. It can also be desirable, in certain embodiments, to cap the ends or excess unreacted reactive groups of the polymer with suitable functional groups.

In particularly preferred embodiments, a polyacrylamide backbone is employed. Recognition Moiety

The multifunctional polymers of preferred embodiments also incorporate moieties or ligands capable of associating with a receptor on the surface of the target cell. As discussed above, vancomycin groups are preferred recognition moieties for target cells including Gram- positive bacteria, which have D- AIa-D- Ala surface groups (Sheldrick, G. M.; Jones, P. G.; Kennard, O.; Williams, D. H.; Smith, G. A., Structure of Vancomycin and its Complex with Acetyl-D-Alanyl-D-Alanine. Nature 1978, 271, 223-225).

Other recognition moieties can be preferred for use with other types of cells. For example, the surface of Enteroaggregative E. coli includes hemagglutinin surface groups which are capable of associating with sialic acid (Qadri, F.; Haque, A.; Faruque, S. M.; Bettelheim, K. A.; Robinsbrowne, R.; Albert, M. J., Hemagglutinating Properties of Enteroaggregative Escherichia-Coli. J. Clin. Microbiol. 1994, 32, 510-514).

Fimbrilated E. coli has FimH adhesin surface groups which can interact with mannose groups (Wu, X. R.; Sun, T. T.; Medina, J. J., In vitro binding of type 1-fimbriated Escherichia coli to uroplakins Ia and Ib: Relation to urinary tract infections. Proc. Natl. Acad. ScL U. S. A. 1996, 93, 9630-9635; Krogfelt, K. A.; Bergmans, H.; Klemm, P., Direct Evidence That the Fimh Protein Is the Mannose-Specific Adhesin of Escherichia-Coli Type-1 Fimbriae. Infect. Immun. 1990, 55, 1995-1998).

S. pneumoniae includes pneumococcal surface adhesin A (PsaA) surface groups capable of bonding to GlcNac groups, choline binding protein A (CpbA) groups capable of bonding to NeuAc and lacto-N-neotetraose groups, and α-enolase groups capable of bonding to plasmin(ogen) groups (Bogaert, D.; de Groot, R.; Hermans, P. W. H., Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect. Dis. 2004, 4, 144- 154; Rosenow, D.; Ryan, P.; Weiser, J. N.; Johnson, S.; Fontan, P.; Ortqvist, A.; Masure, H. R., Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae. MoI. Microbiol. 1997, 25, 819-829; Bergmann, S.; Wild, D.; Diekmann, O.; Frank, R.; Bracht, D.; Chhatwal, G. S.; Hammerschmidt, S., Identification of a novel plasmin(ogen)-binding motif in surface displayed alpha-enolase of Streptococcus pneumoniae. MoI. Microbiol. 2003, 49, 411-423; Bergmann, S.; Rohde, M.; Chhatwal, G. S.; Hammerschmidt, S., alpha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface. MoI. Microbiol. 2001, 40, 1273-1287).

S. aureus includes collagen adhesin surface groups that can associate with collagen (Hudson, M. C; Ramp, W. K.; Frankenburg, K. P., Staphylococcus aureus adhesion to bone matrix and bone-associated biomaterials. FEMS Microbiol. Lett. 1999, 173, 279-284; Bronner, S.; Monteil, H.; Prevost, G., Regulation of virulence determinants in Staphylococcus aureus: complexity and applications. FEMS Microbiol. Rev. 2004, 28, 183- 200; Gurusiddappa, S.; Xu, Y.; Hook, A.; Liang, X.; Hook, M., Identification of binding sites in collagen for the Staphylococcus aureus collagen adhesin. Biopolymers 2003, 71, 377-377; Patti, J. M.; Boles, J. O.; Hook, M., Identification and Biochemical-Characterization of the Ligand-Binding Domain of the Collagen Adhesin from Staphylococcus-Aureus. Biochemistry 1993, 32, 11428-11435; Patti, J. M.; Housepompeo, K.; Boles, J. O.; Garza, N.; Gurusiddappa, S.; Hook, M., Critical Residues in the Ligand-Binding Site of the Staphylococcus-Aureus Collagen-Binding Adhesin (Mscramm). J. Biol. Chem. 1995, 270, 12005-12011; Symersky, J.; Patti, J. M.; Carson, M.; HousePompeo, K.; Teale, M.; Moore, D.; Jin, L.; Schneider, A.; DeLucas, L. J.; Hook, M.; Narayana, S. V. L., Structure of the collagen-binding domain from a Staphylococcus aureus adhesin. Nature Struct. Biol. 1997, 4, 833-838).

P. aeruginosa includes pilus adhesin surface groups that can associate with GlcNac, NeuAc, and lactose (Sheth, H. B.; Lee, K. K.; Wong, W. Y.; Srivastava, G.; Hindsgaul, O.; Hodges, R. S.; Paranchych, W.; Irvin, R. T., The PiIi of Pseudomonas-Aeruginosa Strains Pak and Pao Bind Specifically to the Carbohydrate Sequence Beta-Galnac(l-4)Beta-Gal Found in Glycosphingolipids Asialo-Gm(l) and Asialo-Gm(2). MoI. Microbiol. 1994, 11, 715-723; Hahn, H. P., The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa - A review. Gene 1997, 192, 99-108).

Breast cancer cells include Her2 receptor surface groups capable of associating with AHNP or shorter peptides (Park, B. W.; Zhang, H. T.; Wu, C. J.; Berezov, A.; Zhang, X.; Dua, R.; Wang, Q.; Kao, G.; O'Rourke, D. M.; Greene, M. L; Murali, R., Rationally designed anti-HER2/neu peptide mimetic disables pl85(HER2/neu) tyrosine kinases in vitro and in vivo. Nat. Biotechnol. 2000, 18, 194-198).

Tumor antigens present on the surface of cancer cells can be targeted by aptamers. Aptamers are macromolecular ligands composed of nucleic acid, such as RNA or DNA, which bind tightly to a specific molecular target. The chain of nucleotides that makes up the aptamer forms intramolecular interactions that fold the molecule into a complex three- dimensional shape. The shape of the aptamer allows it to bind tightly against the surface of its target molecule. Because a diversity of molecular shapes exist within the universe of all possible nucleotide sequences, aptamers can be obtained for a wide array of molecular targets, including most proteins and many small molecules. The surface area of interaction between an aptamer and its molecular target is relatively large, so even small changes in the target molecule can disrupt aptamer association. Thus, aptamers can distinguish between closely related but non-identical members of a protein family, or between different functional or confoπnational states of the same protein, hi addition to high specificity, aptamer have very high affinities to their targets. Typically aptamers generated against proteins have affinities in the picomolar to low nanomolar range, hi a preferred embodiment, an RNA aptamer is employed as a recognition moiety that binds to prostate specific membrane antigen (PSMA), a well-known prostate cancer tumor marker that is overexpressed on prostate acinar epithelial cells. See Farokhzad, O. et al. Cancer Res. 64(21), 7668-72 (2004). Recent progress in the identification of human tumor antigens has generated a repertoire of target molecules for use in antigen-specific tumor vaccines. Unique cancer antigens on human cancers including melanoma, breast cancer, colon cancer, and lymphoma have been identified. See Rosenberg, S.A., A new era for cancer immunotherapy based on the genes that encode cancer antigens. Immunity 10, 281-287, (1999); Jager D., Immune response to tumour antigens: implications for antigen specific immunotherapy of cancer. J Clin Pathol, 54: 669-674 (2001). Such antigens can be targeted by the recognition groups in multifunctional polymers of preferred embodiments.

Another target group on cancer cells {e.g., ovarian, endometrial, colorectal, breast, lung, renal cell carcinomas, brain metastases from epithelial cancers, and neuroendocrine carcinomas) is the folate receptor. This receptor can be targeted by folate recognition moieties on a multifunctional polymer. See Sudimack, J. et al., Targeted drug delivery via the folate receptor. Adv. Drug Delivery Rev. 41, 147-162 (2000).

Chlorotoxin, a peptide derived from the venom of the giant Israeli scorpion, binds specifically to a tumor surface marker found in the vast majority of gliomas. See Deshane, J. et al., J. Biol Chem., 278(6), 4135-4144, (2003).

B cells that secrete antibodies that cause an autoimmune disease can also be targeted. For example, a phospholipid (e.g., cardiolipin) can be employed on the multifunctional polymer to bind B cells displaying this IgG associated with antiphospholipid antibody syndrome. For lupus, doubled stranded DNA can be incorporated into the multifunctional polymer to target dsDNA displaying B cells. See, e.g., Merrill, J.T. et al., The Bench to Bedside in Drug Development for SLE. Nature Rev. Drug Discovery 2, 1036-1046 (2004).

Any suitable recognition group can be employed, including known recognition groups, or recognition groups derived from technologies such as phase display. Phase display can be employed to identify a peptide that binds to a minimum degree to a biomarker. See, e.g., Mourez, M. et al. Nat. Biotechnol. 19, 958-961 (2001). Other suitable screening methods can also be employed, e.g., combinatorial chemistry. Antibodies can also function as recognition units, and can be incorporated as such into the multifunctional polymers of preferred embodiments.

As discussed above, it is not necessary that the polymer recognition group(s) have a strong avidity for the target cell surface group. Because a plurality of recognition groups is presented to the target cell surface, even a weak affinity can achieve an effective association between the multifunctional polymer and the target cell. Generally it is preferred that the recognition moiety have an affinity on the order of at least micromolar levels, preferably millimolar levels or higher, hi certain embodiments, however, an affinity of less than micromolar levels can be acceptable, if a sufficient number of recognition groups are presented to the target cell surface. Hapten Moiety

Haptens are low-molecular weight molecules which contain an antigenic determinant but which are not themselves antigenic unless complexed with an immunogenic carrier. The multifunctional polymers of the preferred embodiments incorporate hapten moieties that can enhance opsonization of the target cell. The methods described above for incorporation of recognition moieties into the multifunctional polymer can be adapted to incorporation of hapten moieties, e.g., functionalization of a polymer with hapten moieties, polymerization of hapten-containing monomers, and the like.

Haptens that can recruit endogenous human antibodies include polysaccharides, e.g., polysaccharides capable of binding to anti-alpha(Gal) antibodies.

A variety of haptens of different chemical structure have been shown to induce similar types of immune responses: e.g., dinitrophenyl (DNP); trinitrophenyl (TNP); phosphorylcholine; nickel; and arsenate. Conjugation of a hapten to a cell can, for example, be accomplished by conjugation via ε-amino groups or carboxyl groups. Such haptens include, without limitation, compounds such as halonitrobenzenes (including dinitrofluorobenzene, difluorodinitrobenzene, trinitrofluoro-benzene), N-iodoacetyl-N'-(5- sulfonic-1-naphthyl) ethylene diamine, nitrobenzene sulfonic acids (including trinitrobenzenesulfonic acid and dinitrobenzene sulfonic acid), fluorescein isothiocyanate, arsenic acid benzene isothiocyanate, sulfanilic acid, arsanilic acid, and dinitrobenzene-S- mustard.

Haptens include a group that binds or interacts with an antibody. Examples of such binding groups include, without limitation, dinitrophenyl, trinitrophenyl, fluorescein, other aromatics, phosphorylcholine, peptides, advanced glycosylation end products (AGE), carbohydrates, and the like. The haptens are conjugated to the polymer through a suitable functional group on the polymer backbone, preferably a primary amine group. Groups that can be conjugated to haptens include certain amino acid groups, free carboxylic acid groups, amino groups; thiol groups; hydroxyl groups; imide groups, imidazole groups; and aryl groups.

Hapten reactive groups that can form a covalent bond with primary amines present on a polymer backbone include, but not be limited to, acid chlorides, anhydrides, reactive esters, α,β-unsaturated ketones, imidoesters, and halonitrobenzenes.

Haptens can be bound to the polymer backbone or incorporated into the polymer in any suitable manner, for example, by activating carboxylic acids in the presence of carbodiimides, such as EDC, allowing for interaction with various nucleophiles, including primary and secondary amines. Alkylation of carboxylic acids to form stable esters can be achieved by interaction with sulfur or nitrogen mustards, or haptens containing either an alkyl or aryl aziridine moiety. Interaction of the aromatic moieties associated with certain amino acids can be accomplished by photoactivation of aryl diazonium compound in the presence of the protein or peptide. Thus, modification of the aryl groups, e.g., the aryl side chains of histidine, tryptophan, tyrosine, and phenylalanine, when included as pendant groups on a polymer backbone or as part of a polymer backbone, can be achieved by the use of such a reactive functionality.

There are several reactive groups that can be coupled to sulfhydryl groups. Haptens containing anα,β-unsaturated ketone or ester moiety, such as maleimide, provide a reactive functionality that can interact with sulfhydryl or amino groups.

Reactive disulfide groups include 2-pyridyldithioand 5,5'-dithio-bis-(2-nitrobenzoic acid) groups. Reagents containing reactive disulfide bonds include N-succinimidyl~3-(2- pyridyl-dithio) propionate, sodium S-4-succinimidyloxycarbonyl-alpha-methylbenzyl- thiosulfate, and 4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)-toluene. Some examples of reagents comprising reactive groups having a double bond that reacts with a thiol group include succinimidyl 4-(N-maleimidomethyl)cyclohexahe-l-carboxylate and succinimidyl m-maleimidobenzoate. Other functional molecules include succinimidyl 3- (maleimido)-propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate, sulfo- succinimidyl 4-(N-maleimidomethyl-cyclohexane)- 1 -carboxylate, maleimidobenzoyl-N- hydroxy-succinimide ester. The reactive groups on the polymer backbone used for bonding the hapten moiety can be the same or different. For example, in one embodiment, the same hapten is coupled to the polymer backbone through different reactive groups. For example, dinitrobenzene sulfonic acid, dinitro phenyldiazonium, and dinitrobenzene-S-mustard all form the dinitrophenyl hapten coupled to amino groups, aromatic groups, and carboxylic acid groups, respectively. Similarly, an arsonic acid hapten can be coupled by reacting arsonic acid benzene isothiocyanate to amino groups or azobenzenearsonate to aromatic groups. Haptens that can bind the polymer backbone through an azo linkage include sulfanilic acid, arsanilic acid, and phosphorylcholine.

Any single hapten or combination of different haptens can functionalize the polymer backbones of preferred embodiments. The polymer can be conjugated with haptens in any suitable amount and/or proportion. The haptens can comprise reactive groups that react with different functional groups on the polymer.

The hapten can associate with natural (endogenous) antibodies or synthetic (exogenous) antibodies. Haptens that associate with natural (endogenous) antibodies can be particularly desirable, in that they take advantage of the patient's own immune system. Such haptens can be part of a broadly administered vaccine such as the polio vaccine, with the hapten reacting with the endogenous antibodies. Haptens that recruit endogenous antibodies include sugars for blood type (e.g., A vs. B), HBsAg peptide 139-146 (as described in Deulofeut H et al., Cellular recognition and HLA restriction of a midsequence HBsAg peptide in hepatitis B vaccinated individuals. MoI Immunol. 30(10):941-8 (1993)), and polyribosyl phosphate (as used in vaccines for Haemophilis influenza type B). A particularly preferred hapten to recruit endogenous antibodies is the alpha Gal epitope.

In certain embodiments it can be desirable to employ a hapten that associates only with a synthetic (exogenous) antibody. In such an embodiment, a suitable synthetic antibody to the hapten is administered before, during, and/or after administration of the multifunctional polymer. Likewise, it can be desirable in certain embodiments to administer additional endogenous antibodies. Haptens that can recruit exogenous antibodies can include, for example, small molecules such as fluorescein, DNP, and the like, as listed above. Dosage forms The polymers of preferred embodiments can be administered in any suitable pharmaceutically acceptable form and by any suitable route of administration. Suitable administration routes can include topical, oral, subcutaneous, parenteral, intradermal, intramuscular, intraperitoneal, and intravenous. However, it is particularly preferred to administer the polymer in intravenous form rather than oral form, so as to compensate for any bioavailability issues relating to the polymer's size. The polymer can be formulated into any suitable solid or liquid form, either ready to use or for reconstitution, for example, a lyophilized form that is reconstituted for intravenous administration.

The polymer can advantageously be formulated into liquid preparations for, e.g., oral, nasal, anal, rectal, buccal, vaginal, peroral, intragastric, mucosal, perlingual, alveolar, gingival, olfactory, or respiratory mucosa administration. Suitable forms for such administration include suspensions, syrups, lotions, and elixirs. If nasal or respiratory (mucosal) administration is desired {e.g., aerosol inhalation or insufflation), compositions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or a dose having a particular particle size. Such aerosol forms can be particularly advantageous for treatment of respiratory bacterial infections.

The pharmaceutical compositions containing the polymer are preferably isotonic with blood or other body fluid of the patient. The isotonicity of the compositions can be attained using sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is particularly preferred. Buffering agents can be employed, such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

Viscosity of the pharmaceutical compositions can be maintained at the selected level using pharmaceutically acceptable thickening or thinning agents. Thinning agents can include physiologically acceptable solvents for the polymer. Methylcellulose is a preferred thickener because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener can depend upon the thickening agent selected. An amount is preferably used that can achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increase the shelf life of the pharmaceutical compositions. Benzyl alcohol can be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride can also be employed. A suitable concentration of the preservative is typically from about 0.02% to about 2% based on the total weight of the composition, although larger or smaller amounts can be desirable depending upon the agent selected.

The polymer can be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like, and can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins; 20th edition (June 1 , 2003) and "Remington's Pharmaceutical Sciences," Mack Pub. Co.; 18^th and 19^th editions (December 1985, and June 1990, respectively). Such preparations can include complexing agents, metal ions, additional polymeric materials such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. The presence of such additional components can influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application, such that the characteristics of the carrier are tailored to the selected route of administration.

For oral administration, the polymer can be provided as a tablet, aqueous or oil suspension, dispersible powder or granule, emulsion, hard or soft capsule, syrup or elixir. Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and can include one or more of the following agents: sweeteners, flavoring agents, coloring agents and preservatives. Aqueous suspensions can contain the active ingredient in admixture with excipients suitable for the manufacture of aqueous suspensions.

Formulations for oral use can also be provided as hard gelatin capsules, wherein the polymer is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules. In soft capsules, any active ingredients present can be dissolved or suspended in suitable liquids, such as water or an oil medium, such as peanut oil, olive oil, fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers and microspheres formulated for oral administration can also be used. Capsules can include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the polymer in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.

Tablets can be uncoated or coated by known methods to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate can be used. When administered in solid form, such as tablet form, the solid form typically comprises from about 0.001 wt. % or less to about 50 wt. % or more of active ingredient(s) including the polymer, preferably from about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 wt. % of active ingredients.

Tablets can contain the polymer in admixture with non-toxic pharmaceutically acceptable excipients including inert materials. For example, a tablet can be prepared by compression or molding, optionally, with one or more additional ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free- flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Preferably, each tablet or capsule contains from about 10 mg or less to about 1,000 mg or more of the polymer, more preferably from about 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg to about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900 mg. Most preferably, tablets or capsules are provided in a range of dosages to permit divided dosages to be administered. A dosage appropriate to the patient, the condition to be treated, and the number of doses to be administered daily can thus be conveniently selected. While it is generally preferred to incorporate the polymer and any other therapeutic agent employed in combination therewith in a single tablet or other dosage form, in certain embodiments it can be desirable to provide the polymer and other therapeutic agents in separate dosage forms, e.g., a polymer comprising a synthetic hapten in a dosage form separate from a synthetic antibody to the hapten. Combinations of dosage forms can also be employed, e.g., oral and intravenous.

Suitable inert materials include diluents, such as carbohydrates, mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, starch, and the like, or inorganic salts such as calcium triphosphate, calcium phosphate, sodium phosphate, calcium carbonate, sodium carbonate, magnesium carbonate, and sodium chloride. Disintegrants or granulating agents can be included in the formulation, for example, starches such as corn starch, alginic acid, sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite, insoluble cationic exchange resins, powdered gums such as agar, karaya or tragacanth, or alginic acid or salts thereof.

Binders can be used to form a hard tablet. Binders include materials from natural products such as acacia, tragacanth, starch and gelatin, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, and the like.

Lubricants, such as stearic acid or magnesium or calcium salts thereof, polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes, sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol, starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like, can be included in tablet formulations.

Surfactants can also be employed, for example, anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, cationic such as benzalkonium chloride or benzethonium chloride, or nonionic detergents such as polyoxyethylene hydrogenated castor oil, glycerol monostearate, polysorbates, sucrose fatty acid ester, methyl cellulose, or carboxymethyl cellulose.

Controlled release formulations can be employed wherein the polymer is incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms. Slowly degenerating matrices can also be incorporated into the formulation. Other delivery systems can include timed release, delayed release, or sustained release delivery systems.

Coatings can be used, for example, nonenteric materials such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols, or enteric materials such as phthalic acid esters. Dyestuffs or pigments can be added for identification or to characterize different combinations of active compound doses

When administered orally in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils can be added to the polymer to yield a solution or suspension. Physiological saline solution, dextrose, or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol are also suitable liquid carriers. The pharmaceutical compositions can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, such as olive or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsions can also contain sweetening and flavoring agents.

Pulmonary delivery of the polymer can also be employed. The polymer is delivered to the lungs while inhaling and can traverse across the lung epithelial lining to the blood stream. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be employed, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. These devices employ formulations suitable for the dispensing of polymer. Typically, each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants, and/or carriers useful in therapy.

The polymer and/or other active ingredients are advantageously prepared for pulmonary delivery in particulate form with an average particle size of from 0.1 μm or less to 10 μm or more, more preferably from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 μm to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 μm. Pharmaceutically acceptable carriers for pulmonary delivery of polymer include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations can include dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, distearoyl phosphatidylcholine, and dioleoyl phosphatidylcholine. Natural or synthetic surfactants can be used, including polyethylene glycol and dextrans, such as cyclodextran. Bile salts and other related enhancers, as well as cellulose and cellulose derivatives, and amino acids can also be used. Liposomes, microcapsules, microspheres, inclusion complexes, and other types of carriers can also be employed.

Pharmaceutical formulations suitable for use with a nebulizer, either jet or ultrasonic, typically comprise the polymer dissolved or suspended in an aqueous or other solution at a concentration of about 0.01 or less to 100 mg or more of the polymer (optionally with a synthetic antibody, if necessary) per mL of solution, preferably from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mg per mL of solution. The formulation can also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation can also contain a surfactant, to reduce or prevent surface induced polymer caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device generally comprise a finely divided powder containing the active ingredients suspended in a propellant with the aid of a surfactant. The propellant can include conventional propellants, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons. Preferred propellants include trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, 1,1,1,2- tetrafhioroethane, and combinations thereof. Suitable surfactants include sorbitan trioleate, soya lecithin, and oleic acid.

Formulations for dispensing from a powder inhaler device typically comprise a finely divided dry powder containing the polymer, optionally including a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol, in an amount that facilitates dispersal of the powder from the device, typically from about 1 wt. % or less to 99 wt. % or more of the formulation, preferably from about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. % of the formulation.

When the polymer is administered by intravenous, cutaneous, subcutaneous, parenteral, or other injection, it is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution or oleaginous suspension. Suspensions can be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The preparation of acceptable aqueous solutions with suitable pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for injection preferably contains an isotonic vehicle such as 1,3-butanediol, water, isotonic sodium chloride solution, Ringer's solution, dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution, or other vehicles as are known in the art. In addition, sterile fixed oils can be employed conventionally as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the formation of injectable preparations. The pharmaceutical compositions can also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

The duration of the injection can be adjusted depending upon various factors, and can comprise a single injection administered over the course of a few seconds or less to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more of continuous intravenous administration. Likewise, a single injection can be employed for treatment of certain infections or conditions, or a series of injections spaced over time can be administered.

The polymer can be administered systematically or locally, via a liquid or gel, or as an implant or device. The polymers of the preferred embodiments can additionally employ adjunct components conventionally found in pharmaceutical compositions in their art-established fashion and at their art-established levels. Thus, for example, the compositions can contain additional compatible pharmaceutically active materials for combination therapy (such as antibiotics, chemotherapeutic agents, natural antibodies, synthetic antibodies, and the like), or can contain materials useful in physically formulating various dosage forms of the preferred embodiments, such as excipients, dyes, perfumes, thickening agents, stabilizers, preservatives, and/or antioxidants.

It can be particularly desirable to administer the polymers of the preferred embodiments to the patient in combination with other therapeutic agents (e.g., in a same or different unit dosage form, that is administered at a same or different time), hi the case of the target cell being a bacteria, it can be advantageous to also administer a suitable anti-infective agent, e.g., anthelmintics (mebendazole), antibiotics including aminoclycosides (gentamicin, neomycin, tobramycin), antifungal antibiotics (amphotericin b, fluconazole, griseofulvin, itraconazole, ketoconazole, nystatin, micatin, tomaftate), cephalosporins (cefaclor, cefazolin, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, cephalexin), beta-lactam antibiotics (cefotetan, meropenem), chloramphenicol, macrolides (azithromycin, clarithromycin, erythromycin), penicillins (penicillin G sodium salt, amoxicillin, ampicillin, dicloxacillin, nafcillin, piperacillin, ticarcillin), tetracyclines (doxycycline, minocycline, tetracycline), bacitracin; clindamycin; colistimethate sodium; polymyxin b sulfate; vancomycin; antivirals including acyclovir, amantadine, didanosine, efavirenz, foscarnet, ganciclovir, indinavir, lamivudine, nelfinavir, ritonavir, saquinavir, stavudine, valacyclovir, valganciclovir, zidovudine; quinolones (ciprofloxacin, levofloxacin); sulfonamides (sulfadiazine, sulfisoxazole); sulfones (dapsone); furazolidone; metronidazole; pentamidine; sulfanilamidum crystallinum; gatifloxacin; and sulfamethoxazole/trimethoprim.

If the target cell is a cancer cell, a chemotherapeutic agent used to treat that particular cancer can be co-administered. In the case of breast cancer, suitable chemotherapeutic and hormonal agents can include anthracyclines (doxorubicin, epirubicin), cyclophosphamide, 5- fluorouracil (5-FU), methotrexate (CMF), taxanes (paclitaxel, docetaxel), capecitabine, trastuzumab, vinorelbine, gemcitabine, platinum-based agents (cisplatin, carboplatin, oxaplatin), edatrexate, losoxantrone, anastrozole, letrozole, exemestane, foπnestane, goserelin, fulvestrant, megestrol, and tamoxifen.

If the hapten that functionalizes the polymer associates only with synthetic antibodies, then the synthetic antibody is administered to the patient in conjunction with the polymer (either in the same unit dosage for, or administered in a separate form prior to, during, or after administration of the polymer). Similarly, supplemental natural antibodies can be administered to the patient, if desired, if the hapten can associate with the patient's natural antibodies.

The polymer can be provided to an administering physician or other health care professional in the form of a kit. The kit is a package which houses one or more containers which contain polymer in suitable form and instructions for administering the pharmaceutical composition to a subject. The kit can optionally also contain one or more additional therapeutic agents, e.g., synthetic antibodies for use in conjunction with a polymer comprising synthetic haptens. The kit can optionally contain one or more diagnostic tools and instructions for use, e.g., a diagnostic to characterize a bacterial infection, or identify a malignancy. For example, a kit containing a single composition comprising polymer in combination with one or more additional therapeutic agents can be provided, or separate pharmaceutical compositions containing polymer and additional therapeutic agents can be provided. The kit can also contain separate doses of polymer for serial or sequential administration. The kit can contain suitable delivery devices, e.g., syringes, inhalation devices, and the like, along with instructions for administrating polymer and any other therapeutic agent. The kit can optionally contain instructions for storage, reconstitution (if applicable), and administration of any or all therapeutic agents included. The kits can include a plurality of containers reflecting the number of administrations to be given to a subject.

In a particularly preferred embodiment, a kit for the treatment of a Gram-positive bacterial infection is provided that includes a polymer comprising a synthetic hapten, a synthetic antibody, and instructions for administering them. In another particularly preferred embodiment, a kit for the treatment of Gram-positive bacterial infections is provided that includes polymer and one or more diagnostics or instructions for conducting one or more diagnostics for characterizing the bacterial infection. The kit can also include instructions, an assay, or a diagnostic for determining if a patient has a Gram-positive bacterial infection. Methods of treatment

The polymers and methods of preferred embodiments are suitable for use in enhancing opsonization and subsequent phagocytosis of a variety of target cells, including any cell wherein selective killing of the cell has a benefit to the patient. Preferred target cells include pathogenic bacteria (e.g., Gram-positive bacteria), other pathogens (e.g. viruses such as influenza virus), cancer cells (e.g., breast cancer cells, prostate cancer cells), cells infected with viruses (e.g., HIV infected cells), and disease-causing immune cells. When the target cell is a Gram-positive bacterium, influenza virus, or another type of cell having D-AIa-D-AIa surface groups, the polymer is preferably modified with vancomycin target groups which can associate with D-Ala-D-Ala. However, any suitable target group capable of associating with a specific surface group of the target group can be employed, as described above. Gram-positive Bacteria

Particularly preferred target cells include Gram-positive bacteria. Pyogenic cocci are spherical bacteria that cause various suppurative (pus-producing) infections in animals. They include the Gram-positive cocci Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumonia. Other Gram-positive bacteria include Bacillus anthracis, Bacillus cereus, Clostridium botulimum, Clostridium tetani, Clostridium perfringens, and Clostridium difficil, Listeria monocytogenes, Streptomyces, Mycobacterium tuberculosi, Mycobacterium leprae, and Corynebacterium diphtheriae. Influenza

Influenza is a virus that infects the respiratory tract and causes the disease commonly known as "the flu." The type of influenza that poses the greatest threat to humans is influenza A. Antigenic variation in influenza A comes in a multitude of forms, enabling it to effectively evade the immune system. As with all RNA viruses, the influenza virus lacks a proofreader for replication, allowing the virus to mutate quickly. The host immune system selects for mutants by making antibodies to the original strain of virus. This leads to antigenic drift, whereby the virus slowly changes its form. In humans, changes in certain genes can lead to increasing virulency. Of the three influenza strains (A, B, and C), only A appears to infect humans and animals (birds, swine, horses, and seals). Influenza strains are usually species specific, yet both avian and human influenza strains can infect swine.

Cancer Cells

Her2 (human epidermal growth factor receptor 2), a protein receptor found on the surface of cells, is a key component in regulating cell growth. When the Her2 gene (sometimes referred to as Her2/neu) is altered, extra Her2 receptors may be produced. This over-expression of Her2 causes increased cell growth and reproduction, often resulting in more aggressive tumor cells. Her2 protein over-expression affects 25% to 30% of breast cancer patients. The peptide recognition moiety for the Her2 receptor is described in Park, B. W. Nat. Biotechnol. 18, 194-198 (2000). An RNA aptamer can be employed as a recognition moiety for prostate specific membrane antigen, a prostate cancer tumor marker that is overexpressed on prostate acinar epithelial cells. See Farokhzad, O. et al. Cancer Res. 64(21), 7668-72 (2004). These peptides can be linked to the polymer backbone as the recognition moieties for a multifunctional polymer directed to breast cancer cells or prostate cancer cells, respectively. Synthesis and Characterization of the Bifunctional Polymer (p A- V-F)

The bifunctional polymer, pA-V-F, consists of a polyacrylamide backbone with pendant vancomycin and fluorescein groups (Figure 1). While not wishing to be bound by theory, it is believed that the ordering of the side-chains on both polyacrylamide with pendant vancomycin and fluorescein groups (pA-V-F) and polyacrylamide with pendant fluorescein groups only (pA-F) is random due to the method of synthesis of the polymers. Vancomycin is present on the polymer at 5 sidechain % and fluorescein is present at 1 side-chain % (as determined by iH NMR and UV- Vis spectroscopy), hi this context, the "side-chain %" of a group represents the percentage of side chains in the polymer containing that group. A control polymer, pA-F, consisting of a polyacrylamide backbone with only pendant fluorescein groups (Figure 1).

In order to bind to the exterior surface of the microorganism, these polymers did not need to diffuse through the peptidoglycan layer (a three-dimensional structure with substantial porosity and an exclusion limit of -100 kDa) (Schrerrer, R. & Gerhardt, P. Molecular sieving by the Bacillus megaterium cell wall and protoplast. J. Bacteriol. 107, 718-735 (1971)) to the membrane surface (where the antibiotic activity of vancomycin is manifested); they could, therefore, be large.

It was confirmed that pA-VF does not serve as an antibiotic at the concentrations used in these experiments through two control studies: i) S. aureus treated with pA-V-F formed a number of colonies similar to that formed by bacteria that were not treated with polymer, and ii) E. faecalis grew normally in brain-heart infusion with (or without) pA-V-F. The average degree of polymerization for the polyacrylamide backbone was -900; this value corresponds to an average of 45 vancomycin groups and nine fluorescein groups per molecule of pA-V-F, a molecular weight of 136 kDa after functionalization, and an extended, end-to-end length of the polymer of approximately 100 nm. pA-V-F Bound Specifically to the Gram-Positive Bacteria S. aureus, S. epidermidis, and E. faecalis

Binding of polyacrylamide with pendant vancomycin and fluorescein groups (pA-V- F) to Gram-positive bacteria was demonstrated fir S. epideπnidis (Figure 3) and S. aureus (data not shown). A suspension of S. epidennidis with a solution of pA-V-F (10 μM in vancomycin, 2 μM in fluorescein) and the cell-permeable dye for nucleic acids Syto-63 (5 μM), in phosphate-buffered saline (PBS). The bacteria were then washed with PBS and examined by confocal fluorescence microscopy using an Ar-Kr laser for excitation of the fluorophores. The stained nucleic acid of the S. epidermidis was clearly visible when Syto-63 was selectively excited (Figure 3). For a large fraction of bacteria, fluorescence was observed from fluorescein (on pA-V-F) that co-localized with the fluorescence from Syto-63 (Figure 3); this result suggests a high labeling efficiency of the bacteria by pA-V-F. Binding of the polymer to S. aureus (data not shown) was also investigated. Suspensions of this bacterium were incubated in a solution of pA-V-F, washed, and examined using fluorescence microscopy. Syto-63 was omitted in order to eliminate any possibility of interference from strongly fluorescent complexes of Syto-63 and nucleic acid. Similar levels of fluorescence from fluorescein were observed from the pA-V-F-labeled S. aureus, S. epidermidis, and E. faecalis. A control experiment with S. aureus and pA-F, a polymer that does not have vancomycin side chains, showed no detectable fluorescence from fluorescein on the bacteria (data not shown). An additional control experiment using the Gram-negative bacterium, E. coli, incubated with Syto-63 and pA-V-F revealed fluorescence from Syto-63 but not from fluorescein (Figure 3). The results from these control experiments support the inference that binding of pA-V-F to the surface of bacteria requires vancomycin-mediated molecular recognition.

The binding of p A- V-F to vancomycin-resistant E.faecalis VanB was investigated to determine whether the polymer could be effective against these clinically important bacteria. While pA-V-F bound to vehicle (PBS)-treated VanB, it did not bind as effectively to VanB that had been grown overnight in 5 μM vancomycin (a concentration sufficient to induce expression of the vancomycin-resistance genes, which replace D-AIa-D-AIa at the surface of the bacteria with D-Ala-D-Lac) (Figure 3) (Baptista, M., Depardieu, F., Courvalin, P. & Arthur, M. Specificity of induction of glycopeptide resistance genes in Enterococcus faecalis. Antimicrob. Agents Chemother. 40, 2291-2295 (1996)). This result suggests that pA-V-F is not able to form a kinetically stable complex with the bacterial surface displaying D-AIa-D- Lac (presumably due to the low affinity of vancomycin for this sequence, KA ~ mM), and confirms the specificity of the vancomycin/D-Ala-D-Ala interaction in the binding of pA-V-F to the surfaces of Gram-positive bacteria, hi a second experiment, VanB was grown in the presence of pA-V-F (at the concentration used in binding experiments) in order to determine whether the polymer would induce active expression of the vancomycin-resistance genes. Figure 3 shows that pA-V-F bound to the surface of these treated bacteria (after rinsing with PBS and re-treating with pA-VF); this result suggests that pA-V-F does not induce expression of these genes {i.e., the bacteria were still displaying D-AIa-D-AIa on their surfaces). The observation that (monomelic) vancomycin triggers VanB to become resistant to binding both vancomycin and pA-V-F, while pA-V-F does not induce such a resistant phenotype, suggests that pA-V-F may have advantages over (monomelic) vancomycin for treating infections caused by VanB (and potentially, by other vancomycin-resistant bacteria) using the strategy described below.

Opsonization of Polymer-Labeled Bacteria by Antibodies

The ability of anti-fluorescein antibodies to bind to S. epidennidis, S. pneumoniae, and S. aureus that had been labeled with polymer was investigate. For the initial studies, fluorescence microscopy was used to examine qualitatively the binding of fluorescently labeled anti-fluorescein antibodies to the pA-V-F-labeled bacteria (Figure 4). As a control experiment, S. epidermidis was incubated with Syto-63 and pA-V-F (as for polymer labeling studies). S. epidermidis were incubated sequentially with Syto-63 (a dye for nucleic acids used to visualize the bacteria), pA-V-F, and IgG(anti-fluor)phyco and then rinsed with PBS (phosphate-buffered saline) to remove any material not associated with a cell. IgG(anti- fluor)phyco or pA-VF was omitted for control experiments. The treated bacteria were examined by fluorescence microscopy using an Ar-Rr laser to excite the fluorophores.

The bacteria displayed fluorescence from Syto-63 and fluorescein (on pA-V-F) but not from R-phycoerythrin (phyco) (Figure 4); this result demonstrates that Syto-63 and fluorescein were not fluorescent at the wavelengths used to excite and detect phycoerythrin. S. epidermidis that was incubated with pAV- F, Syto-63, and anti-fluorescein antibody labeled with R-phycoerythrin (IgG(anti-fluor)phyco) exhibited phycoerythrin (phyco) fluorescence (from the bound IgG(anti-fluor)phyco) that colocalized with fluorescence from fluorescein (and from Syto-63) (Figure 4). Bacteria that were exposed to IgG(anti-fluor)phyco and Syto-63 but not pA-V-F did not display any phycoerythrin fluorescence (Figure 4). These results demonstrate that pA-V-F must be bound to the bacteria to mediate binding by the antibody, and that every bacterium labeled with pA-V-F was recognized by at least one molecule of antibody. Flow cytometry measured the opsonization of pA-V-F-labeled bacteria (S. aureus and S. pneumoniae) by anti-fluorescein antibodies. This technique quantitatively and rapidly determines the fluorescence intensity of some thousands of bacteria. In addition, flow cytometry experiments do not require any dye for nucleic acids, because bacteria are identified by their ability to scatter light. The bacteria were incubated with polymer (pA-V-F or pA-F) and a primary antibody in a manner similar to that used in studies using microscopy. For flow cytometry, however, an unlabeled monoclonal IgG2a antibody (mouse anti- fluorescein antibody (IgG(anti-fluor))) was used, so that the same primary antibody could be used in studies of both opsonization and phagocytosis. For control experiments, a mouse isotype-control IgG2a antibody directed towards an unrelated hapten (IgG(control)) was used to probe for non-specific interactions of the antibody {e.g., the Fc region) with the polymer- labeled bacteria. Finally, incubation with an anti-mouse secondary antibody labeled with R- phycoerythrin (goat IgG (anti-mouse)phy.o) facilitated detection of primary antibody bound to the bacterial surface. Four groups of bacteria were examined: untreated bacteria (no polymer or antibody) (1), pA-F / IgG(anti-fluor) / IgG(anti-mouse)phyco (2b), pA-V-F / IgG(control) / IgG(anti-mouse)phyco (3b), and pA-V-F / IgG(anti-fluor) / IgG(anti-mouse)phyco (4b) (Figure 5).

The changes in phycoerythrin fluorescence of the different groups of bacteria were quantitated by determining the mean fluorescence intensity and the percentage of bacteria in each group that were more fluorescent than a threshold. The threshold was set to the highest fluorescence intensity observed for untreated bacteria (Figure 6). Bacteria (S. aureus (a) or S. pneumoniae (b)) were incubated sequentially with polymer (pA-V-F or pA-F), mouse anti- fluorescein IgG (IgG(antifluor)) or mouse isotype-control IgG (IgG(control)), and goat R- phycoerythrin-conjugated secondary IgG (IgG(anti-mouse)phyoo). Bacteria were also left untreated to serve as a control (untreated bacteria). Bacterial populations were identified and gated based on 90° light scatter, and -10,000 events were analyzed for phycoerythrin fluorescence intensity. There was a shift to higher phycoerythrin fluorescence of the entire population for both types of pA-V-F labeled bacteria when incubated with IgG(anti-fluor) (4b) than when left untreated (1). There was no such shift in fluorescence for the population of pA-V-F labeled bacteria that were incubated with IgG(control) (group 3b). There was a small shift towards higher phycoerythrin fluorescence for the S. pneumoniae that were incubated with pA-F and IgG(anti-fluor) (4b) than when S. pneumoniae were left untreated (1) (a). Such an increase in this control group (4b) was not present for S. aureus (b) in this experiment.

Treatment of bacteria with pA-F / IgG(anti-fluor) / IgG(anti-mouse)phyco (2b) controlled for any non-specific (i.e., independent of vancomycin) binding of the bifunctional polymer to the bacterial surface. This control group for both S. aureus and 5. pneumoniae demonstrated a slight increase in phyco fluorescence intensity (mean fluorescence increased by ~2-fold, Table 1), and in the fraction of bacteria above the threshold (-16%, Table 1), relative to untreated bacteria. The possibility of non-specific binding of the antibodies to the bacterial surfaces was eliminated with a control experiment, described below. These increases are larger than the uncertainties in the measurements, and are consistent with a low level of non-specific binding of the polymer to the surfaces of both bacteria by interactions that are independent of vancomycin. Bacteria treated with pA-V-F / IgG(control) / IgG(anti- mouse)phyco (3b) displayed the same mean fluorescence intensity as untreated bacteria (within error) for both S. aureus and S. pneumoniae. The percentage of bacteria in this control group (IgG(control)) with fluorescence greater than the threshold was negligible (within error) for both bacteria.

Table 1. Summary of Flow Cytometry Data for Opsonization of Bacteria

Mean Percentage

Fluorescence of Bacteria

Intensity¹ Above

Threshold³'¹³

S. aureus

Bacteria (untreated) (1) 4.5 ± 0.3

+ pA-F / IgG(anti-fiuor) / IgG(anti-mouse)phyco (2b) 9 ± 4 17 ± 16 + pA-V-F / IgG(control) / IgG(anti-mouse)phyco (3b) 6 ± 2 6 ± 5 + pA-V-F / IgG(anti-fluor) / IgG(anti-mouse)phyco (4b) 90 ± 40" 76 ± 9" S. pneumoniae

Bacteria (untreated) (1) 4.3 ± 0.2

+ pA-F / IgG(anti-fluor) / IgG(anti-mouse)ph_yco (2b) 8 ± 3 15 ± 11 + pA-V-F / IgG(control) / IgG(anti-mouse)phyco (3b) 4.5 ± 0.2 o±o + pA-V-F / IgG(anti-fluor) / IgG(anti-mouse)ph_yco (4b) 80 ± 30" 92 ± 6^** ^a Uncertainties represent standard errors of the mean from three independent experiments. ^b Threshold was set based on the maximum fluorescence observed for untreated bacteria (and is shown as dotted line in Figure 6). Percentages above threshold in group 1 are zero by definition.

** p < 0.05 vs. other polymer-treated groups. Comparisons were performed using a one-way analysis of variance (ANOVA) followed by a Duncan post-hoc test. When data did not pass Levine's test for normality and equal variance, they were logarithmically transformed (base 10) to meet ANOVA requirements.

The absence of phyco fluorescence (over background) in these control experiments eliminated the possibility of nonspecific binding to the bacterial surfaces of the antibodies: the Fc region of the IgG2a primary antibodies (IgG(control) and IgG(anti-fluor)) and the secondary antibody (IgG(anti-mouse)phyco). Both S. aureus and S. pneumoniae treated with pA-V-F / IgG(anti-fiuor) / IgG(antimouse) Phyco (4b) had mean phyco fluorescence intensities between 10- and 20-fold greater than control groups (2b and 3b) based on statistical analysis. The percentage of bacteria that were above the fluorescence threshold was high in this group for both bacteria (S. aureus, 76% ± 9%; S. pneumoniae, 92% ± 6%). These values were much higher than those values observed in any of the control groups. For S. pneumoniae, the data from flow cytometry indicate that every bacterium is labeled by pA-V-F and IgG(anti- fluor) (the percentage of bacteria with fluorescence above the threshold was -100%).

For S. aureus, the data for the group incubated with pA-V-F / IgG(anti-fluor) show a high degree of labeling of bacteria by antibody (76% of bacteria have phyco fluorescence above threshold). While not wishing to be bound by any particular theory, it is possible that some fraction of the population of S. aureus did not bind to either IgG(fluor) or pA-V-F, given the overlap of the histograms for control and experimental groups (Figure 6a).

From both the flow cytometry and fluorescence microscopy results, it is concluded that the polymer must have both vancomycin and fluorescein side-chains to opsonize S. aureus and S. pneumoniae: vancomycin, to adsorb the polymer to the bacterial surface via binding to D-AIa-D-AIa in the cell wall (cf. 2b and 4b, Figure 6 and Table 1), and fluorescein, to localize anti-fluorescein antibodies to the bacterial surface (cf. Figure 3b and 4b, Figure 6 and Table 1) (Figures 2b and 2c).

Association with Macrophages and Phagocytosis of Opsonized S. aureus.

Having demonstrated that antibodies recognize haptens (fluorescein) on the polymers bound to bacteria, we explored the ability of the bound IgG antibodies to interact with macrophages and to promote phagocytosis of the bacteria (Figures 2d and 2e). Flow cytometry allowed us to follow the association of the fluorescent polymer-labeled bacteria with macrophages (Figure 7). We incubated S. aureus that were pre-treated with pA-V-F and primary antibody (IgG(anti-fluor): 4a, or IgG(control): 3a) with cultured J774 cells (a mouse macrophage-like cell line, which we refer to below as macrophages). After incubation, we washed the macrophages and quantitatively examined macrophages associated with pA-V-F labeled bacteria using flow cytometry (gating on the macrophages and detecting fluorescence from fluorescein, based on forward and 90° light scatter, and fluorescence intensity from fluorescein was measured for 5000 events of the macrophage population) (Figure 7).

The macrophages were also observed qualitatively by optical microscopy (Figure 8), wherein macrophages were treated with pA-V-F and IgG(anti-fluor). In the flow cytometry experiments, bacteria could only be observed that were labeled with pA-V-F because fluorescence was detected from fluorescein (on p A- V-F). The macrophages were gated on to exclude signals from free pA-V-F and pA-V-F-labeled bacteria that were not associated with the macrophages. The control experiments for phagocytosis, therefore, used fluorescent pA- V-F-labeled bacteria treated with IgG(control) (3a) to estimate the antibody-independent phagocytosis of S. aureus. (Thomas, C. A. et al. Protection from lethal gram-positive infection by macrophage scavenger receptor-dependent phagocytosis. J. Exp. Med. 191, 147- 155 (2000); Stuart, L. M. et al. Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain. J. Cell Biol. 170, 477- 485 (2005)).

The control experiments for opsonization had demonstrated that IgG(control) does not bind to pA-V-F-labeled bacteria (Figure 6).

In the control phagocytosis experiment with IgG(control), approximately one-half (51% ± 3%) of the macrophages had fluorescence from fluorescein above the threshold, which was set as the maximum fluorescence intensity observed for macrophages interacting with untreated S. aureus {i.e., background fluorescence); the mean fluorescence of the macrophages in this group was 2.5 ± 0.2. These results suggest a moderate level of association between pA-V-F-labeled S. aureus and macrophages independent of specific antibodies. These data are consistent with previous reports demonstrating that macrophages have antibody-independent mechanisms for phagocytosis of S. aureus (Thomas, C. A. et al. Protection from lethal gram-positive infection by macrophage scavenger receptor-dependent phagocytosis. J. Exp. Med. 191, 147-155 (2000); Stuart, L. M. et al. Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH- terminal cytoplasmic domain. J. Cell Biol. 170, 477-485 (2005)).

There was a statistically significant increase in the percentage of macrophages above the fluorescence threshold (from 51% ± 3% to 84% ± 1%) when the pA-V-F labeled S. aureus were incubated with IgG(anti-fluor) (4a) as compared to being incubated with IgG(control) (3 a) (Figure 7b). This increased percentage of fluorescent macrophages represents an increase in the percentage of macrophages that are associated with pA-V-F- labeled bacteria in a specific antibody (IgG(anti-fluor))-dependent manner. The high background level of association (antibody-independent) between macrophages and pA-V-F- labeled S. aureus (Figure 7) limited the maximum effect that could be observed (theoretical maximum of 100% fluorescent macrophages). The results, nevertheless, demonstrate that pA-V-F is able to increase the percentage of macrophages that are associated with S. aureus in a manner that requires opsonization by anti-fluorescein antibodies. The greater than two-fold increase in mean fluorescence for macrophages in the IgG(antifluor) group (4a) as compared to those in the IgG(control) group (3 a) (Figure 7c) indicates an increase in the average number of pA-V-F labeled bacteria associated with each macrophage when there is IgG(anti-fluor) present on the surface of the bacteria. While not wishing to be bound by any particular theory, it is believed that this increase in mean fluorescence underestimates the interaction between the pA-V-F-labeled bacteria and macrophages in the IgG(anti-fluor) group due to the quenching of fluorescence from fluorescein upon binding by IgG(anti-fluor).

It was determined that the addition of an excess of IgG(anti-fluor) quenched the fluorescence of pA-V-F by -88% (data not shown). It is not known as to the quantitative amount that this quenching reduces the observed interaction between the pA-V-labeled bacteria and macrophages in the IgG(anti-fluor) group because no estimate of the number of free (not bound by antibody) fluorescein moieties per molecule of polymer associated with bacteria is available. Regardless, this quenching does not occur in the control samples incubated with IgG(control). The results with IgG(anti-fluor), even with the likely underestimation due to quenching of fluorescence by binding of antibody, demonstrate an increased association of pA-V-F-labeled bacteria with macrophages in a manner that is dependent upon an IgG with specificity towards the hapten (fluorescein) introduced by the polymer.

Macrophage studies included both one and two hour time points, in order to evaluate whether the enhanced phagocytosis of the IgG(anti-fiuor) group relative to the IgG(control) group persisted. Similar to the earlier time point, IgG with specificity for the fluorescein hapten on pA-V-F (IgG(anti-fluor)) (4a) significantly increased the percentage of macrophages associated with bacteria (Figure 7b) and the number of bacteria per macrophage (Figure 7c) relative to non-specific IgG (IgG(control)) (3 a) after two hours of incubation. The percentage of macrophages above the fluorescence threshold and the fluorescence per macrophage changed little between one and two hours for the IgG(anti-fluor) group; this result suggests that peak or near peak bacterial uptake in the hapten-specifϊc antibody group was achieved by one hour. The association of pA-V-F-labeled bacteria with macrophages achieved after two hours of incubation with IgG(control) failed to reach the levels achieved after only one hour of incubation with IgG(anti-fluor). Taken together, these findings suggest that the bifunctional polymer strategy (mediated by hapten-specific IgG) increases the net amount and the rate of bacteria association with macrophages for a significant time period relative to innate phagocytosis (independent of specific IgG) by the macrophages.

Flow cytometry does not distinguish between bacteria that are actually internalized and those that are merely associated with the periphery of the cell membrane of the macrophages. To address this issue, bacteria-associated macrophages (prepared as above) were examined using optical microscopy (Figure 8). Bacteria (diameter of 0.8 μm) are visible both inside of the macrophage in phagosomes (Figure 6a; thin black arrow) and associated with the external cell membrane (Figure 8a; thick black arrow). Moreover, overlaying a phase-contrast image with a fluorescence image (Figure 8b) suggested an intracellular location of a fraction of the pA-V-F labeled, fluorescent S. aureus. These images strongly suggest that the data from flow cytometry (increased mean fluorescence and increased percentage of events above threshold relative to control groups) for macrophages exposed to pA-V-F labeled S. aureus and IgG(anti-fluor) is attributable to increases in both the amount of bacteria bound to and internalized by the macrophages, i.e., some fraction of the macrophage-associated bacteria is phagocytosed. Phagocytosis of Complexed Cells

A bifunctional polymer, pA-V-F (Figure 1) was used to form complexes with the surfaces of several representative Gram-positive bacteria (S. aureus, S. epidermidis, S. pneumoniae, and E. faecalis) and to "decorate" these bacteria with a synthetic hapten (fluorescein); this molecule served as a hapten that was recognized by antibodies (IgG(anti- fluor)) in a second step. These antibodies, which were bound to the bacterial surfaces, interacted with macrophages (presumably via interactions with the Fc region of the antibody) and promoted phagocytosis of the opsonized, polymer-labeled bacteria (Figure 2).

There are several strengths to this approach for treating Gram-positive bacterial infections. For the specific system studied here, vancomycin gives the polymer a broad, but specific target (all Gram-positive bacteria). The bifunctional polymer is modular in design: one component is the recognition element that binds the polymer to the surface, and the other component is the "functional" element. This modularity makes this strategy a general approach to the design of a wide range of polymers with different specificities (by varying the recognition element) and functions (by varying the secondary or "functional" element).

These bifunctional polymers function sequentially: i) the polymer binds to the surface of the target mediated by one type of side-chain on the polymer; ii) antibodies bind to the other side-chain functionality on the polymer; iii) the opsonized bacteria are recognized and ingested by macrophages. This step-wise mode of action is in contrast to previous approaches using monomeric bifunctional molecules {e.g., chimeric proteins (Capon, D. J. et al. Designing CD4 immunoadhesins for AIDS therapy. Nature 337, 525-531 (1989); Traunecker, A., Schneider, J., Kiefer, H. & Karjalainen, K. Highly efficient neutralization of HIV with recombinant CD4-immuglobulin molecules. Nature 339, 68-70 (1989)) or pre- assembled complexes (Bertozzi, C. R. & Bednarski, M. D. Antibody Targeting to Bacterial- Cells Using Receptor-Specific Ligands. J. Am. Chem. Soc. 114, 2242-2245 (1992); Bertozzi, C. R. & Bednarski, M. D. A Receptor-Mediated Immune-Response Using Synthetic Glycoconjugates. J. Am. Chem. Soc. 114, 5543-5546 (1992)) and has potential benefits in future applications of these polymers (described below). hi the specific systems of preferred embodiments, pA-V-F cannot target Gram- negative bacteria, viruses, or other cell types and is thus not general for all pathogens. The use of fluorescein as the functional element {i.e., hapten) requires using anti-fluorescein, an antibody directed towards a synthetic hapten, in the opsonization step. Systems can be tailored to use for other pathogens by selecting recognition group(s) that bind to a particular functional group characteristic of the surface of the pathogen or cell of interest. Likewise, synthetic and/or natural haptens can be selected depending upon the antibody to be associated with the target pathogen or cell.

Bifunctional polymers (like all polymers) are subject to certain limitations in therapeutic applications: they are not orally bioavailable and their innate polydispersity has hampered their FDA approval (Duncan, R. The dawning era of polymer therapeutics. Nat. Rev. Drug Discovery 2, 347-360 (2003)). Polymers, however, are particularly effective in places where their large size (and thus, low oral bioavailability) is an advantage rather than a disadvantage. Examples include administration to appropriate compartments, such as the digestive tract, respiratory system, eye, superficial soft tissue infections, and vagina, where retaining the polyvalent ligand in that organ or structure is useful, and where release into the systemic circulation may be undesirable. The polymers of preferred embodiments can be useful therapeutically in the clearance from appropriate biological compartments of bacterial infections caused by Gram-positive bacteria.

The methods and polymers of the preferred embodiments can also be suitable for treatment of bacterial infections in the respiratory system. As a general approach, bifunctional polymers can be used therapeutically to target antibodies to pathogens or cancer cells to accelerate their destruction. An appropriate recognition element for the target of interest can be incorporated into the polymer and, given the polyvalent nature of the interaction, weak monovalent interactions can be acceptable in this role.

The functional element on the polymer can be either a synthetic or a natural hapten {e.g., part of a vaccine). A synthetic hapten requires the subsequent administration of antibodies directed towards that hapten, while a natural hapten allows antibodies of the host to target the polymer-labeled target. Bifunctional polymers can also be useful in analytical applications. Binding to cells or viruses by a molecule that has both a tunable recognition component and a tunable labeling component can be useful in approaches designed to quantitate specific populations of cells (Disney, M. D., Zheng, J., Swager, T. M. & Seeberger, P. H. Detection of bacteria with carbohydrate-functionalized fluorescent polymers. J. Am. Chem. Soc. 126, 13343-13346 (2004)) {e.g., by flow-cytometry).

The binding of pA-V-F to E. faecalis VanB after overnight incubation with pA-V-F (but not after incubation with monomelic vancomycin itself), and preliminary results which suggest that pA-V-F was able to mediate opsonization of some fraction of induced VanB by antifluorescein antibodies, suggest that the bifunctional polymer approach using vancomycin as the recognition moiety can be successful in targeting even these vancomycin-resistant bacteria, which pose a serious public health problem, for phagocytosis in vitro and in vivo. Bifunctional polymers can also target microbes for antibody-mediated immunity. By appropriate selection of the recognition moiety, this polyvalent approach can allow the selective destruction of pathogens (or other cells) of interest by coating their surfaces with hapten, to which antibody-mediated host defenses can be targeted.

All references cited herein are incorporated herein by reference in their entirety. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

The term "comprising" as used herein is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it coyer all modifications and alternatives coming within the true scope and spirit of the invention as embodied in the attached claims.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for facilitating opsonization of a target cell or a target organism in a host, the composition comprising: a polymeric substance, the polymeric substance comprising a plurality of first functional groups and a plurality of second functional groups, wherein the first functional group is capable of specific binding to the target cell or the target organism, and wherein the second functional group comprises a hapten capable of causing an antibody to associate with the target cell or the target organism; and a pharmaceutically acceptable excipient.

2. The pharmaceutical composition of claim 1, wherein the hapten is capable of causing an antibody naturally occurring in the host to associate with the target cell or the target organism.

3. The pharmaceutical composition of claim 1, wherein the hapten is capable of causing only a synthetic antibody or an antibody not naturally occurring in the host to associate with the target cell or the target organism.

4. The pharmaceutical composition of claim 1, wherein the polymeric substance comprises a functionalized polyacrylamide.

5. The pharmaceutical composition of claim 1, wherein the first functional group comprises a sialic acid moiety capable of specific binding to a hemagglutinin moiety on the target cell or the target organism.

6. The pharmaceutical composition of claim 1, wherein the first functional group comprises a mannose moiety capable of specific binding to a FimH adhesin moiety on the target cell or the target organism.

7. The pharmaceutical composition of claim 1, wherein the first functional group comprises a Glc-Nac moiety capable of specific binding to a pneumococcal surface adhesin A moiety or a pilus adhesin moiety on the target cell or the target organism.

8. The pharmaceutical composition of claim 1, wherein the first functional group comprises a Neu-Ac moiety or a lacto-N-neotetraose moiety capable of specific binding to a choline binding protein A moiety on the target cell or the target organism.

9. The pharmaceutical composition of claim 1, wherein the first functional group comprises a Neu-Ac moiety capable of specific binding to a pilus adhesin moiety on the target cell or the target organism.

10. The pharmaceutical composition of claim 1, wherein the first functional group comprises a plasmin moiety or a plasminogen moiety capable of specific binding to an α- enolase moiety on the target cell or the target organism.

11. The pharmaceutical composition of claim 1, wherein the first functional group comprises a collagen moiety capable of specific binding to a collagen adhesin moiety on the target cell or the target organism.

12. The pharmaceutical composition of claim 1, wherein the first functional group comprises a lactose moiety capable of specific binding to a pilus adhesin moiety on the target cell or the target organism.

13. The phaπnaceutical composition of claim 1, wherein the first functional group comprises a vancomycin moiety capable of specific binding to a D-AIa-D-AIa moiety on the target cell or the target organism.

14. The pharmaceutical composition of claim 1, wherein the first functional group comprises an anti-Her2 peptide moiety capable of specific binding to a Her2 receptor moiety on the target cell or the target organism.

15. The pharmaceutical composition of claim 1, wherein the second functional group comprises a polysaccharide capable of specific binding to an anti-alpha(Gal) antibody.

16. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is in unit dosage form.

17. The pharmaceutical composition of claim 1, for use in treating an infection by a pathogenic bacterium.

18. The pharmaceutical composition of claim 1, for use in treating breast cancer or prostate cancer.

19. The phaπnaceutical composition of claim 1, for use in killing the target cell or the target organism.

20. The pharmaceutical composition of claim 19, wherein the target cell is a cancer cell or a B cell.

21. The pharmaceutical composition of claim 19, wherein the target organism is selected from the group consisting of enteroaggregative E. coli, fimbrilated E. coli, S. pneumoniae, P. aeruginosa, S. aureus, S. epidermidis, and E. faecalis.

22. The pharmaceutical composition of claim 1, for use in facilitating opsonization and subsequent phagocytosis of the target cell or the target organism.