BACTERIAL SURFACE HOMING MOIETIES CONJUGATED TO HAPTENS

FIELD OF THE INVENTION

The present invention relates to compounds which are useful to treat individuals who have bacterial infections, particularly those infections involving drug resistant or otherwise difficult to treat bacteria. Methods of treating individuals infected with bacteria, particularly those infections involving drug resistant or otherwise difficult to treat bacteria are provided.

BACKGROUND OF THE INVENTION

The human immune system has powerful mechanisms in place to prevent the entry and colonization of most pathogens. Once bacterial pathogens escape detection, however, they often extensively colonize the patient, which can result in severe symptoms and even death in the absence of medical intervention. The rapid surge in drug-resistant bacterial infections has now become one of the primary public health crises of the 21st century. Problematic infections are caused by drug resistant bacteria. Discovery and development of drug leads against the most serious pathogenic bacteria are desperately needed to reinvigorate the antibiotic pipeline.

Gram-positive bacteria such as for example those belonging to the genera Staphylococcus, Streptococcus, Corynebacterium, Listeria, Bacillus, Clostridium and others include pathogenic species and some such species have developed stains that are resistant to many currently available therapies or are otherwise difficult to treat. Likewise, Gram-negative bacteria such as for example those belonging to the genera as Acinetobacter, Pseudomonas, Klebsiella, Legionella, Neisseria, Moraxella, Haemophilus, Chlamydia, Yersinia, Proteus, Enterobacter, Serratia, Helicobacter, Salmonella, Salmonella, Escherichia and others include pathogenic species and some such species have developed stains that are resistant to many currently available therapies or are otherwise difficult to treat. Treatment of individuals infected with pathogenic bacteria can present therapeutic challenges. There remains a need for compounds that are useful in methods to treat individuals infected with pathogenic bacteria. There remains a need for strategies and methods of treating infection by pathogenic bacteria that can engage additional antibacterial modalities. SUMMARY OF THE INVENTION

Compounds comprising a surface homing moiety, a linker and a hapten are provided. In some embodiments, the surface homing moiety is selected from the group consisting of Vancomycin, Teicoplanin and derivatives thereof. In some embodiments, the surface homing moiety is selected from the group consisting of: polymyxins, Lipid A binding polymyxin derivatives, linear antimicrobial peptides and GP12. The hapten may be 2,4-dinitrophenol (DNP), rhamnose, a-Gal, fluorescein isothiocyanate (FITC), amino acids 110-121 of Hepatitis A, amino acids 117-128 of Hepatitis B, aminobenzene, o- aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, urushiol, hydralazine, fluorescein, biotin, or digoxigenin.

Method of treating an individual identified as having a Gram-positive bacterial infection are provided. The methods comprise administering to the individual, a therapeutically effect amount of a compound comprising a surface homing moiety, a linker and a hapten. The surface homing moiety is Vancomycin, Teicoplanin and derivatives thereof. The hapten is 2,4-dinitrophenol (DNP), rhamnose, a- Gal, fluorescein isothiocyanate (FITC), amino acids 110-121 of Hepatitis A, amino acids 117-128 of Hepatitis B, aminobenzene, o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, urushiol, hydralazine, fluorescein, biotin, or digoxigenin.

Method of treating an individual identified as having a Gram-negative bacterial infection are provided. The methods comprise administering to the individual, a therapeutically effect amount of a compound comprising a surface homing moiety, a linker and a hapten. The surface homing moiety is selected from the group consisting of polymyxins, Lipid A binding polymyxin derivatives, linear antimicrobial peptides and GP12. The hapten is 2,4-dinitrophenol (DNP), rhamnose, a-Gal, fluorescein isothiocyanate (FITC), amino acids 110-121 of Hepatitis A, amino acids 117-128 of Hepatitis B, aminobenzene, o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, urushiol, hydralazine, fluorescein, biotin, or digoxigenin.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A depicts a compound comprising a surface homing moiety connected to an antibody recruiting hapten by a linker.

Figure IB depicts a bacterial cell without any conjugated compound associated with it and the same cell after exposure to the conjugated compound whereby the surface homing moiety associates with a component near the cell surface and the hapten is exposed, thereby tagging the cell. Figure 2 shows primary modes of pathogen clearance of a bacterial cell targeted with a conjugated compound based on immunomodulators.

Figure 3 shows the chemical structure of the series of PM BN modified with DNP units. Each member of this series varies in the number of PEG units in the tether connecting DNP to PM BN

Figures 4A-4D are related to the discussion of antibody recruitment by PDn series using E. coli disclosed in Example 2. Figure 4A: Chemical structures of the PDn series. Figure 4B: Cartoon representation of the assay used to determine antibody recruitment to the surface of E. coli cells based on the PDn series of agents. E. coli WT (Figure C) and K12 (Figure 4D) were incubated for 2 h with 40 μΜ of PDn followed by incubation with FITC-conjugated anti-DNP antibodies and analyzed using flow cytometry. Data are represented as mean + SD (n =3). Inset, confocal microscopy imaging of E. coli treated with PF3.

Figures 5A-5C are related to the discussion of the effect of LPS mutations in Example 2. Five designated E. coli mutants were incubated for 2 h with 40 μΜ of PD6 (Figure 5A) followed by incubation with FITC-conjugated anti-DNP antibodies or incubated for 2 h with 40 μΜ of PF6 (Figure 5B) and analyzed using flow cytometry. Data are represented as mean + SD (n =3). (Figure 5C) Cartoon representations of the LPS mutants used in this study.

Figures 6A-6D are related to the discussion of the induction of bactericidal activity by PD6 in Example 2. (Figure 6A) E. coli were incubated with PD6 alone, rifamycin alone, or in combination of the two agents at varying concentrations. Optical density at 600 nm was analyzed to assess bacteria viability. (Figure 6B) E. coli were treated with either PD6, PHS, PM BN, their combinations at 10 μΜ, or media to determine complement dependent cytotoxicity (CDC). Colony counts were measured the next day. (Figure 6C) E. coli were incubated with PD6tail at varying concentrations. Optical density at 600 nm was analyzed to assess bacteria viability. (Figure 6D) E. coli cells were incubated for 30 min with the stated concentrations of PD6 or PD6tail followed by incubation with FITC-conjugated anti-DNP antibodies and analyzed using flow cytometry. Data are represented as mean + SD (n =3).

Figures 7A-7C relate to the discussion of recruitment across Gram-negative bacterial species in Example 2. Figure 7A shows data from experiments in which specified bacteria were incubated for 2 h with 40 μΜ of PD6 followed by incubation with FITC-conjugated anti-DNP antibodies and analyzed using flow cytometry. Figure 7B shows data from experiments in which P. aeruginosa were incubated for 2 h with 40 μΜ of PDn followed by incubation with FITC-conjugated anti-DNP antibodies and analyzed using flow cytometry. Data are represented as mean + SD (n =3). Figure 7C shows data from experiments in which P. aeruginosa were incubated with PD6tail at varying concentrations. Optical density at 600 nm was analyzed to assess bacteria viability.

Figures 8A-8C refer to Example 10. Figure 8A depicts association of vancomycin with the d-Ala- d-Ala motif on lipid I I. Figure 8B depicts binding of vancomycin to PG scaffold on cell surface. Figure 8C illustrates strategy to graft immune-cell attracts on the surface of S. aureus based on PG targeting by vancomycin.

Figures 9A-9C refer to Example 10. Figure 9A shows the chemical structures of VancCdnp and VancNdnp. Figures 9B shows the scheme of the assay to measure anti-DNP recruitment. Figures 9C shows data from experiments in which S. aureus cells were incubated for 30 min with 10 μΜ of either VancCdnp or VancNdnp followed by incubation with FITC-conjugated anti-DNP antibodies and analyzed using flow cytometry. Data are represented as mean + SD (n = 3).

Figures 10A-10E refer to Example 10. Figure 10A is a scheme showing the combination of vancomycin with SrtA to anchor DNP epitopes onto bacterial PG. Figure 10B shows the basic chemical structure of FITC-labeled constructs. S. aureus cells were treated with FITC-based constructs with variable PEG spacers at Y position (Figure IOC) or the X position (Figure 10D) and analyzed using flow cytometry. Data are represented as mean + SD (n = 3). Figure 10E shows designation of variants with the common SrtA substrate recognition peptide.

Figures 11A-11B refer to Example 10. Figure 11A shows data from experiments in which specified bacteria were treated with Sortl and Sort 2 (5 μΜ ) overnight and fluorescence was measured using flow cytometry. Data are represented as mean + SD (n = 3). Figure 11B shows images of C. elegans infected with S. aureus (expressing mCherry) were treated with Sortl (50 μΜ), washed, anesthetized, mounted on a bed of agarose, and imaged using confocal microscopy. Scale bar presents 20 μιη.

Figures 12A-12B refer to Example 10. Figures 12A shows data from experiments in which S. aureus cells were incubated overnight with 5 μΜ of Sort3 followed by incubation with FITC-conjugated anti-DN P antibodies and analyzed using flow cytometry. Data are represented as mean + SD (n = 3). Figures 12B refers to phagocytosis of bacterial cells that was evaluated by treating S. aureus cells in the presence or absence of 5 μΜ of Sort3. Untreated or opsonized cells (with anti-DNP antibody) were incubated with J774A.1 macrophages for 30 min in the absence or the presence of calcein-AM labeled S. aureus cells. Fluorescence was measured by flow cytometry. Data are represented as mean + SD (n = 3).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Compounds are provided that are useful in methods to treat individuals infected with pathogenic bacteria. Such compounds are useful in strategies and methods of treating infection by pathogenic bacteria that can engage additional anti-bacterial modalities. Many anti-bacterial agents are transported to and bind or otherwise associate with bacterial components exposed at or accessible at the bacterial surface. These agents are employed as surface homing moieties is heterobifunctional compounds that further comprise additional moieties, antibody-recruiting haptens, which can be recognized by immunological components. As depicted in Figure 1A. the surface homing moieties is connected to the hapten with a linker. Conjugating such surface homing moieties with haptens produces conjugated compounds which associate with bacterial components exposed at or accessible at the bacterial surface and which can be recognized by antibodies and immunological components such as those present in an infected individual's immune system. The conjugated compounds tag or decorate the bacteria (Figure IB) so it can be attacked and eliminated by immune processes (Figure 2).

Examples of bacteria that include pathogenic bacterial strains include Gram-positive bacteria and Gram- negative bacteria. In some embodiments, compounds are provided which comprise surface homing moieties that associate with bacterial components exposed at or accessible way of the bacterial surface of Gram positive bacteria. In some methods, such compounds are useful to treat individual who are infecting with Gram positive bacteria. In some embodiments, compounds are provided which comprise surface homing moieties that associate with bacterial components exposed at or accessible way of the bacterial surface of Gram negative bacteria. In some methods, such compounds are useful to treat individual who are infecting with Gram negative bacteria.

In some embodiments, compounds that target gram positive bacteria are provide for use in treating individual suffering from gram positive bacterial infections. Gram-positive bacteria include pathogenic genera such as the sphere-shaped cocci Staphylococcus and Streptococcus, and rod-shaped bacilli which include non-spore formers Corynebacterium and Listeria and spore formers Bacillus and Clostridium. Species of gram-positive bacteria that are particularly medically important include Streptococcus pneumoniae, Streptococcus mutans, Staphylococcus aureus and Streptococcus sanguinis. Bacillus subtilis has also been widely studied. Compositions for and methods of treating infections by gram positive bacteria, such as for example those listed above, are provided.

In some embodiments, compounds that target gram positive bacteria are provide for use in treating individual suffering from gram negative bacterial infections. Gram-negative pathogens such as Acinetobacter baumanii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Legionella pneumophila Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Chlamydia trachomatis, Yersinia pestis, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi and Escherichia coil can present therapeutic challenges in infected individuals, particularly when strains of such pathogens have resistance to many of the known antibiotics. The need for new agents to treat infections by these pathogens as well as carbapenem-resistant Enterobacteriaceae (C E), multidrug-resistant (M DR) Acinetobacter, and MDR P. aeruginosa is great and the conjugated compounds disclosed herein are useful to treat such infections. Other gram-negative bacteria that can be infectious include strains of Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, as well as cyanobacteria, spirochaetes, green sulfur, and green non-sulfur bacteria.

Compositions for and methods of treating infections by gram negative bacteria, such as for example those listed above, are provided.

Bacterial cells may be targeted for destruction via compounds which comprise hapten conjugated to moieties that specifically home to bacterial cell surfaces. These compounds can mobilize the immune system to target poorly immunogenic bacterial pathogens. In some embodiments, the compounds bind to and tag Gram-positive bacteria for destruction. In some embodiments, the compounds bind to and tag Gram-negative bacteria for destruction. Tagging the bacteria allows the patients to detect and attack the bacteria resulting in a reversal of disease progression and elimination of the bacterial pathogens from the system.

The cell wall structure of Gram-positive bacteria cell surface includes a thick peptidoglycan layers covering the cytoplasmic membrane. The cytoplasmic membrane includes lipids such as Lipid II molecules. The peptidoglycan layers include pentapeptides that comprise d-Ala-d-Ala dipeptide units.

The cell envelope of Gram-negative bacteria is composed of an inner membrane, periplasm, and outer membrane (OM). The OM displays an unusual asymmetry in which phospholipids populate the inner leaflet and lipopolysaccharides (LPS) make up the outer leaflet. Lipid A, an essential anchor of LPS to the OM, is composed of a phosphorylated diglucosamine unit connected to lipid chains. The natural product antibiotics polymyxins, particularly polymyxin B (PMB) and polymyxin E (colistin) are among the few small molecules that associate with lipid A with high affinity and specificity. PM B and colistin are proposed to impart their antibacterial activity by binding to lipid A and destabilizing the OM layer - although the exact mechanism has yet to be fully elucidated.

By exploiting surface exposed features (e.g., lipid II and d-Ala-d-Ala dipeptide units of Gram positive bacteria; lipid A of Gram-negative bacteria), heterobifunctional agents composed of a surface homing moiety linked to a hapten can tag the bacterial cell surfaces with the hapten and engage components of the immune system (e.g., antibodies and primary immune cells) (Figure ID) to kill the bacteria. The surface homing moiety that targets lipid II and d-Ala-d-Ala dipeptide may be used in compounds useful against Gram positive bacteria. The surface homing moiety that targets Lipid A and other components of the lipopolyscaccharide (LPS) may be used in compounds useful against Gram negative bacteria.

In some embodiments, surface homing moieties inherently possess antimicrobial activity. The use of surface homing moieties that inherently possess antimicrobial activity represents a significant advance due to the duel anti-bacterial activity that includes the direct bactericidal activity of the surface homing moieties and the engagement of immune system through the use of the hapten linked to the surface homing moieties. Combined, these agents target pathogenic bacteria in two distinct ways.

Gram-positive surface homing moieties: The antibiotics vancomycin and teicoplanin are glycopeptide antibiotics which inhibit cell wall synthesis in Gram positive bacteria. These drugs bind to d-Ala-d-Ala dipeptide units of the peptidoglycans. Vancomycin also binds to lipid II. Representative non-limiting examples of Vancomycin derivatives are disclosed in US Patent No. 7,273,845, US Patent No. 7,368,422, US Published Application US 2004/0018582, US Published Application US 2016/0200768, the PCT Published Applications WO 2003/016908, WO 2008/076483, WO 2015/024389, WO

2016/134622, and WO 2017/186110A1, European Patent No. EP 0145484 Bl, European Patent No. EP 2208732 Bl, European Application No. EP 2688580 Al and European Application No. EP3037431A1, each of which is incorporated herein by reference. Representative non-limiting examples of Teicoplanin derivatives are disclosed in US Patent No. 5,135,857, US Patent No. 5,185,320, US Patent No. 5,194,424, US Patent No. 5,500,410, US Patent No. 5,869,449, US Patent No. 5,916,873 and US Patent No.

5,936,074, the PCT Published Applications WO 1990/011300, WO 1992/010516, and WO 1998/046579, European Patent No. EP 0448940 Bl, European Application No. EP 0204179 Al, European Application No. EP 0290922 A2, European Application No. EP 0301247 A2, European Application No. EP 0351597 A2 and European Application No. EP 0873997 Al, each of which is incorporated herein by reference.

Gram negative surface homing moieties: The class of antibiotics referred to as polymyxins bind to lipid A. Polymyxins have two principal structural features: (1) a cationic cyclic peptide that binds to lipid A on bacterial OMs and (2) an aliphatic fatty acid chain to anchor and disrupt the OM. Polymyxin derivatives useful as surface homing moieties include those with the cyclic peptide portion of the molecule that functions to bind to lipid A. Examples polymyxins used in some embodiments include polymyxin B (PMB) and polymyxin E (colistin) and lipid A biding derivatives thereof. One such polymyxin derivative useful as surface homing moiety is referred to PMBN - this derivative contains the cationic cyclic peptide of polymyxin B that binds to Lipid A but does not contain the aliphatic fatty acid chain of polymyxin B. Representative non-limiting examples of polymyxin derivatives are disclosed in US Patent No. 3,450,687, US Patent No. 6,380,356, US Patent No. 7,807,637, US Patent No. 8,329,645, US Patent No. 9,763,996, US Published Application US 2009/0239792A1, US Published Application US

2014/0162937A1, and the PCT Published Applications WO2008017734A1, WO2009098357A1,

WO2012168820A1, WO2013072695A1, WO2014188178A1, WO2015135976A1, WO2015149131A1, WO2017054047A1, WO2017147958A1 and WO2018108154A1, each of which is incorporated herein by reference. In some embodiments, the compounds that target the surface of Gram-negative pathogens comprise polymyxins and lipid A biding derivatives thereof, such as for example polymyxin B (PMB) or polymyxin E (colistin). In some embodiments, the Gram-negative surface homing moieties comprise antimicrobial peptides (AMPs), referred to as LL-37, KLAKLAK2, and WLBU2, which have the sequences set forth in SEQ ID NOs: 1-3, respectively. These peptides have been evaluated in live animals and are known to bind to LPS. LL-37 (SEQ ID NO:l - LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) is a peptide adopts an a-helical structure with an overall amphipathic configuration. It is naturally found throughout the human body as part of the innate immune system and has broad activity against microbial pathogens. LL-37 has been shown to have potent anti-infective immunity in vivo. KLAKLAK2 (SEQ ID NO:2 - KLAKLAKKLAKLAK) was originally a de novo designed, idealized amphipathic peptide that has potent antimicrobial activity and low mammalian hemolytic activity. WLBU2 (SEQ ID NO:3 - RRWVRRVRRWVRRVVRVVRRWVRR) is a de novo designed peptide that has the ability to target bacterial cells in live and bind to LPS. In some embodiments, the Gram-negative surface homing moiety comprise the trimeric short tail fiber from T4 phage GP12. See Accession Numbers: P10930.3, CAA39905.1, NP_899594.1, NP_899593.1, AAQ64417.1, AAQ64416.1, NP_932502.1, AAQ81466.1, YP_238874.1, NP_049770.1, AAD42417.1, AAA32495.1, YP_004324409.1, YP_004324404.1, YP_195259.1,

YP_195254.1, AD099139.1, AD099134.1, CAF34289.1 and CAF34284.1 which are each, including all sequences, incorporated herein by reference. SEQ ID NO:6 corresponds to the sequence in P10930.3 and is a representative sequence.

GenPeptldentical ProteinsFASTAGraphics. Bacteriophage T4 initiates the infection of E. coli by recognizing and binding to features on the cell surface. T4 phages associate with the LPS headgroups to engage with the surface and deliver the genetic material into the target cells. Structurally, the short tail fiber GP12 is the unit responsible for LPS binding. GP12 forms the primary contact with LPS. GP12 or LPS binding fragments thereof may serve as a unique and highly specific mode of surface tagging of Gram-negative pathogens with antigenic epitopes. GP12 may be chemically modified using a cysteine to provide sites for conjugation of haptens. The unmodified GP12 was recently shown to have potent activity in an LPS-challenge using live mice infected with E. coli. These results point to a possibility of a secondary mode of ameliorating symptoms based on a GP12 vehicle. The larger molecular weight of GP12 and its natural ability to tightly associate with LPS may have specific advantages relative to PM B and short synthetic lipid A-binding peptides in terms of circulation half-life and hapten accessibility within the cell surface.

The surface of bacteria is "decorated" or "tagged" using surface homing moieties conjugated to haptens that home to and associate with bacterial cell surfaces. In embodiments directed to Gram positive bacteria, the surface homing moieties comprise vancomycin and teicoplanin. In embodiments directed to Gram negative bacteria, the surface homing moieties comprise: polymyxins, Lipid A binding polymyxin derivatives, linear antimicrobial peptides (AM Ps) comprising SEQ ID Nos: 1-3, the trimeric short tail fiber from T4 phage GP12 or an LPS binding fragment thereof. These various surface binding moieties are conjugated to haptens by linkers.

The haptens include 2,4-dinitrophenol (DNP), rhamnose, a-Gal, fluorescein isothiocyanate (FITC), amino acids 110-121 of Hepatitis A (SEQ ID NO: 4; FWRGDLVFDFQV), and amino acids 117-128 of Hepatitis B (SEQ ID NO: 5; STGPCKTCTTPA). Other haptens include aminobenzene, o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, urushiol, hydralazine, fluorescein, biotin, and digoxigenin. In some embodiments, the hapten used is selected based upon the known presence of antibodies in the patient's serum that will recognize the hapten when the conjugated compound is associated with a bacterium. Some hapten may be used because some antibodies against some haptens are expected to be present in most individuals. Some hapten may be used because some antibodies against some haptens are expected to be present in particular individuals such as those vaccinated with antigens, i.e. the HepA or HepB vaccines, corresponding to the hapten. In some embodiments, the patient is screened to confirm the presence of antibodies in the patient's serum that will recognize the hapten including recognition of the hapten present on a conjugated compound that is associated with a bacterium. Methods of screening are provided as are methods of treating which include a prescreening step. Some embodiments provide kits which comprise reagents for pre-screening such as positive and negative controls and reagents for detecting antibody binding to confirm the presence of antibodies in the serum. In some embodiments, the patient is known to lack antibodies in the patient's serum that will recognize the hapten. In such embodiments, the patient may be administered antibodies that will recognize the hapten as part of the method of treating the patient with the conjugated compound. Patients may lack endogenous anti-FITC antibodies. When a conjugated composition is provided which includes FITC as its hapten, the patient is co-administered anti-FITC antibodies as part of the method of treatment, before, during and/or after administration of the conjugated compound. In some embodiments, anti-FITC antibodies are monoclonal antibodies. In some embodiments, anti-FITC antibodies are human or humanized monoclonal antibodies. In some embodiments, anti-FITC antibodies are monoclonal antibodies, including human or humanized monoclonal antibodies, generated or derived from antibodies generated to recognize the FITC-hapten containing conjugated compound associated with the bacteria. In some embodiments, anti-FITC antibodies are monoclonal antibodies, including human or humanized monoclonal antibodies, generated or derived from antibodies generated to recognize the FITC-hapten containing conjugated compound associated with the bacteria but which do not recognize the FITC-hapten containing conjugated compound when not associated with the bacteria.

Linkers used to conjugate the surface homing moiety to the hapten are typically 5-50 nm or greater such as up to 100 nm, biocompatible and stable. In some embodiments, the linker is up to 50 nm. In some embodiments, the linker is up to 40 nm. In some embodiments, the linker is 15-40 nm. In some embodiments the linker is a molecular bond connecting the surface homing moiety to the hapten. In some embodiments, the linker is a PEG polymer. In some embodiments, the linker is an amino acid polymer such as those having the formula Surface homing moiety-(GS)x-hapten, Surface homing moiety- (GGS)x-hapten, Surface homing moiety-(GGGS)x-hapten where G is glycine, S is serine, and x represents the number of units wherein X may be the number that yields a linker up to 140 amino acids in length. In some embodiments, the linker is an amino acid polymer such as those having the formula Surface homing moiety-(P)x-hapten Surface homing moiety-(PO)x-hapten Surface homing moiety-(PPOO)x- hapten where P is proline, O is hydroxyl proline, and x represents the number of units and O is hydroxyproline wherein X may be the number that yields a linker up to 140 amino acids in length. For hydroxyproline, the position and stereochemistry of the hydroxyl can vary. US Patent No. 6,251 ,382, US Patent No. 7,919,118 and US Patent No. 8,263,062, which are each incorporated herein by reference describe examples of spacer linkers.

Linkers may be attached to the surface binding moieties and haptens using routine and well- known processes such as those for example disclosed in the Examples. Other processes include cysteine-maleimide, click chemistry including Copper-mediated azide alkyne cycloaddition, copper-free strained alkyne click chemistry with azide, tetrazine-transcyclooctane, amide coupling between amino groups and carboxylic acids. The immunomodulation strategy provides first-in-class agents against bacteria, particularly poorly immunogenic pathogenic bacteria. The compounds tag cell surfaces with antigenic epitopes. The targeted pathogens are highly relevant, as drug leads against them are urgently needed. The unique modes of immunomodulation provided herein that operate by targeting common features of Gram-positive bacteria provide broad coverage against most Gram-positive pathogens and the unique modes of immunomodulation provided herein that operate by targeting common features of Gram- negative bacteria provide broad coverage against most Gram-negative pathogens.

Preparation, formulation and administration of compositions

The compounds may be provided in compositions that further comprise physiologically acceptable carrier or diluent.

The compounds and compositions may be administered by oral, intraperitoneal, intramuscular and other conventional routes of pharmaceutical administration. Pharmaceutical compositions of the present invention may be administered either as individual therapeutic/prophylactic agents or in combination with other agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. Usually a daily dosage of active ingredient can be about 0.0001 to 1 gram per kilogram of body weight, in some embodiments about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily dosages are in the range of 0.5 to 50 milligrams per kilogram of body weight, and preferably 1 to 10 milligrams per kilogram per day. In some embodiments, the pharmaceutical compositions are given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results. Dosage forms (composition) suitable for internal administration generally contain from about 1 milligram to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5- 95 by weight based on the total weight of the composition.

For parenteral administration, the compound can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques. Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro et al. Eds., Mack Publishing Co., Easton, Pa., 1990, a standard reference text in this field.

Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are used. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. The pharmaceutical preparations according to the present invention are preferably provided sterile and pyrogen free.

One of skill in the art of pharmaceutical formulations, e.g., having an advanced degree in Pharmaceutics or Pharmaceutical Sciences, can prepare a variety of appropriate dosage forms and formulations for the compositions of the invention with no more than routine experimentation. A number of texts in the field, e.g., Remington's Pharmaceutical Sciences and The U.S.

Pharmacopoeia/National Formulary, latest editions, provide considerable guidance in this respect.

A pharmaceutically acceptable formulation will provide the active ingredient(s) in proper physical form together with such excipients, diluents, stabilizers, preservatives and other ingredients as are appropriate to the nature and composition of the dosage form and the properties of the drug ingredient(s) in the formulation environment and drug delivery system.

Subsequent to initial administration, individuals may receive multiple subsequent

administrations. In some preferred embodiments, multiple administrations are performed. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten or more administrations are administered.

EXAMPLES

Example 1

Computer-aided linker design provides an ability to model and simulate the complex outer membranes (OMs) of Gram-negative bacteria with bacterial specific lipopolysaccharides (LPS) using CHARM M-GU I LPS Modeler (http://www.charmm-gui.org/input/lps) and Membrane Builder

(http://www.charmm-gui.org/input/membrane). Membrane Builder provides an intuitive web-based graphical user interface (GU I) to automate the building process of heterogeneous lipid bilayers with or without proteins, and to setup simulation input files with well-established simulation protocols for CHARM M, NAM D, GROMACS, AM BER, GEN ESIS, LAM M PS, Desmond, and OpenM M. This building procedure, is used to build any LPS-containing membranes. Results in E. coli K12 membranes are encouraging and to provide novel insight into designed agents binding to the lipid A/inner core regions of K12 LPS and the exposure of hapten for antibody recruitment.

Example 2

Molecules that decorate bacterial cell surfaces with the goal of re-engaging components of the immune system towards Gram-negative bacteria such as for example Escherichia coli and Pseudomonas aeruginosa have been designed. Conjugates were assembled using a surface homing moiety, specifically polymyxin B, an antibiotic that inherently attaches to the surface of Gram-negative pathogens, and a hapten, i.e. antigenic epitopes that recruit antibodies found in human serum. The surface homing moiety was conjugated to the hapten using a linker, also referred to as a spacer. The spacer length has a role in hapten display within the bacterial cell surface. Specific killing of bacteria by our agent was observed in the presence of human serum. By enlisting the immune system, these agents provide novel antimicrobial modality.

Results and Discussion

A fragment of PM B known as PM B nonapeptide (PM BN) was identified as a polymyxin derivative for use as a surface homing moiety for targeting a hapten to the surface of Gram-negative bacteria. PM BN is devoid of the membrane-disrupting fatty acid tail, but retains the cyclic hexapeptide that is responsible for association to lipid A. PM BN was used as a surface homing moiety to prepare a series of conjugated compounds whereby PM BN was linked to the hapten DNP by varying PEG linker lengths shown in Figure 3. Deprotected PM BN was generated by papain-mediated removal of the acyl chain along with the N-terminal residue from the parent PM B. Next, the tetra-Boc protected PM N B was produced using the reagent Boc-ON, which yielded an unprotected N-term inus amino group and all the amino sidechains Boc-protected. This common Boc-protected PM BN building block was used for the synthesis of PM B derivatives.

PM BN was initially modified with a fluorescein moiety at the N-terminus by reacting the free amino group on PM BN with fluorescein isothiocyanate (FITC) followed by the deprotection of the sidechain Boc groups (PF). The goal was to synthesize a PM BN derivative that could be used to establish the initial working parameters for cell surface tagging. To quantify surface labeling of live bacteria, Escherichia coli (E. coli) cells were incubated with increasing concentrations of PF and cellular fluorescence was measured by flow cytometry. Results showed that cellular fluorescence levels increased with increasing concentrations of PF, reaching levels 86-fold over background by 40 μΜ . The same cells were analyzed by confocal microscopy to delineate the cellular organization of the fluorescence signal. Labeling was observed primarily on the outer periphery, which is also the site of LPS within the OM. Moreover, cellular fluorescence levels were found to be relatively stable; cells retained 76% of fluorescence levels after 2 hours following an initial washing step, which highlights the avidity of PM BN to the cell surface. Results were consistent with previous reports of fluorescently-labeled PMBN that used dansyl as the fluorescent handle. Most importantly, these results confirmed that PMBN conjugates effectively label the surface of Gram-negative bacteria.

PM BN derivatives containing hapten units to direct the recruitment of antibodies were built. Given the location of lipid A within LPS, it was important to consider the display of haptens within the surface of Gram-negative bacteria. The inner core, outer core, and O-antigen segments extend away from lipid A units within LPS, which can potentially obscure binding of small molecules and antibodies. The spacer connecting the hapten and PM BN must be of sufficient length to facilitate the availability of haptens to interact with antibodies from the extracellular space. Flexible and polar polyethylene glycol (PEG) spacers of defined lengths were used to empirically select the most appropriate spacer. 2,4- dinitrophenol (DNP) was chosen as the hapten due to the high abundance of endogenous anti-DNP antibodies in human serum.

With the first series of PDn (n represents the number of diethyleneoxy units, Figure 4A) agents in hand, their ability to induce bacterial opsonization by anti-DNP antibodies was determined. Antibody recruitment was assessed by treating E. coli (WT with intact O-antigen) with each individual agent, followed by an incubation period with fluorescently labeled anti-DNP antibodies, and antibody recruitment was measured by flow cytometry (Figure 4B). Fluorescence levels reflect the level of anti- DNP antibodies associated with bacterial cell surfaces. Treatment of E. coli WT with PDn agents resulted in high levels of anti-DNP recruitment (Figure 4C). From these results, it is evident that spacer length optimization is possible. PD3 may represent a balance in spacer length by optimizing both permeation into the LPS matrix and availability of DNP epitopes. A similar molecule to PD3 was synthesized in which a fluorescein handle was installed in place of DNP (PF3). Based on the confocal microscopy analysis (Figure 4C), it is evident that despite the inclusion of the PEG spacer the modified PMBN fragment was able to label bacterial cell surfaces similar to PF.

The role of O-antigens within LPS was investigated by performing a similar antibody recruitment assay using E. coli (K12), which lacks O-antigen segments. There was a general increase in fluorescence levels across the entire PDn series relative to untreated bacteria. In contrast to E. coli WT, treatment of K12 cells with the smaller sized PEG units (PD1, PD2, PD3, and PD6) resulted in significant recruitment levels over background. These results suggest that accessibility and hapten display control opsonization of tagged cells (Figure 4D). There was a shift in the most efficient opsonization inducer towards PD6. In an attempt to disentangle the contribution of surface binding and DNP availability, the two types of E. coli were treated with the fluorescein-modified PF3 and PF6. While PF3 labeled both strains equally well, the larger PF6 labeled E. coli K12 cells nearly 50% better than E. coli WT. These results are supportive of permeability through the LPS being a factor in determining labeling efficiency.

To explore the influences of PEG spacer length on the DNP distribution and availability on E. coli K12 cell surfaces, molecular dynamics (MD) simulations of three different PDn (PD1, PD3, and PD6) agents in K12 LPS bilayers were performed. The E. coli K12 LPS modeling, its assembly to a bilayer, and simulation protocols followed the CHA MM-GUI LPS Modeler and Membrane Builder step-by-step protocol, which were successfully generalized and applied in the previous LPS simulation. The positively charged PM BN of PDn stays in between the phosphorylated glucosamine residues of lipid A and the K12 inner core containing the carboxyl (in Kdo) and phosphate (in Hep) groups. DNP in PD1 was most fluctuating in the outer region of the K12 outer core and the PEG spacer clearly does not provide sufficient length for DNP to be well displayed to anti-DNP antibodies although it can be exposed to the region within the space in between outer core sugars. As the PEG spacer length increases, MD simulations clearly showed that DNP epitopes became more exposed to the bulk medium and thus showed broader distribution above the K12 core for anti-DNP antibody recruitment PD3 and PD6, respectively). When the 90% confidence interval was used to measure solvent-exposed DNP epitopes above the K12 core (i.e., above z = 43 A in Figure S3A), the populations of solvent-exposed DNP epitopes are 22.9% (PD1), 46.1% (PD3), and 61.5% (PD6). When normalized by PD6, these exposed populations correspond to a ratio of 0.37:0.75:1, which is well correlated with a ratio of 0.39:0.80:1 based on the fluorescence levels of PD1, PD3, and PD6 in the E. coli K12 cell (Figure 4D; normalized by the PD6 value).

To further explore the effect of surface composition on antibody recruitment, six additional E. coli mutants were tested for both antibody recruitment and surface labeling with PD6 and PF6, respectively. Treatment of E. coli mutants with PD6 demonstrated how size, charge, and composition of LPS can dictate antibody recruitment. Data is shown in Figures 6A-5C. Antibody recruitment was higher with the loss of LPS segments. Segments that extend away from lipid A may hinder the availability of the hapten. Surface labeling by the similarly sized fluorescent PF6 resulted in more constant labeling levels across LPS mutants.

The role of LPS binding by PDn agents was studied by performing a competition experiment with exogenous LPS. A near 2-fold and 8-fold decrease in anti-DNP recruitment was observed in E. coli treated with PF6 in the presence of 0.5 mg/mL and 2 mg/mL of LPS, respectively. These results suggest LPS being the primary target of PF6 based on the PMBN homing moiety. Antibody recruitment was also evaluated directly from pool human serum (PHS). Immunotherapeutic approaches against Gram- negative pathogens based on PD6 must rely on opsonization of bacterial cells by anti-DNP antibodies from PHS, which is a complex mixture of biomacromolecules and diverse antibodies. For this assay, detection of anti-DNP recruitment was performed using a FITC-labeled anti-human antibody. Treatment of E. coli with PD6 led to a significant increase in cellular fluorescence, which is indicative of anti-DNP recruitment directly from serum.

Cytotoxicity towards mammalian cells was evaluated to establish the potential therapeutic window of PD6. No loss of cellular viability was observed in the presence of up to 100 μΜ of PD6, which is likely reflective of the lack of lipid A in mammalian cells. The co-incubation of PD6 with rifamycin was tested. The OM provides a barrier for the translocation of most polar antibiotics, which can be a major cause of intrinsic resistance by Gram-negative pathogens to entire classes of potent antibiotics. For these reasons, rifamycin displays weak antibacterial properties against Gram-negative pathogens. While PD6 alone displayed a minimum inhibitory concentration (MIC) value >25 μg/mL and the MIC for rifamycin alone was 12.5 μg/mL, the combination of sub-lethal concentrations of PD6 reduced the MIC for rifamycin to 0.78 μg/mL (Figure 6A). This significant decrease in the MIC value indicates that the PM BN fragment within PD6 retains the ability to promote the entry of polar antibiotics. In addition to its immunomodulatory properties, PD6 also potentiated co-therapeutics.

Opsonization of foreign and potentially dangerous entities triggers a series of steps within the innate immune system. In complement dependent cytotoxicity (CDC), antigen display on bacteria drives the recruitment of antibodies. Subsequently, opsonized cells elicit a complement cascade that leads to the formation of membrane attack complexes that ultimately lyse target cells. Having shown that PD6 promoted the recruitment of anti-DNP antibodies directly from PHS, its ability to induce the killing of Gram-negative bacteria was evaluated. Results showed that treatment of E. coli with PHS, PMBN, or PD6 alone led to no significant change in colony counts (Figure 6B). Similarly, the co-incubation of PM BN (10 μΜ) and PHS resulted in minimal decrease in colony counts. Treatment of cells with PD6 plus PHS resulted in a greater than one log reduction in colony counts. This increase in bacterial lysis is suggestive of DNP mediated induction of CDC. To further confirm the role of anti-DNP antibodies, a similar assay was performed with PHS depleted of antibodies. When cells were treated with antibody-depleted PHS, there was no induction in cell lysis upon co-incubation with PD6. In addition, there was no increase in cell killing upon the heat-mediated depletion of complement proteins from PHS.

Initial design of surface homing agents focused primarily on PMBN, which is devoid of the fatty acid tail on the N-terminus that is proposed to be responsible for OM disruption. The absence of the fatty acid tail made measuring antibody recruitment and isolating the effect of opsonization less challenging. Having successfully demonstrated the ability of PD6 to graft DNP antigens onto the surface of Gram-negative bacteria, experiments were done with the fatty acid tail included. For the design of this agent, the DNP epitope was conjugated on the sidechain of a lysine residue that bridged PMBN and the fatty acid tail (PD6tail, Figure 6C inset). The inherent antimicrobial activity of PD6tail (M IC ~ 3.25 μg/mL) was similar to the parent PMB (M IC ~ 1 μg/mL) and considerably lower than PD6 (Figure 6C). These results show that the re-introduction of the fatty acid tail recovers most of the antimicrobial activity of the parent PM B. A near 2-fold increase in cellular fluorescence was observed for cells treated with PD6tail at 10 μΜ over background fluorescence (Figure 6D). Using similar conditions, there was no observable increase in cellular fluorescence in cells treated with PD6. These results may reflect higher surface retention by PD6tail relative to PD6 due to better membrane anchorage, which should reduce the concentration required to decorate cell surfaces with DNP epitopes. The PD6tail may operate in two distinct antimicrobial modes. Bacteria treated with PD6tail that are not initially destroyed by direct cell lysis would, in turn, be targeted by the immune system.

Antibody recruitment strategy was assessed with respect to other Gram-negative pathogens. More specifically, Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter baumannii (A. baumannii), and Klebsiella pneumoniae (K. pneumoniae) were chosen. There was a wide range in opsonization levels across all four species treated with PD6 (Figure 7A). Reduced cellular fluorescence was observed for both K. pneumoniae and A. baumannii despite their well-established sensitivity to PM B. This difference may be attributed to the diversity in cell surface architecture between Gram-negative bacteria despite the common lipid A anchor. I n the case of P. aeruginosa, cells treated with PD6 resulted in fluorescence levels 11-fold higher than untreated cells. Given the possibility that the surface composition of E. coil and P. aeruginosa are unique to each species, this difference may also reflect on the optimum PEG spacer length and epitope availability. For these reasons, the entire PDn panel of agents was tested against P. aeruginosa to empirically identify the most appropriate spacer length (Figure 7B). Similar to the pattern of recruitment to E. coil, PD6 was the most efficient inducer of opsonization against P. aeruginosa. PD6 may represent an idealized combination of a spacer long enough to display DNP epitopes while minimizing steric interference that is common for longer PEG spacers, thus providing the best induction in antibody recruitment regardless of surface composition. Alternatively, differences in surface composition between the two types of bacteria may favor the same spacer length for unrelated reasons. The sensitivity of P. aeruginosa against the most efficient opsonizing agents was evaluated (Figure 7C). PD6 displayed a lower M IC value than the parent PM BN. P. aeruginosa was considerably more sensitive to PD6 than E. coli displaying a M IC value of 12.5 μg/ml. As expected based on our results with E. coli, the introduction of the fatty acid tail in PD6tail further lowers the MIC value to 6.25 μg/ml. Additionally, PD6tail displayed no toxicity towards mammalian cells at all concentrations tested. The specificity endowed by PMBN towards lipid A could be exploited within a living host to target Gram- negative bacteria for imaging and therapeutic interventions. The nematode Caenorhabditis elegans (C. elegans), a widely used model animal to study bacterial pathogenesis, was used. C. elegans (at primarily L4 larval stage) were pre-incubated with P. aeruginosa to establish bacterial colonization. Residual bacteria were gently washed away and P. aeruginosa infected C. elegans were treated with PF6. At the end of the assay, the worms were anesthetized and mounted on a bed of agarose to be imaged via confocal fluorescence microscopy. Remarkably, bacterial cells were readily visualized inside the host C. elegans. These results represent the first use of polymyxins to track bacterial infection in a live host. Most importantly, they demonstrate the homing properties of our designed agents towards Gram-negative bacteria. Given the demonstrated ability of our agents to target bacteria in culture and in vivo, we anticipate that PD6 (or similar scaffolds) can guide the design of potential drug leads.

The immunotherapeutic agents described herein exploit the lipid A binding scaffold of polymyxins to decorate the surface of Gram-negative bacteria with haptens. The most potent members of this panel trigger the opsonization of E. coli and P. aeruginosa. The lead agent induced CDC-based killing of E. coli. Re-introduction of the membrane disrupting fatty acid tail restored its inherent antimicrobial activity. This agent can target and label the surface of Gram-negative pathogens in a live host.

Example 3

Experimental Design (AMPs): Hapten-displaying AMPs (PM B, LL-37, KLAKLAK, and WLBU2) are synthesized using solid phase chemistry (or semi-synthesis from PMB). In most designs, the spacer and the happen are installed onto the peptide during solid phase synthesis using standard coupling reactions. In instances when the tether may be much longer or difficult to install using solid phase chemistry, an alternative approach may be used. The AM P is modified with a click chemistry partner (azido functional group). The tether is constructed separately and modified with an alkyne handle. Conjugation of the two click partners will be accessed via Copper-catalyzed azide-alkyne cycloaddition (CuAAC). For all constructs, both termini are sampled as candidate sites for hapten modification. All intermediate and final constructs are isolated by RP-HPLC. The final molecules are characterized by NM R (1H and 13C) and high-resolution MS (purity >99% as verified by analytical RP-HPLC).

Prelim inary work on KLAKLAK-based peptides demonstrate its suitability. Several features of this particular peptide make it an excellent delivery vehicle for hapten presentation. Its synthesis is compatible with solid phase chemistry, it has been shown to be effective in the D-enantiomeric form (resistant to proteolytic degradation), and it displays two-orders of magnitude selectivity in cytotoxicity towards bacterial cells compared to mammalian cells. Moreover, it was recently shown that (KLAKLAK) ₂ can specifically deliver agents to bacterial cell surfaces even in the presence of human red blood cells.72 We have synthesized both the L- and the D-versions of (KLAKLAK) ₂ using standard Fmoc-based solid phase peptide chemistry and conjugated the hapten DN P to the N-terminus. KLAKLAK-DN P conjugates are effective in inducing anti-DNP recruitment to the surface of E. coli.

Example 4

Experimental Design (GP12). GP12 is recombinantly expressed in E. coli. Briefly, the gene encoding GP12 from the T4 short tail fiber is cloned into the expression plasmid pCDF-Duetl (Novagen, USA) by Genscript with an N-terminus Hig-tag for purification purposes along with the GP12 chaperone GP57. E. coli carrying the expression plasmid is induced with IPTG and bacteria are harvested after overnight incubation. GP12 from harvested bacteria is isolated using standard Ni-NTA purification methods followed by ion exchange chromatography. Protein purity is verified using SDS-PAGE and protein concentration will be quantified by BCA Protein Assay Kit. LPS from the protein stock is removed using EndoTrap Blue. Native GP12 has three cysteine residues that are covalently modified to display hapten epitopes with spacers of various lengths and compositions. The tether characteristics are initially established using computational modeling to optimize hapten display and availability.

Attachment of hapten onto GP12 is performed using a cysteine reactive iodoacetamide. Conjugated proteins are isolated from the hapten reagents using membrane dialysis (10 kDa cut-off membranes) and extent of conjugation is monitored via high resolution MALDI-TOF. Alternatively, the native cysteine residues are m utated to either alanine or serine using standard cloning techniques and a cysteine residue will be introduced at the N-terminus. Cysteine-reassigned GP12 is tested for its ability to bind LPS using a standard Limulus Amebocyte Lysate assay. The N-terminus end of GP12 is expected to be the most exposed part of the protein to the extracellular medium, which should provide greater engagement with antibodies. The protein is expressed, isolated, and characterized using the same methods as the native GP12. Hapten-displaying moieties are synthesized using a combination of solution phase and solid phage chemistries. The final molecules are characterized by N M (1H and 13C) and high-resolution MS.

If the cysteine-iodoacetamide conjugation with GP12 proves to be more complicated than anticipated or the native cysteine become essential for LPS binding (cell targeting), GP12 variants will be constructed using genetically encoded unnatural amino acids with orthogonal chemical reactivity. An amino acid displaying an azido functional group (lysine analog) will be installed at the N-terminus. A click chemistry compatible handle (terminal alkyne) is also installed in place of the iodoacetamide group. Copper mediated ligation will then be performed to form a stable triazole bond. The final product is purified and characterized as described above. Multiple haptens at the same site of the surface-homing agent.

M ultiple haptens within the same modification site are useful if it is necessary to increase the level of antibody recruitment. This may be done by building hapten-dendrons. Dendrons (similar to dendrimers) are well-defined, homogeneous, and usually symmetrical polymers with sizes and physiochemical properties that resemble proteins. Peptide-based dendrons can be readily assembled using solid-phase chemistry, providing access to predictable and modular ranges of epitope displays. We will decorate the terminal end of dendrons with haptens. Dendrons (composed of bis-Fmoc-L-Lys(Fmoc)- OH) will be synthesized using standard solid-phase chemistry and will be flanked with N-terminus com plementary conjugation handles (maleimide or alkyne).

Example 5

Antibody recruitment and induced toxicity (antibody dependent cell-mediated cytotoxicity and com plement-dependent cytotoxicity) may be evaluated in three primary pathogens: Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coll. Lipid A-binding agents will home to the surface of Gram-negative pathogens to present haptens, which will trigger the recruitment of antibodies. Following the opsonization stage, pathogens will be targeted for complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).

Experimental Design. DNP-based agents are evaluated for their ability to induce anti-DNP recruitment to bacterial cell surfaces. Preliminary results demonstrate that DNP haptens conjugated to PM BN induce the recruitment of anti-DNP antibodies to the surface of E. coil and P. aeruginosa.

Antibody recruitment by DN P-modified agents (PM B derivatives, AM Ps, and GP12) in E. coli, K.

pneumoniae, and P. aeruginosa is evaluated. Recruitment is assessed using FITC-labeled anti-DNP and quantified by flow cytometry. Next, recruitment of antibodies is analyzed directly from pooled human serum (PHS). The use of PHS to opsonize bacterial cells serves two purposes. It is primarily used to evaluate recruitment of anti-rhamnose antibodies, which are not commercially available. It also demonstrates the ability of agents to induce opsonization in a more physiologically-relevant condition (recruitment of antibodies directly from serum). From these results, a single lead construct is selected for further testing for CDC and ADCC. E. coli, K. pneumoniae, and P. aeruginosa cells are first treated with the hapten-displaying constructs (at sub-lethal concentrations) and subsequently incubated with PHS (commercially available from Innovative Research) to assess CDC or peripheral blood mononuclear cells (PBMCs, commercially available from AllCells) to assess ADCC. Briefly, bacterial cells (~10 ⁶ cells per cell type) are incubated with hapten-displaying agent or DMSO (control) in PBS for 2 h at 37° C and gently washed with PBS. Fresh PHS (1, 5, and 10 v/v%) or PBMCs (1:1, 10:1, and 100:1 as effector: target) are co-incubated with bacterial cells and gently spun to induce co-localization and incubated at various empirically-defined times. Bacteria are serially diluted, plated onto solid medium containing agar, and incubated overnight at 37° C. Colony forming units (CFU) are measured and the change in CFU determined based on hapten-displaying agent treatment.

Gram-negative pathogens are decorated by hapten-displaying agents based on the LPS composition of their OM. In the case of PMB, the re-introduction of the fatty acid tail may improve the inherent antimicrobial activity of the constructs. Antibody recruitment by PM B-based conjugates may be efficient at lower concentrations. The ability of the fatty acid tail to imbed within the OM may result in better affinity towards lipid A and longer residency time of the entire construct on bacterial cell surfaces. The opsonization of Gram-negative pathogens by our constructs may lead to robust CFU reduction by CDC and/or ADCC. Gram-negative bacteria are susceptible to both mechanism of immune protection following opsonization. By elevating the levels of antibodies on the bacteria surface, bacterial lysis may be induced by the serum-components and directly by human immune cells. Selection criteria: Levels of antibody recruitments by antigen-displaying agents are determined from at least three independent experiments. These results are compared to results obtained with unmodified agent (no hapten) and anti-antigen antibody alone through multiple comparisons analyses using either the Dunnett (at 95% confidence intervals) or the Holm-Sidak tests (Prism for Mac). Antigen-modified conjugates will be selected to advance based on antibody recruitment (>10,000 antigens per cell). Example 6

Computational optimization of the spacer length for the lipid A-binding construct that results in the largest antibody recruitment and bacterial killing to improve binding to lipid A and hapten display. Modeling and simulation of the complex OMs and their interactions with lipid A-binding constructs provide simulation results which combined with the experimental findings allow for design better spacer lengths for optimal antibody recruitments to various bacterial cell surface.

LPS membranes of K. pneumoniae, P. aeruginosa, and E. coli. Based on the LPS types (i.e., the core type and O-antigen type) of experimentally-chosen Gram-negative bacteria, corresponding LPS membrane systems are built using CHA MM-GUI Membrane Builder (now containing LPS Modeler) and equilibrate the systems using the well-validated Membrane Builder protocols.

Modeling and simulation of AMPs (PMB, LL-37, KLAKLAK, and WLBU2) and GP12 in LPS membranes. While these molecules are known to bind to lipid A of LPS, their interactions with and locations/orientations in LPS layers are evaluated. Before adding the linker and hapten DNP to these lipid A binders, free energy calculations are performed along the membrane normal to find the free energy minimum locations/ orientations in LPS layers. The window exchange umbrella sampling molecular dynamics (WEUSM D), a variant of Hamiltonian replica exchange M D, can significantly improve conformational sampling in transmembrane helix assembly. Therefore, using the equilibrated LPS membrane systems, windows are generated for WEUSMD free energy simulations by pulling each lipid A binder 1 A every 500 ps to the lipid A head group region from the bulk side. Then, each window with a 1- A interval is simulated for 10 ns and the simulations are extended if necessary for better convergence. The available crystal structure for GP12 (PDB ID: 1H6W) may be used and helical structures for LL-37, KLAKLAK, and WLBU2 assumed during the window generation and let the structures relax in each window during the WEUSM D simulation. The calculated free energy profiles along the membrane normal will provide insight into the influences of the LPS type and the binder's size and charge states on the locations/orientations of each binder in LPS layers. The decomposition of the total free energy into the contributions from the interactions between each distinct chemical moiety of each binder with individual environmental components such as water, ions, lipid A, core, and O-antigen is performed., revealing quantitative information on each component's energetic contribution to the free energy minima and maxima along the binding pathway. This information may suggest any further chemical modifications or mutations of each binder to improve the binding affinity to the lipid A region.

PEG spacer length variations. Using the free energy minimum locations/orientations of each binder in each LPS system, PEG spacers are linked with various lengths to lipid A binders and then link hapten DNP to the other end of PEG. After short minimization to avoid bad contacts, each system may be simulated for 200 ns (with three replicates) and the simulations extended if necessary for better convergence. The distribution of DNP with respect to the LPS layer in each system may be characterized. Also, each DNP's accessibility from and exposure to the bulk solution is quantified and compared with Example 7

Additional antigen conjugates within the selected agents replacing DN P with two additional endogenous antigens (rhamnose and a-Gal) and an exogenous antigen (FITC).

Endogenous Antibodies. Endogenous antibodies are germ-line encoded IgG, IgM, and IgA that circulate in the serum of healthy humans. Pre-existing antibodies in human serum are exploited as initiators of bacterial clearance. DNP has the advantage of being extremely synthetically tractable. However, there are two other haptens (the a-gal trisaccharide (a-Gal) and rhamnose) for which humans have extensive levels of antibodies. There is a greater abundance of endogenous anti-rhamnose antibodies compared to anti-DN P antibodies in human serum. Human serum contains ~8-times more endogenous anti-rhamnose antibodies relative to anti-DN P. Additionally, anti-rhamnose opsonization can induce more effective pathogen killing.

Exogenous Antibodies. The use of exogenous haptens (haptens for which we have no known antibodies in circulation and display low immunogenicity) can serve as orthogonal routes of targeting bacterial cell surfaces. FITC has been used to decorate the surface cancer cells and is under clinical evaluations.1 The major advantages of using exogenous haptens are: finer control on the level of antibodies in circulation, temporal control on introduction of antibodies, and lack of basal

immunogenicity.

Example 8

The following experiment is useful to test conjugates for reduction in bacterial burden and survival in a model in mice (pre-immunized against hapten or administered monoclonal antibody against hapten) in both immuno-competent and neutropenic animals and computationally optimize the spacer length and character of the top candidates to improve binding to lipid A and hapten display.

Testing in murine intraperitoneal challenge model is performed as described above with the exception that the agents being administered will be modified with rhamnose, a-Gal, or FITC. In the case of FITC-modified agents, anti-FITC are administered post injection of the FITC-modified agent. Testing in neutropenic mice is performed. Neutropenia is chemically induced via cyclophosphamide (CPM) administration. CPM powder is dissolved in distilled USP water for injection to a final concentration of 20 mg/m L. Animals are subjected to CPM treatment at a total dose of 250 mg/kg by two 0.5 mL intraperitoneal injections scheduled at day 1 (150 mg/kg) and day 4 (100 mg/kg). Similar experiments are carried out with drug sensitive and resistant strains of E. coli, K. pneumoniae, and A. baumannii. Using the free energy minimum locations/orientations of selected lipid A binder in each LPS systems, PEG spacers with various lengths are linked to lipid A binders and then linked to rhamnose, a- Gal, or FITC at the other end of PEG.

Example 9

Evaluate reduction in bacterial burden and survival in intranasal challenge model in mice (a pneumonia model) followed by intranasal challenge in rabbits and evaluate toxicity.

Experimental Design.

Murine intranasal challenge model for P. aeruginosa infection:

Six-week-old wild-type BALB/c mice are housed under pathogen-free conditions and fed autoclaved rodent feed and water. P. aeruginosa acute pneumonia infection models will be performed following published procedures with minor modifications. DiGiandomenico, A. et al. J Exp Med 2012, 209, 1273, which is incorporated herein by reference, discloses P. aeruginosa acute pneumonia infection models procedures with minor modifications described herein. In the acute pneumonia model, BALB/c mice are infected with P. aeruginosa strains suspended in a 50 μί inoculum instilled intranasally according to procedures described in Warrener, P et al. Antimicrob Agents Chemother 2014, 58, 4384, which is incorporated herein by reference. Hapten-conjugated agents, anti-FITC antibodies, or PBS are administered intraperitoneally (i.p.) at 24 h before infection (prophylaxis) or intravenously (i.v.) at 1 h post-infection (treatment) at empirically defined concentrations to assess the most efficacious levels of administration. For organ burden experiments, acute pneumonia is induced in mice followed by harvesting of lungs, spleens, and kidneys 24 h after infection for determination of CFU. Similar experiments are carried out with drug sensitive and resistant strains of E. coli, K. pneumoniae, and A. baumannii.

Experimental Design for Toxicology Assessment.

Possible off-target toxicity is evaluated by drawing blood from treated mice not harboring bacterial infections that were injected intravenously with the lead antigen-based conjugates (2 mg/Kg, q2d for a total of 4 injections) and the vehicle (n=3 in each cohort). Blood samples are harvested before treatment with hapten-conjugated agents as well as 3 and 14 days after treatment. Serum-based clinical chemistry evaluation (Antech Diagnostic Laboratories) of systemic toxicity is focused on liver and kidney functions (e.g., alkanine phosphatase, bilurin, urea nitrogen, creatinine, phosphorus, glucose).

Circulating white and red blood cells count and mouse mass are also assessed throughout duration of the study. T-cell activation is assessed by ELIspot.

Example 10 Agents with novel mechanisms of action are needed to complement traditional antibiotics. Towards these goals, the surface-homing properties of vancomycin was exploited to tag the surface of Gram-positive pathogens with immune cell attractants in two unique modes. Vancomycin was conjugated to the small molecule hapten 2,4-dinitrophenol (DNP) to promote bacterial opsonization. The tagging specificity and mechanism of incorporation was then improved by coupling it to a sortase A substrate peptide

Vancomycin was used to decorate Gram-positive bacteria with 2,4-dinitrophenol (DNP) epitopes to re-enlist components of the immune system. In addition, a coupled sortase-A surface-remodeling strategy was used to improve activity and selectivity towards drug-sensitive and methicillin-resistant strains of S. aureus.

Vancomycin binds lipid II molecules at the cytoplasmic membrane to halt PG biosynthesis but can also associate with d-Ala-d-Ala dipeptide units within the full length pentapeptide found on mature peptidoglycan (PG) scaffolds (Figures 8A and 8B). For the problematic human pathogen S. aureus, PG d- Ala-d-Ala content is ~20%, with the rest being composed of shorter stem peptides processed by carboxypeptidases or transpeptidases. Hapten-conjugated vancomycin effectively graft antigenic epitopes onto bacterial cell surfaces that display of d-Ala-d-Ala on PG scaffolds of Gram-positive bacteria at such level (Figures 8C).

Results and discussion

Vancomycin-based design

Semi-synthetic derivatives of vancomycin have been shown to label at the septal region of Bacillus subtilis (B. subtilis), which has a higher density of lipid II molecules, and also throughout the bacterial sidewalls. Vancomycin can also target Gram-positive bacteria in live hosts. Labeling experiments using BODIPY ^® FL vancomycin in B. subtilis, S. aureus, and Enterococcus faecium (E.

faecium) was performed and cells were found to be labeled both at the septal region and the sidewalls. Based on these results and the well-established ability of vancomycin to target Gram-positive bacteria, hapten-modified vancomycin derivatives were tested to effectively tag bacterial cell surfaces.

Two derivatives of vancomycin were synthesized, VanNdnp and VanCdnp that incorporated DNP epitopes (Figure 9A) at the amino group and carboxylic acid sites, respectively. S. aureus cells were incubated with both vancomycin derivatives, exposed to FITC-conjugated anti-DNP antibodies, and antibody recruitment was measured by flow cytometry (Figure 9B). Due to the nature of our assay, it was important to minimize non-specific binding of antibodies by surface anchored protein A. By using a protein A-deletion mutant S. aureus strain, fluorescence levels reflect the specific recruitment of anti- DNP antibodies onto cell surfaces. Treatment ofS. aureus cells with VancCdnp and VancNdnp resulted in 10.3-fold and 2.7-fold increases in fluorescence levels, respectively (Figure 9C). Similarly, VancCdnp led to greater antibody recruitment in E. faecium and B. subtilis. These results indicated that anti-DNP recruitment can be specifically induced by DNP-modified vancomycin.

Sortase-vancomycin conjugates

VancCdnp was redesigned to (1) increase surface display of haptens in pathogens that have reduced d-Ala-d-Ala content on mature PG scaffolds and (2) covalently incorporate haptens within the PG scaffold. Towards these goals, vancomycin-DNP constructs were conjugated to the substrate peptide sequence from S. aureus sortase A (SrtA). SrtA transpeptidase is a surface-bound enzyme that attaches bacterial proteins onto the PG scaffold (Figure 10A). This mode of surface modification is critically important for entry and colonization of the host organism. The transpeptidase recognizes a SrtA specific peptide sequence (LPXTG motif, where X is any amino acid) and catalyzes the acyl-transfer onto lipid II of S. aureus. As the PG monomeric unit from lipid II is loaded onto the existing PG scaffold, so will the anchored protein.

Experiments were performed to test whether the covalent conjugation of vancomycin to LPXTG would bring the substrate peptide to its partner (lipid II) based on the ability of vancomycin to associate with the neighboring d-Ala-d-Ala dipeptide on lipid II (Figure 10A). The higher effective concentration should result in covalent PG labeling at physiologically relevant concentrations. The combination of SrtA and vancomycin will decouple the tagging step from availability of d-Ala-d-Ala on the PG scaffold.

Therefore, high tagging levels should be achievable irrespective of the levels of d-Ala-d-Ala on surface exposed PG.

The cell labeling experiments were repeated using SrtA sequences alone to establish baseline values. The three primary FITC modified peptide sequences were: K(FITC)LPETG, K(FITC)LPMTG, and K(FITC)MGTLP. Bacterial cells were separately treated with all three peptides (incubated overnight at 1 mM). S. aureus cells displayed a 20-fold increase in fluorescence at 1 mM using K(FITC)LPETG, relative to unlabeled cells. The methionine containing K(FITC)LPMTG peptide resulted in an additional ~2-fold increase in surface labeling. Attenuated cellular fluorescence was observed for the control scrambled sequence peptide. Molecules that combined SrtA substrate sequence and vancomycin (Figure 10B) were assembled. The linker between LPMTG and vancomycin was systematically varied to establish the optimum linker length to bridge binding to d-Ala-d-Ala and SrtA. The tether length was empirically established by evaluating a small panel of polyethylene glycol (PEG) spacers. Shorter linkers are preferred (Figure IOC). The covalent attachment of vancomycin to each one of these constructs led to a major increase in surface tagging at 5 μΜ (up to 200-fold increase). The PEG spacer between LPMTG and FITC was sampled at two different lengths (Figure 10D). A longer PEG linker was preferred.

Confocal microscopy imaging was performed to delineate the localization of cellular labelling of Sortl. Fluorescence was observed throughout the entire cell surface with pronounced labeling at the septal region. The role of vancomycin within Sortl in mediating cellular labeling was also probed.

The co-incubation of S. aureus cells with Sort2 and vancomycin led to fluorescence signals near background levels. Based on these results it is not enough to treat cells with both vancomycin and Sort2. The covalent conjugation is necessary to induce a large increase in cellular labeling. The addition of free vancomycin to cells treated with Sortl resulted in a 34% increase in cellular fluorescence. This finding is points to the potential of co-treatment with vancomycin in future in vivo testing, which should target bacterial cells in two complementary ways.

To evaluate the labeling across different bacterial species, cellular labeling was measured in S. aureus, Staphylococcus epidermidis (S. epidermidis), Listeria monocytogenes (L. monocytogenes), B. subtilis, and Escherichia coli (E. coli). Fluorescence levels were highest for S. aureus treated with Sortl followed by S. epidermidis (Figure 11A). B. subtilis were sensitive to Sortl presumably due to the presence of vancomycin. Gram- negative E. coli showed background fluorescence levels, which is consistent with the mode of incorporation and lack of vancomycin accessibility to the PG layer. L.

monocytogenes labeled ~20-fold less than S. aureus. The substrate sequence (NAKTN) for SrtB labeled the surface of L. monocytogenes cells. To test this idea, FITC(PEG)-NAKTN was synthesized and incubated with L. monocytogenes cells, which led to high levels of cellular fluorescence. Constructs label the surface of bacteria with defined specificity and can potentially be generalized based on sortase substrate sequence.

Labeling in live host animal

Bacterial surface labeling in Caenorhabditis elegans (C. elegans), which is a powerful model animal for bacterial pathogenesis, was studied. C. elegans (L4 larval stage) were incubated with S.

aureus to establish bacterial colonization.

After removing residual bacteria, S. aureus infected C. elegans were treated with Sortl. Bacteria were clearly labeled in vivo post infection (Figure 11B). This is an example of metabolic labeling of bacterial PG in vivo. Exogenous epitopes can be selectively grafted onto the surface of S. aureus in a live host. Distinguishing PG features of Sortl treated S. aureus cells were readily visible in the green channel consistent with the mode of incorporation. Localization of S. aureus cells was accomplished under the constitutive cytosolic expression of mCherry. Co-localization of green and red signals were consistently observed in all the samples analyzed. Treatment of S. oureus-infected C. elegans with Sort2 (lacking the vancomycin moiety) resulted in no observable green signal. The results demonstrated the ability to target bacterial cells in live hosts.

A construct composed of DNP in place of FITC (Sort3) was synthesized using the same synthetic route. The hapten DNP was chosen due to the naturally high abundance of endogenous anti-DNP antibodies in human serum. S. aureus cells were exposed to Sort3 and anti-DNP recruitment was analyzed similar to VanCdnp (Figure 12A). A clear increase in cellular fluorescence was observed at sub- micromolar concentrations indicative of anti-DNP recruitment. At 5 μΜ, fluorescence levels were 14.8- fold above untreated cells. Cell treatment with Sort4 led to background fluorescence levels; vancomycin is critical for surface labeling. Recruitment of anti-DNP was also observed directly from pooled human serum, indicating that Sort3 can potentially operate in physiologically relevant conditions. Experiment was performed using the pathogenic and widely disseminated methicillin-resistant S. aureus (MRSA). Protein A on the surface of MRSA can be disruptive to assay read-out. To circumvent this, MRSA cells were pre-treated with mock IgGs (lacking a FITC label) to mimic the anticipated occupancy of protein A by antibodies from serum. Treatment of MRSA cells with Sort3 led to similar fluorescent levels to protein A-deleted strains. In addition, pre-incubation of MRSA cells with pooled human IgGs, which include anti-DNP antibodies, effectively blocked binding of FITC-labeled anti-DNP.

The inherent toxicity of Sort3 towards S. aureus cells was evaluated. Exposing S. aureus to concentration > 100 μΜ led to no significant change in cell density. The covalent anchoring of vancomycin within the stem peptide may lead to segregation from its lethal lipid II target. Toxicity was also minimal towards mammalian cells at all concentrations tested, a finding that was expected based on the polarity and size of Sort3 and lack of cognate binding partners. The recognition and phagocytosis of opsonized bacteria by macrophages was evaluated. Bacteria treated with Sort3 and exposed to anti- DNP antibodies led to a 2-fold increase in phagocytosis compared to treatment of Sort3 alone and anti- DNP alone (Figures 12B). The surface of bacterial cells remodeled with Sort3 display antigenic epitopes that are available to engage with immune cells.

Conclusions

Two classes of vancomycin conjugates that tag S. aureus cell surfaces by non-covalent association with the PG scaffold and covalent integration within bacterial PG were described.

Fluorescent-based constructs were synthesized to optimize incorporation levels. The surface of S. aureus was shown that it could metabolically tagged with unnatural epitopes in live C. elegans hosts. Hapten- based constructs were synthesized and bacterial opsonization was demonstrated, which resulted in the induction of phagocytosis by macrophages. Combined, this class of agents represents a promising immune-modulatory strategy to combat bacterial infections.

SEQUENCES

SEQ ID N0:1 - LLGDFF KSKEKIGKEFK IVQ IKDFL NLVP TES (LL-37)

SEQ ID N0:2 - KLAKLAKKLAKLAK (KLAKLAK2)

SEQ ID N0:3 - RRWVRRVRRWVRRVVRVVRRWVRR (WLBU2)

SEQ ID N0:4 - FWRGDLVFDFQV (Hepatitis A amino acids 110-121)

SEQ ID N0:5 - STGPCKTCTTPA (Hepatitis B amino acids 117-128)

SEQ ID N0:6 (representative example of GP12)

1 msnntyqhvs nesryvkfdp tdtnfppeit dvqaaiaais pagvngvpda ssttkgilfi 61 pteqevidgt nntkavtpat latrlsypna tetvygltry stndeaiagv nnessitpak 121 ftvalnnafe trvstessng vikisslpqa lagaddttam tplktqqlai kliaqiapse 181 ttatesdqgv vqlatvaqvr qgtlregyai spytfmnsss teeykgvikl gtqsevnsnn 241 asvavtgatl ngrgsttsmr gvvkltttag sqsggdassa lawnadviqq rggqiiygtl 301 riedtftian gganitgtvr mtggyiqgnr ivtqneidrt ipvgaimmwa adslpsdawr 361 fchggtvsas dcplyasrig tryggnpsnp glpdmrglfv rgsgrgshlt npnvngndqf 421 gkprlgvgct ggyvgevqiq qmsyhkhagg fgehddlgaf gntrrsnfvg trkgldwdnr 481 syftndgyei dpesqrnsky tlnrpelign etrpwnisln yiikvke