EP4701639A1 - Molecules targeting infected tissues and methods of use thereof - Google Patents

Abstract

Polypeptides that selectively target and bind to infected tissue and/or microbial pathogens in vivo have been discovered. Antimicrobial agents and diagnostic agents such as dyes that are conjugated to the infected tissue homing molecules are effectively targeted selectively to infected tissues and/or microbial pathogens in vivo. Compositions and methods of infected tissue binding molecules for selectively homing to infected tissues are provided. In some forms, the compositions include nanoparticles, such as silver nanoparticles, conjugated to the infected tissue homing peptides and loaded with antimicrobial agents. Typically, the amount of antimicrobial that is required to treat or prevent infection by pathogens such as Mycobacterium tuberculosis (Mtb) when delivered within the nanoparticles is less than the amount of the same antimicrobial delivered alone.

Description

MOLECULES TARGETING INFECTED TISSUES AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Application No. 63/470,002 filed May 31, 2023, which is hereby incorporated by reference in its entirety.

REFERENCE TO THE SEQUENCE LISTING

The Sequence Listing XML submitted as a file named “Tartu_P229_PCT_ST26.xml”, having a size of 37,562 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

FIELD OF THE INVENTION

The invention is generally directed to compositions and methods of peptides that target and bind to tissues infected by microbial pathogens , particularly peptides that selectively target and bind to tissues infected by bacteria, such as Mycobacterium tuberculosis.

BACKGROUND OF THE INVENTION

Tuberculosis (TB) is a treatable disease caused by the bacterium Mycobacterium tuberculosis (Mtb~). TB is a difficult infection to treat, treatment of even uncomplicated, drug-susceptible tuberculosis (TB) is lengthy, requiring 6 to 9 months with a 40-year-old four-drug regimen for which extensive clinical drug resistance exists. One of the main obstacles is poor penetration of drugs to the lesions harboring the bacteria: the need for prolonged treatment is that the pathogen often resides in hard to treat lesions called “granulomas,” which slow and impede the ability of antibiotics to reach the pathogen (Ernest et al., 2020, Strydom et al., 2019, Irwin et al., 2016, Sarathy et al., 2016). Poor drug penetration may also contribute to the emergence and expansion of drug resistance as it enables periods in which local populations of bacilli experience suboptimal drug levels, or even monotherapy, especially in human TB patients with complex pulmonary lesions. New methods to overcome the problem of suboptimal antibiotic delivery are urgently needed.

Targeting of chemotherapeutics to cancer cells and tissues has been achieved with some initial success, and there are many similarities between infected and cancerous tissue, including suboptimal drug penetration of some drugs into tumor-rich tissues (Simon-Gracia et al., 2022). However, no peptides with drug delivery potential that recognize the TB bacilli in infected tissues are currently available.

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45652286 Therefore, it is an object of the invention to provide compositions that specifically recognize and home to tissues infected with microbial pathogens in vivo.

It is a further object of the invention to provide compositions that specifically recognize and home to Mycobacterium tuberculosis bacteria and TB-infected tissues in vivo.

It is a further object of the invention to provide compositions that can penetrate the tissue barriers protecting the pathogen in vivo from effective delivery of drugs to treat and prevent microbial infections.

It is another object of the present invention to provide formulations targeting drugs to microbial pathogens in vivo with minimal systemic toxicity.

It is another object of the present invention to provide formulations and methods for delivering antibiotics selectively to sites of infection in dosages that are safe and effective for use in humans.

SUMMARY OF THE INVENTION

Peptides, non-peptidic polymers and other molecules that selectively target and bind to tissues infected with microbial pathogens, such as Mycobacterium sp., including Mycobacterium tuberculosis (Mtb) bacteria have been developed. Compositions including the infected tissue homing molecules in combination with one or more active agents, such as antimicrobial agents, and optionally a delivery vehicle are provided. The compositions selectively target microbial pathogens and infected tissues in vivo and therefore selectively deliver the associated one or more active agents, such as antimicrobial agents, to microbial pathogens, such as cells of Mtb and/or infected tissues, such as tissues infected with Mtb.

Methods for treatment or prevention of an infection by a microbial pathogen in a subject in need thereof are provided. In some forms, the methods include administering to the subject a pharmaceutical formulation including (a) an infected tissue homing molecule; and (b) an antimicrobial agent, wherein the antimicrobial agent is conjugated or complexed with the infected tissue homing peptide, and wherein the antimicrobial agent is in an amount effective to treat or prevent one or more symptoms of the infection in the subject.

Methods for identification of an infection by a microbial pathogen in a subject, including administering to the subject a pharmaceutical formulation including (a) an infected tissue homing molecule; and (b) an imaging or diagnostic agent, wherein the imaging or diagnostic agent is in an amount effective to bind to infected tissue and/or pathogens and label or otherwise identify the infection in the subject. In some forms, the

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45652286 infected tissue homing molecule is or includes a peptide. Exemplary infected tissuehoming peptides include a peptide having an amino acid sequence of any one or more of SEQ ID NOs:l-37, or a functional variant thereof having at least 70% sequence identity to any one of SEQ ID NOs: 1-37. hi some forms, the infected tissue homing molecule is a peptide having an amino acid sequence PPRRGLIKLKTS (SEQ ID NO: 1). In other forms, the infected tissue homing molecule is a function variant of the peptide of SEQ ID NO: 1 , for example, having at least 70% sequence identity to PPRRGLIKLKTS (SEQ ID NO: 1). In certain forms, the infected tissue homing molecule is a peptide that consists of PPRRGLIKLKTS (SEQ ID NO: 1). In some forms, the infected tissue homing molecule is or includes a peptide having an amino acid sequence AGRGRLVR (SEQ ID NO:2). In other forms, the infected tissue homing molecule is a function variant of the peptide of SEQ ID NO:2, for example, having at least 70% sequence identity to AGRGRLVR (SEQ ID NO:2). In certain forms, the infected tissue homing molecule is a peptide that consists of AGRGRLVR (SEQ ID NOG). In some forms, the infected tissue homing molecule is a peptide having an amino acid sequence GRP ARP AR (SEQ ID NO:3). In other forms, the infected tissue homing molecule is a function variant of the peptide of SEQ ID NO:3, for example, having at least 70% sequence identity to GRP ARP AR (SEQ ID NOG). In certain forms, the infected tissue homing molecule is a peptide that consists of GRP ARP AR (SEQ ID NOG). In some forms, the infected tissue homing molecule is a peptide having an amino acid sequence CKRDLSRRC (SEQ ID NOG). In other forms, the infected tissue homing molecule is a function variant of the peptide of SEQ ID NOG, for example, having at least 70% sequence identity to CKRDLSRRC (SEQ ID NOG). In certain forms, the infected tissue homing molecule is a peptide that consists of CKRDLSRRC (SEQ ID NOG). In some forms, the infected tissue homing molecule is a peptide having an amino acid sequence AKRGARSTA (SEQ ID NOG). In other forms, the infected tissue homing molecule is a function variant of the peptide of SEQ ID NOG, for example, having at least 70% sequence identity to AKRGARSTA (SEQ ID NOG). In certain forms, the infected tissue homing molecule is a peptide that consists of AKRGARSTA (SEQ ID NOG).

In some forms, the methods administer a formulation that further includes one or more infected tissue homing molecules, such as polypeptides or other amino acid sequences, for example, whereby the polypeptides or other amino acid sequences are contiguous with the amino (NH) or carboxyl (COOH) terminus of the infected tissue homing peptide. In some forms, the methods administer a formulation that further includes

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45652286 one or more additional molecules selected from the group including a carbohydrate, a lipid, a small organic molecule, a polymer, and a nucleic acid.

In some forms, the methods administer a formulation including an antimicrobial agent selected from bedaquiline, pretomanid, sutezolid, delamanid, isoniazid, vancomycin, rifampin, rifabutin, pyrazinamide, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, ethionamide, aminosalicylic acid, and cycloserine. In certain forms, the antimicrobial is vancomycin. In some forms, the antimicrobial agent is attached to the peptide via a linker, such as a cleavable linker.

In some forms, the methods administer a formulation including a delivery vehicle. An exemplary delivery vehicle is a particle, such as a microparticle or a nanoparticle. In some forms, the delivery vehicle is selected from a citric acid silver nanoparticle, a liposome, a micelle, a viral capsid, a polymeric particle, a dendrimer, a porous silicon particle, a metal particle, and an iron oxide nanoparticle. In certain forms, the delivery vehicle is a citric acid nanoparticle. Typically, when the methods include administering a formulation including a particle, the infected tissue homing molecule is conjugated to, or dispersed within the outer surface of the particle. In some forms, the infected tissue homing molecule is attached to the surface of the particle via a linker, such as a cleavable linker. In an exemplary form, the infected tissue-targeting molecule is attached to the surface of the particle via a polyethylene glycol (PEG) linker. In an exemplary form, the PEG has a molecular weight of between about 1,000 Da and about 10,000 Da such as about 5,000 Da. In some forms, the antimicrobial agent is encapsulated or embedded within one or more cavities and/or pores within the particle. Generally, the particle has a mean hydrodynamic diameter of between about 40 nm and about 80 nm, inclusive, optionally wherein the particle has a mean diameter of about 60 nm. Typically, the particle does not include another targeting or homing agent.

Typically, the particle is conjugated to between one and about 10,000 molecules of the infected tissue homing molecule. In some forms, the formulation is in a form selected from the group including a solution, a dry powder, micelles, colloids, nanodroplets, nanostructured hydrogel, nanocrystals, and a nanosuspension.

In some forms, the microbial pathogen is one or more Mycobacterium sp. selected from the group including M. tuberculosis, M. africanum, M. canetti, M. bovis, M. caprae, M. microti, M. pinnipedii, M. mungi, M. leprae, M. avium, M. ulcerans, M. xenopi, M. kansasii, M. abscessus, M. chelonae, M. fortuitum and M. orygis. In some forms, the

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45652286 microbial pathogen is M. tuberculosis. In some forms, the microbial pathogen is an antibiotic resistant strain that is resistant to one or more antibiotics when administered in vivo alone at a safe dose. In some forms, the microbial pathogen is a Mycobacterium sp. that is an antibiotic resistant strain that is resistant to one or more antibiotics when administered in vivo alone at a safe dose. For example, in some forms, the Mycobacterium sp. is resistant to one or more antibiotics selected from the group including bedaquiline, pretomanid, sutezolid, delamanid, isoniazid, vancomycin, rifampin, rifabutin, pyrazinamide, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, ethionamide, aminosalicylic acid, and cycloserine, either alone (i.e., monoresistant) or in combination (polyresistant including multi-drug and Extensively-drug resistant forms of TB).

In some forms, the antibiotic resistant strain is susceptible to the same antibiotic when administered conjugated with the infected tissue homing peptide. In certain forms, the subject has been diagnosed as having, or as being at risk of having tuberculosis (TB) selected from pulmonary TB, CNS TB, pericardial TB, pleural TB, Lymphadenitis, Abdominal TB, skeletal TB, genitourinary TB and miliary TB. In some forms, the methods are effective to reduce, eliminate, or prevent one or more symptoms of Mtb infection selected from the group including fever, chest pain, wheezing or difficulty breathing, cough, fatigue, and unintentional weight loss. Typically, the pharmaceutical formulation is administered to the subject via a route selected from oral administration, intramuscular injection, intravenous injection, sub-cutaneous injection, and intra-articular injection. In some forms, the effective amount of the antimicrobial agent administered in the formulation is less than the amount of the same therapeutic agent that is required to be effective when administered alone.

Dosage forms for treatment of an infection by a microbial pathogen are also described. Dosage forms for treatment of an infection by Mycobacterium tuberculosis (Mtb) in a subject in need thereof, are also described. In some forms, the dosage form includes (a) an infected tissue homing molecule including an amino acid sequence selected from SEQ ID NOs:l-37; and (b) an antimicrobial agent, whereby the antimicrobial agent is in an amount effective to treat or prevent one or more symptoms of the infection in the subject. In some forms, the effective amount of therapeutic agent administered in the formulation is less than the amount of the same therapeutic agent that is required to be effective when administered alone.

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45652286 BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1T are photo-micrograph images showing distribution of receptors of the candidate homing peptides in the TB-infected necrotic and cellular lesions. Images show TB-infected tissue sections stained with antibodies to identify the location of receptors in infected cellular lesions (Figs. 1A-1J) and infected necrotic lesions (Figs. 1K- 1P), respectively. Fluorescence immunostaining corresponds with presence of receptors including p32 (Figs. 1A, IK); RXRB (Figs. IB, IL); RelB (Figs. 1C, IM); Laminin (Figs. ID, IN); FN-EDB (Figs. IE, IO); HAS3 (Figs. IF, IP); TNC-C (Figs. 1G, IQ); NRP-1 (Figs. 1H, 1R); Fibrin (Figs. II, IS); and Alpha-v integrin (Figs. 1J, IT), respectively.

Figures 2A-2B are graphs of data from in vivo play-off experiments in TB- infected or healthy mice. Fig. 2A shows the ratio of each of the targeting peptides (x-axis) to a control peptide (y-axis; 0 tolOO fold over G7 control phage) in each of TB-infected lung (dark bars) and non-infected lung (light bars) tissue, respectively Fig. 2B shows the ratio of peptides (y-axis; 0-10 infected/noninfected lung phage representation) of each of the targeting peptides (x-axis), respectively.

Figures 3A-3C are diagrams depicting peptide-functionalized silver nanoparticles (AgNP). Fig. 3A is a graph of Absorbance [a.u.] over Wavelength [nm], with a photomicrograph of the particles also depicted. Scale bar is 200 nm; Fig. 3B is a bar graph of Volume [%] over Hydro-dynamic Diameter [nm]; Fig. 3C is a diagram showing specifications of citric AgNPs used in the homing study, showing the silver (Ag) core covered with NeutrAvidin-PEG(5K)-thiols (boxes with lines) and Iipoic-PEG(1K)-NH2 (lines), respectively. Targeting peptides are attached to NeutrAvidin and the payload is conjugated to the free NH2 groups of the lipoic-PEG(lK)-linkers.

Figure 4 is a flow chart depicting the experimental schema for in vivo homing studies using peptide-functionalized silver nanoparticles in Mycobacterium tuberculosis infected C3HeB/FeJ mice.

Figures 5A-5B are photo-micrograph images showing staining of infected tissue with FAM-labelled PL1 peptide (Fig. 5A); and with control peptide (Fig. 5B), respectively, in cellular lesions in Mycobacterium tuberculosis Erdman infected C3HeB/FeJ mice.

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45652286 DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

Ranges can he expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The terms “high,” “higher,” “increases,” “elevates,” or “elevation” refer to increases above basal levels, e.g., as compared to a control. The terms “low,” “lower,”

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45652286 “reduces,” or “reduction” refer to decreases below basal levels, e.g., as compared to a control.

As used herein, the terms "including", "includes", "having", "has", "with", or variants thereof, are intended to be inclusive similar to the term "including."

The term “inhibit” means to reduce or decrease in activity or expression. This can be a complete inhibition of activity or expression, or a partial inhibition. Inhibition can be compared to a control or to a standard level. Inhibition can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,

35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,

59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,

83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

The term “nanoparticle,” as used herein, generally refers to a structure of any shape having a diameter from about 1 nm up to, but not including, about 1 micron, more preferably from about 5 nm to about 500 nm, most preferably about 100 nm.

Nanoparticles having a spherical shape are generally referred to as “nanospheres.” Nonlimiting examples of nanoparticles include virus-like particles, particles formed of high- order structures of proteins, and soft nanoparticles, e.g., micelles, colloids, liposomes, vesicles, nanodroplets nano-structured hydrogel, nanocrystals, and nanosuspension. Soft nanoparticles generally dissolve or dissemble to release agents.

The terms “Mtb” and “M. tuberculosis” are used interchangeably herein, to refer to the organism Mycobacterium tuberculosis, including all strains and variants thereof.

The term “Mycobacterium sp.” as used herein refers to multiple species of the bacteria Mycobacterium. The term “Mycobacterium sp.,” as used herein, refers to a single, but unspecified species of the bacteria Mycobacterium.

The term “selectively target” refers to the capability of a referenced component to bind to, target (e.g., for binding), or home to (e.g., in a subject) to the referenced component’s binding target preferentially, to a greater extent, and/or at higher affinity to the referenced component’s binding target than other components, compounds, and/or materials in a milieu of interest (e.g., in a subject). For example, the disclosed microbial pathogen-targeting peptides bind to microbial pathogens and to pathogen-infected tissues in vivo to a greater extent than to uninfected “control” cells and tissues of the same animal in vivo. In an exemplary form, the disclosed microbial pathogen -targeting peptides bind to Mycobacterium bacteria to a greater extent than to cells and tissues of animals. Similarly,

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45652286 the disclosed microbial pathogen -targeting peptides target and home to Mycobacterium bacteria present in a subject to a greater extent than to cells and tissues of the subject.

The term “polymer” refers to a chemical entity with a plurality of repeating units generally bonded covalently. In some forms, a polymer has a molecular weight greater than 500 or 1,000, or more. Non- limiting exemplary polymers include poly-amino acids, naturally occurring, and synthetic chemical compounds.

The term “pharmaceutically acceptable,” as used herein, refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration.

The terms “biocompatible” and “biologically compatible,” as used herein, generally refer to materials that are, along with any metabolites or degradation products thereof, generally non-toxic to the recipient, and do not cause any significant adverse effects to the recipient. Typically, biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient.

The term “hydrophilic,” as used herein, refers to the property of having affinity for water. For example, hydrophilic polymers (or hydrophilic polymer segments) are polymers (or polymer segments) which are primarily soluble in aqueous solutions and/or have a tendency to absorb water. In general, the more hydrophilic a polymer is, the more that polymer tends to dissolve in, mix with, or be wetted by water.

The term “hydrophobic,” as used herein, refers to the property of lacking affinity for or repelling water. For example, the more hydrophobic a polymer (or polymer segment), the more that polymer (or polymer segment) tends to not dissolve in, not mix with, or not be wetted by water.

The term "therapeutic agent" refers to an agent that can be administered to prevent or treat a disease or disorder. Therapeutic agents can be a nucleic acid, a nucleic acid analog, a small molecule, a peptidomimetic, a protein, peptide, carbohydrate or sugar, lipid, or surfactant, or a combination thereof.

The terms “treating” and “retarding development of’ a disease, disorder, or condition occurring in an animal which has or may be predisposed to the disease, disorder and/or condition mean inhibiting the disease, disorder or condition, e.g.. impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the

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45652286 disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. In some forms, the animal has been diagnosed with the disease or disorder. In other forms, the animal has not yet been diagnosed as having the disease or disorder. The terms “preventing” and “preventing development of’ used in the context of a disease, disorder, or condition in an animal mean inhibiting the initiation or development of the disease, disorder or condition, e.g., stopping the animal from developing the disease, disorder or condition, or impeding its progress; and/or preventing the advancement or continuation of at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as preventing pain in a subject by administration of an analgesic agent, even though such agent does not treat the cause of the pain.

The term "targeting moiety" as used herein refers to a moiety that localizes to or away from a specific locale. The moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. The entity may be, for example, a therapeutic compound such as a small molecule, or a diagnostic entity such as a detectable label. The locale may be a tissue, a particular cell type, or a subcellular compartment.

The term “therapeutically effective amount” means an amount of the therapeutic agent that, when incorporated into and/or onto particles described herein, produces some desired effect at a reasonable benefit/risk ratio applicable to any treatment. The effective amount may vary depending on such factors as the disease or condition being treated, the particular formulation being administered, the size of the subject, or the severity of the disease or condition.

The terms “incorporated” and “encapsulated” means incorporating, formulating, or otherwise including an agent into and/or onto a composition, regardless of the manner by which the agent or other material is incorporated.

The term “fragment” means a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

The terms “isolated,” “purified,” or “biologically pure” mean material that is free to varying degrees from components which normally accompany it as found in its native

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45652286 state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation.

The term “polypeptides” includes proteins and fragments thereof. By “protein” or “polypeptide” or “peptide” is meant any chain of more than two natural or unnatural amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring or non-naturally occurring polypeptide or peptide, as is described herein. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

“Variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

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45652286 In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 + 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (- 1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity,

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45652286 hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Vai: He, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.

“Identity,” as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” can also mean the degree of sequence relatedness of a polypeptide compared to the full-length of a reference polypeptide. “Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure.

By way of example, a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from: at least one amino acid deletion,

13

45652286 substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide.

By "subject" is meant a mammal, including, but not limited to, a human or nonhuman mammal, such as a bovine, equine, canine, ovine, or feline. The subject is preferably a mammal in need of treatment, e.g. , a subject that has been diagnosed with a disease or a predisposition thereto. The mammal is any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a horse, as well as livestock or animals grown for food consumption, e.g., cattle, sheep, pigs, chickens, and goats. In a preferred form, the mammal is a human.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

II. Compositions

It has been discovered that several molecules recognize and bind to tissues infected with microbial pathogens in vivo, as well as to microbial pathogens, such as Mycobacterium tuberculosis (Mtb) bacteria. As set forth in the Examples, it has been established that peptide receptors that are selectively expressed and/or up-regulated in microbial-infected tissues include p32, NRP-1, Fn-EDB, CD206, Retinoid X receptor beta (RXR-beta), alpha-V-integrin, hyaluronan, heparin sulphate and TNC-C. Therefore,

14

45652286 molecules such as peptides, non-peptidic polymers and other molecules that selectively target and/or bind to one or more of p32, NRP-1, Fn-EDB, CD206, Retinoid X receptor beta (RXR-beta), alpha- V-integrin, hyaluronan, heparin sulphate and TNC-C are provided.

Molecules, including peptides and non-peptidic polymers that selectively target and bind to tissues infected with microbial pathogens in vivo, as well as to microbial pathogens such as Mycobacterium tuberculosis, have been developed, and compositions thereof are provided. Typically, the infected tissue-targeting molecules are conjugated with, complexed with, or otherwise are associated with one or more active agents, such as antimicrobial agents. In some forms, the infected tissue-targeting molecule/active agent conjugates are incorporated into or onto a delivery vehicle. Typically, the infected tissuetargeting molecules are distributed at or near the solvent-exposed surfaces of the delivery vehicle. Typically, the number, type, distribution and density of infected tissue-targeting molecule(s) associated with a delivery vehicle are effective to drive homing of the vehicle and any active agents to the site of pathogenic bacteria or infected tissues in vivo. In some forms, infected tissue-targeting molecules are embedded within or otherwise attached to the outer surface of a hollow or porous particle, for example, a porous particle encapsulating one or more active agents for delivery selectively to a site of pathogenic bacteria in vivo. The compositions are typically formulated for delivery in vivo.

The term “infected tissue binding molecule” as used herein, means a molecule, such as a polypeptide or a non-peptidic polymer, that is capable of binding to infected tissue in vivo, and/or to pathogenic microbes, such as bacterial cells, in the absence of another molecule that targets, chaperones or otherwise mediates the binding of the molecule to the infected tissues and/or bacterial cells. In other forms, the infected tissue binding molecules specifically bind to one or more substances that are extruded from pathogenic microorganisms, such as bacterial cells in the region of the infected tissue.

Infected tissue binding molecule s, and compositions thereof are provided. In some forms, the compositions of infected tissue-binding peptides include one or more active agents. In some forms, the compositions of infected tissue-binding peptides include one or more delivery vehicles. In some forms, the compositions of infected tissue-binding peptides include one or more linkers or coupling agents. In some forms, the compositions of infected tissue-binding peptides are formulated with one or more reagents into a formulation.

15

45652286 A. Mycobacterium sp.

Peptides that selectively target and bind to tissues infected with Mycobacterium sp. in vivo, as well as to cells of Mycobacterium sp., for example, cells of Mycobacterium tuberculosis, and compositions thereof are provided.

Typically, the peptides are conjugated with, complexed with, or otherwise are associated with one or more active agents, such as antimicrobial agents. In some forms, the Mycobacterium sp.-infected tissue targeting peptide/active agent conjugates are incorporated into or onto a delivery vehicle. Typically, the Mycobacterium sp. -infected tissue targeting peptides are distributed at or near the solvent-exposed surfaces of the delivery vehicle.

Typically, the number, type, distribution and density of Mycobacterium sp.- infected tissue targeting peptide(s) associated with a delivery vehicle are effective to drive homing of the vehicle and any active agents to the site of Mycobacterium tuberculosis bacteria and/or infected tissues in vivo. In some forms, Mycobacterium sp. -infected tissue targeting peptides are embedded within or otherwise attached to the outer surface of a hollow or porous particle, for example, a porous particle encapsulating one or more active agents for delivery selectively to a site of Mycobacterium tuberculosis bacteria in vivo. The compositions are typically formulated for delivery in vivo.

The term “Mycobacterium tuberculosis binding peptide” as used herein means a polypeptide that is capable of binding to a Mycobacterium tuberculosis cell in the absence of another molecule that targets, chaperones or otherwise mediates the binding of the peptide to a Mycobacterium tuberculosis cell. In other forms, the Mycobacterium tuberculosis binding peptides specifically bind to one or more substances that are extruded from Mycobacterium tuberculosis cells in the region associated with the Mycobacterium tuberculosis cells, such as infected tissues or organs.

B. Infected tissue-targeting Molecules

Molecules that selectively target and bind to the tissues infected with microbial pathogens in vivo, as well as to microbial pathogens themselves have been developed, and compositions thereof are provided. In some forms, the molecule is a peptide that selectively targets and binds to the extracellular matrix of infected tissues, in vivo.

The compositions are typically formulated for delivery in vivo.

In some forms the infected tissue binding molecules specifically bind to or near to the surface of pathogenic microbial cells, such as Mycobacterium tuberculosis cells. The infected tissue binding molecules include at least one infected tissue binding region.

16

45652286 Preferably, the infected tissue binding molecules are non-toxic in vivo and do not cause damage or otherwise disrupt the integrity of tissue/cells they contact, or any other structure in the body.

1. Peptides and Variants

Polypeptides that selectively target and bind to tissues infected with bacteria have been discovered. As described herein, these polypeptides and variants of these polypeptides can be combined with other useful materials and compositions and can be used in various methods.

Therefore, in some forms, peptides that bind to p32, NRP-1, Fn-EDB, CD206, Retinoid X receptor beta (RXR-beta), alpha- V-integrin, hyaluronan, heparin sulphate and/or TNC-C are described as infected tissue-binding peptides. Peptides that selectively target and bind to infected tissue and/or microbial pathogens, such as Mycobacterium tuberculosis bacteria include polypeptides, such as polypeptides having the amino sequence of any one or more of PPRRGLIKLKTS (“PL1”; SEQ ID NOT); AGRGRLVR (“PL3”; SEQ ID NO:2); GRPARPAR (“RPARPAR”; SEQ ID NO:3); CKRDLSRRC (“IP3”; SEQ ID NO:4); and AKRGARSTA (“LinTTl”; SEQ ID NO:5); CSPGAKVRC (SEQ ID NO:6); CSPGAK (SEQ ID NO:7); CRVLRSGSC (SEQ ID NO:8); YEQDPWGVKWWY (SEQ ID NO:9); KFRKAFKRFF (SEQ ID NOTO); CGNKRTR (SEQ ID NO: 11); CRGDKGPDC (SEQ ID NO: 12); CSGRRSSKC (SEQ ID NO: 13); CAQK (SEQ ID NO: 14); TSKQNSR (SEQ ID NO: 15); AGRGRLVRSAGGSVA (SEQ ID NO:16); CARSKNKDC (SEQ ID NO:17); CREKA (SEQ ID NO:18); CGLSGLGVA (SEQ ID NO: 19); CVGTNCY (SEQ ID NO:20); GGSRRVISRAKLAAAL (SEQ ID NO:21); TFFYGGSRGKRNNFKTEEY (SEQ ID NO:22); CAGALCY (SEQ ID NO:23); CGGGGGGGC (SEQ ID NO:24); SSVDKLAAALE (SEQ ID NO:25); NAVPNLRGDLQVLAQKVART (SEQ ID NO:26); TFATHGKHWAAP (SEQ ID NO:27); CNWMINKEC (SEQ ID NO:28); DRWVARDPASI (SEQ ID NO:29); GDGNSVLKPGNW (SEQ ID NO:30); TFPDGFLWAVGSAAYQTEGGWRQHGKG (SEQ ID NOG I); CGLIIQKNEC (SEQ ID NO:32); CRNGRGPDC (SEQ ID NO:33); CGNSNPKSC (SEQ ID NO:34); CSSTRESAC (SEQ ID NO:35); CLHQSPHIC (SEQ ID NO:36); and CKRGARSTC (SEQ ID NO:37). The infected tissue binding polypeptides typically include from about eight to about twelve amino acid residues, inclusive. Therefore, each amino acid residue within the polypeptide is located at a position from 1 to 12, typically annotated from the amino (NH2) to the carboxyl (COOH) terminus. The disclosed polypeptides can also mediate targeting and delivery of compounds and

17

45652286 compositions coupled to, associated with, conjugated to, or even co-administered with the polypeptide.

The PL1 polypeptide was derived from a 26 amino acid polypeptide discovered using peptide phage biopanning. In particular, disclosed are polypeptides including an amino acid sequence including (a) the sequence PPRRGLIKLKTS (SEQ ID NO: 1) or a variant of SEQ ID NO:1. Peptides related to PPRRGLIKLKTS (SEQ ID NO:1) can be referred to as PL1 peptides. Analysis revealed that many of the amino acids can be substituted with the peptide retaining useful binding ability. Amino acid positions 6 and 9 appear to be more important for binding. Thus, some forms of PL1 peptides can include an amino acid sequence including the sequence PPRRGLIKLKTS (SEQ ID NO: 1) or a variant of the sequence PPRRGLIKLKTS (SEQ ID NO:1) with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions, wherein position 6 remains leucine and position 11 remains threonine. In some forms, the amino acid sequence can include the sequence PPRRGLIKLKTS (SEQ ID NO:1) or a variant of the sequence PPRRGLIKLKTS (SEQ ID NO:1) with one, two, three, four, five, six, seven, or eight amino acid substitutions. In some forms, the amino acid sequence can include the sequence PPRRGLIKLKTS (SEQ ID NO:1) or a variant of the sequence PPRRGLIKLKTS (SEQ ID NO:1) with one, two, three, four, five, or six amino acid substitutions. In some forms, the amino acid sequence can include the sequence PPRRGLIKLKTS (SEQ ID NO:1) or a variant of the sequence PPRRGLIKLKTS (SEQ ID NO:1) with one, two, three, or four amino acid substitutions. In some forms, the amino acid sequence can include the sequence PPRRGLIKLKTS (SEQ ID NO:1) or a variant of having at least 50% sequence identity with PPRRGLIKLKTS (SEQ ID NO: 1); or a variant having at least 58% sequence identity with PPRRGLIKLKTS (SEQ ID NO:1); or a variant having at least 66% sequence identity with PPRRGLIKLKTS (SEQ ID NO:1); or a variant having at least 75% sequence identity with PPRRGLIKLKTS (SEQ ID NO:1); or a variant having at least 83% sequence identity with PPRRGLIKLKTS (SEQ ID NO: 1); or a variant having at least 91% sequence identity with the sequence PPRRGLIKLKTS (SEQ ID NO:1). Peptides related to PPRRGLIKLKTS (SEQ ID NO: 1) can be referred to as PL1 peptides.

Another type of disclosed peptide is based on the amino acid sequence AGRGRLVR (SEQ ID NO:2). Peptides related to AGRGRLVR (SEQ ID NO:2) can be referred to as PL3 peptides. Analysis revealed that many of the amino acids can be substituted with the peptide retaining useful binding ability. Amino acid positions 3, 5, 6,

18

45652286 and 8 appear to be important for binding. Thus, some forms of PL3 peptides can include an amino acid sequence including the sequence AGRGRLVR (SEQ ID NO:2) or a variant of the sequence AGRGRLVR (SEQ ID NO:2) with one, two, three, four, five, six, or seven amino acid substitutions, wherein position 3 remains arginine. In some forms, the amino acid sequence can include the sequence AGRGRLVR (SEQ ID NO:2) or a variant of the sequence AGRGRLVR (SEQ ID NO:2) with one, two, three, four, five, or six amino acid substitutions, wherein position 6 remains leucine and/or position eight remains arginine. In some forms, the amino acid sequence can include the sequence AGRGRLVR (SEQ ID NO:2) or a variant of the sequence AGRGRLVR (SEQ ID NO:2) with one, two, three, four, or five amino acid substitutions, wherein position 6 remains leucine, position eight remains arginine, and position 5 remains arginine. In some forms, the amino acid sequence can include the sequence AGRGRLVR (SEQ ID NO:2) or a variant of the sequence AGRGRLVR (SEQ ID NO:2) with one, two, three, or four amino acid substitutions. In some forms, the amino acid sequence can include the sequence AGRGRLVR (SEQ ID NO:2) or a variant of the sequence AGRGRLVR (SEQ ID NO:2) with one, two, or three amino acid substitutions. In some forms, the amino acid sequence can include the sequence AGRGRLVR (SEQ ID NO:2) or a variant of the sequence AGRGRLVR (SEQ ID NO:2) with one or two amino acid substitutions. In some forms, the amino acid sequence can include the sequence AGRGRLVR (SEQ ID NO:2) or a variant of the sequence AGRGRLVR (SEQ ID NO:2) having at least 25% sequence identity with AGRGRLVR (SEQ ID NO:2); or a variant having at least 37% sequence identity with AGRGRLVR (SEQ ID NO:2); or a variant having at least 50% sequence identity with AGRGRLVR (SEQ ID NO:2); or a variant having at least 62% sequence identity with the sequence AGRGRLVR (SEQ ID NO:2); or a variant of having at least 75% sequence identity with the sequence AGRGRLVR (SEQ ID NO:2); or a variant having at least 87% sequence identity with the sequence AGRGRLVR (SEQ ID NO:2). Peptides related to AGRGRLVR (SEQ ID NO:2) can be referred to as PL3 peptides.

In some forms, the (a) the amino acid sequence can include the formula X1-X2-X3- X4-X5-X6-X7-X8-X9-X10-X11-X12, (b) the amino acid sequence can include the formula X13-X14-X15-X16-X17-X18-X19, (c) the amino acid sequence includes the formula X20-X21- X22-X23-X24-X25-X26-X27, or (d) combinations thereof, wherein X₍₁ is leucine, wherein X7 is isoleucine, leucine, or valine, wherein X9 is leucine, isoleucine, or valine, wherein Xu is threonine, wherein X19 is arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine, wherein Xis is serine, alanine, glycine, asparagine, or threonine,

19

45652286 wherein X22 is arginine, lysine, or histidine, wherein X25 is leucine, isoleucine, valine, or alanine, wherein X27 is arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine, and wherein Xi, X2, X3, X4, X5, Xs, X10, X12, X13, X14, X15, Xi6, X17, X20, X21, X23, X24, and X26 are each, independently, any amino acid. Peptides of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12 are PL1 peptides. Peptides of the formula X20-X21-X22-X23-X24-X25-X26-X27 are PL3 peptides.

Therefore, in some forms, the PL1 peptide has an amino acid sequence including the formula XXXXXL(X₇)X(X₉)XTX (SEQ ID NO:40), whereby each X is, independently, any amino acid; X7 is any one of isoleucine, leucine, or valine; and X9 is any one of leucine, isoleucine, or valine. In some forms, the peptide has an amino acid sequence including the formula XXXXX(Xis)(Xi9) (SEQ ID NO:41), whereby each X is, independently, any amino acid; Xis is any one of serine, alanine, glycine, asparagine, or threonine; and X19 is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine. In some forms, the PL3 peptide has an amino acid sequence including the formula XX(X22)XX(X2s)X(X27) (SEQ ID NO:42), whereby each X is, independently, any amino acid; X22 is any one of arginine, lysine, or histidine, wherein X25 is any one of leucine, isoleucine, valine, or alanine; and X27 is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine.

PL1 peptides can also be described in terms of an amino acid sequence including the formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12, wherein X& is leucine, wherein X7 is isoleucine, leucine, or valine, wherein X₉ is leucine, isoleucine, or valine, wherein Xu is threonine, and wherein Xi, X2, X3, X4, X5, Xs, X10, and X12, are each, independently, any amino acid, i.e., as set forth in (SEQ ID NO:40). For example, in some forms, the PL1 peptide has an amino acid sequence including the formula (XI)(X2)XX(X₅)L(X₇)X(X9)XTX (SEQ ID NO:43), whereby each X is, independently, any amino acid; Xi is any one of proline, glycine, alanine, serine, or asparagine; X2 is any one of proline, glycine, alanine, serine, or asparagine; X5 is any one of glycine, alanine, valine, leucine, or isoleucine; X7 is any one of isoleucine, leucine, or valine; and X9 is any one of leucine, isoleucine, or valine. In some forms, the PL1 peptide has an amino acid sequence including the formula (Xi)(X2)XX(X5)LLXLXTX (SEQ ID NO:40), whereby each X is, independently, any amino acid; Xi is any one of proline, glycine, alanine, serine, or asparagine; X2 is any one of proline, glycine, alanine, serine, or asparagine; and X5 is any one of glycine, alanine, valine, leucine, or isoleucine. In some forms, X2 can be proline. For example, in some forms, the PL1 peptide has an amino acid sequence

20

45652286 including the formula XPXXXL(X₇)X(X₉)XTX (SEQ ID NO:45), whereby each X is, independently, any amino acid; X7 is any one of isoleucine, leucine, or valine; and X9 is any one of leucine, isoleucine, or valine. In some forms, X3 can be arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine, wherein X4 can be arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine, wherein Xs can be alanine, lysine, histidine, arginine, glutamate, glutamine, tyrosine, or tryptophan, wherein X10 can be alanine, lysine, histidine, arginine, glutamate, glutamine, tyrosine, or tryptophan, and wherein X12 can be serine, alanine, glycine, asparagine, threonine, glutamine, aspartate, or proline. For example, in some forms, the PL1 peptide has an amino acid sequence including the formula XP(X3)(X4)XLX(Xs)X(Xio)T(Xi2) (SEQ ID NO:46), whereby each X is, independently, any amino acid; X is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine; X4 is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine; Xs is any one of alanine, lysine, histidine, arginine, glutamate, glutamine, tyrosine, or tryptophan; X10 is any one of alanine, lysine, histidine, arginine, glutamate, glutamine, tyrosine, or tryptophan; and X12 is any one of serine, alanine, glycine, asparagine, threonine, glutamine, aspartate, or proline. In some forms, Xi can be proline, glycine, or alanine, wherein X3 can be arginine, lysine, or histidine, wherein X4 can be arginine, lysine, or histidine, wherein X5 can be glycine, alanine, or valine, wherein Xs can be alanine, lysine, histidine, or arginine, wherein X10 can be alanine, lysine, histidine, or arginine, and wherein X12 can be serine, alanine, glycine, asparagine, or threonine. For example, in some forms, the PL1 peptide has an amino acid sequence including the formula (Xi)P(X3)(X₄)(X₅)LXXX(Xio)T(Xi2) (SEQ ID NO:47), whereby each X is, independently, any amino acid; Xi is any one of proline, glycine, or alanine; X3 is any one of arginine, lysine, or histidine; X4 is any one of arginine, lysine, or histidine; X5 is any one of glycine, alanine, or valine, wherein Xs can be alanine, lysine, histidine, or arginine; X10 is any one of alanine, lysine, histidine, or arginine; and X12 is any one of serine, alanine, glycine, asparagine, or threonine. In some forms, any amino acid substitution at X7 and X9 are conservative amino acid substitutions. In some forms, any amino acid substitutions are conservative amino acid substitutions. In some forms, the amino acid sequence can include the sequence PPRRGPL1KLKTS (SEQ ID NO:1).

PL3 peptides can also be described in terms of an amino acid sequence including the formula X20-X21-X22-X23-X24-X25-X26-X27, wherein X22 is arginine, lysine, or histidine, wherein X25 is leucine, isoleucine, valine, or alanine, wherein X27 is arginine, lysine,

21

45652286 histidine, glutamate, glutamine, aspartate, asparagine, or alanine, and wherein X20, X21 , X23, X24, and X26 are each, independently, any amino acid. For example, in some forms, the PL3 peptide has an amino acid sequence including the formula XX(X22)XX(X2s)X(X2?) (SEQ ID NO:48), whereby each X is, independently, any amino acid; X22 is any one of arginine, lysine, or histidine; X25 is any one of leucine, isoleucine, valine, or alanine; and X27 is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine. In some forms, X24 can be arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine. For example, in some forms, the PL3 peptide has an amino acid sequence including the formula XX(X22)X(X24)(X2s)X(X27) (SEQ ID NO:49), whereby each X is, independently, any amino acid; X22 is any one of arginine, lysine, or histidine; X24 is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine; X25 is any one of leucine, isoleucine, valine, or alanine; and X27 is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine. In some forms, X21 can be glycine, alanine, valine, leucine, or isoleucine, X23 can be glycine, alanine, valine, leucine, or isoleucine and wherein X26 can be valine, leucine, isoleucine, glycine, or alanine. For example, in some forms, the PL3 peptide has an amino acid sequence including the formula X(X₂i)(X22)(X23)(X24)(X2₅)(X26)(X27) (SEQ ID NO:50), whereby each X is, independently, any amino acid; X21 is any one of glycine, alanine, valine, leucine, or isoleucine; X22 is any one of arginine, lysine, or histidine; X23 is any one of glycine, alanine, valine, leucine, or isoleucine X24 is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine; X25 is any one of leucine, isoleucine, valine, or alanine; X26 is any one of valine, leucine, isoleucine, glycine, or alanine; and X27 is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine. In some forms, X20 can be alanine, glycine, valine, leucine, or isoleucine. (X2o)(X2i)(X22)(X₂3)(X24)(X25 (X₂6)(X27) (SEQ ID NO:51), whereby X20 is any one of alanine, glycine, valine, leucine, or isoleucine X21 is any one of glycine, alanine, valine, leucine, or isoleucine; X22 is any one of arginine, lysine, or histidine; X23 is any one of glycine, alanine, valine, leucine, or isoleucine X24 is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine; X25 is any one of leucine, isoleucine, valine, or alanine; X26 is any one of valine, leucine, isoleucine, glycine, or alanine; and X27 is any one of arginine, lysine, histidine, glutamate, glutamine, aspartate, asparagine, or alanine. In some forms, X22 can be arginine or lysine, wherein X25 can be leucine, isoleucine, or valine, wherein X27 can be arginine, lysine, or histidine. In some

22

45652286 forms, X24 can be arginine, lysine, or histidine. In some forms, X24 can be arginine or lysine. In some forms, X22 can be arginine, wherein X25 can be leucine, wherein X27 can be arginine. In some forms, X24 can be arginine. For example, in some forms, the PL3 peptide has an amino acid sequence including the formula (X2o)(X2i)R(X23)(X24)L(X26)R (SEQ ID NO:52), whereby X20 is any one of alanine, glycine, valine, leucine, or isoleucine X21 is any one of glycine, alanine, valine, leucine, or isoleucine; X23 is any one of glycine, alanine, valine, leucine, or isoleucine; and X26 is any one of valine, leucine, isoleucine, glycine, or alanine. In some forms, any amino acid substitution at X22, X25, and X27 are conservative amino acid substitutions. In some forms, any amino acid substitutions are conservative amino acid substitutions. In some forms, the amino acid sequence can include the sequence AGRGRLVR (SEQ ID NO:2). In some forms, the amino acid sequence can include the sequence AGRGRLVRAKLAAALE (SEQ ID NO:38).

In some forms, the peptide can be less than 20 amino acids in length. In some forms, the peptide can be less than 15 amino acids in length. In some forms, the peptide can be 12 amino acids in length. In some forms, the peptide can include the sequence PPRRGLIKLKTSSNTKENSVVASLRP (SEQ ID NO:39). In some forms, the peptide is linear. In some forms, the peptide is cyclic. In some forms, the peptide is a modified peptide. In some forms, the peptide is a methylated peptide. In some forms, the methylated peptide can include a methylated amino acid segment. In some forms, the peptide is N- or C-methylated in at least one position.

The disclosed peptides preferably include the sequences of (1) one or more PL1 peptides, (2) one or more RPARPAR peptides, (3) one or more PL3 peptides, (4) one or more PL1 peptides and one or more RPARPAR peptides, (5) one or more PL1 peptides and one or more PL3 peptides, (6) one or more RPARPAR peptides and one or more PL3 peptides, or (7) one or more PL1 peptides, one or more RPARPAR peptides, and one or more PL3 peptides. Such peptides (i.e., peptides having any of these combinations) can be referred to as “LRPARPAR” peptides. The disclosed compositions preferably include (1) one or more PL1 peptides, (2) one or more RPARPAR peptides, (3) one or more PL3 peptides, (4) one or more PL1 peptides and one or more RPARPAR peptides, (5) one or more PL1 peptides and one or more PL3 peptides, (6) one or more RPARPAR peptides and one or more PL3 peptides, or (7) one or more PL1 peptides, one or more RPARPAR peptides, and one or more PL3 peptides.

Any of the disclosed peptides (such as PL1, and/or PL3, and/or RPARPAR peptides) can be composed of, for example, amino acids, amino acid analogs, peptide

23

45652286 analogs, amino acid mimetics, peptide mimetics, etc. Although structures, design, etc. of the disclosed peptides is described herein in terms of amino acids and peptides composed of amino acids for convenience, it is understood that analogous analogs, mimetics, modified forms, etc. of amino acids and peptides can also be used as the disclosed peptides and designed using similar principles.

Any component, such as the components disclosed herein, can overlap, be adjacent to, and/or be upstream, downstream, or both of a peptide, such as an PL1 and/or PL3 and/or RPARPAR peptide. Examples of such components include accessory molecules, homing molecules, protease cleavage sites, etc. It is useful to have some components coupled to or associated with a peptide, such as an PL1 and/or PL3 and/or RPARPAR peptide to be downstream (C-terminal) of the peptide. For example, activatable peptide having an accessory protein or a homing peptide downstream of the peptide (and thus downstream from the cleavage site for activation) will be separated from the peptide when it is activated. As another example, activatable peptides having an accessory molecule or a homing molecule downstream of the peptide (and thus downstream from the cleavage site for activation) will be separated from the peptide when it is activated. This can have some advantages such as making the peptide function more efficient or reducing the chance for extraneous effects of the eliminated component.

In some forms the peptides include changes, such as addition, deletion or substitution of one or more amino acids. Therefore, in some forms, the infected tissue binding molecules are variants of any one of SEQ ID NOs:l-37 that include or consist of 7, 8, 9, 10, 11 of more than 11 amino acids.

Generally, a variant of any one of SEQ ID NOs:l-37 is considered to have the function of selectively target and bind to infected tissue and/or microbial pathogens, such as Mycobacterium tuberculosis bacteria if the variant has at least 70%, 80%, 90%, 95%, 100%, or more than 100%, such as 105%, 110% or more of the binding affinity for infected tissue and/or microbial pathogens, such as Mycobacterium tuberculosis bacteria of the non-variant sequence from which it is derived.

Exemplary variants include one or more peptides having an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, or 99% identical to one or more of the respective amino acid sequences of SEQ ID NOs:l-37. For example, variants can include one or more peptides having an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, or 99% identical to one or more of the respective amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some forms, the variants include one

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45652286 or more peptides having an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, or 99% identical to the amino acid sequence of SEQ ID NO:1. In other forms, the variants include one or more peptides having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1. In other forms, the variants include one or more peptides having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2. In other forms, the variants include one or more peptides having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:3.

Therefore, in some forms, the infected tissue and/or microbial pathogen-targeting peptide includes the amino acid sequence of any one of PPRRGLIKLKTS (SEQ ID NO:1); AGRGRLVR (SEQ ID NO:2) and GRPARPAR (SEQ ID NO:3); CKRDLSRRC (“IP3”; SEQ ID NO:4); or AKRGARSTA (“LinTTl”; SEQ ID NO:5), or a functional variant thereof having at least 80% sequence identity to any one of SEQ ID NOs:l-5, whereby the peptide selectively binds to Mycobacterium bacteria.

In some forms, the infected tissue and/or microbial pathogen- targeting peptides include one or more additional polypeptides contiguous with the peptide of any one of SEQ ID NOs:l-37, for example, having the schematic structure:

(SEQ ID NO:l-37)-(additional polypeptide 1); or (additional polypeptide 1)-(SEQ ID NO: 1-37); or (additional polypeptide 1)-(SEQ ID NO:l-37)-(additional polypeptide 2). Typically, when the infected tissue and/or microbial pathogen binding peptides include one or more additional polypeptides, the additional peptide(s) form a peptide bond with the residue at position 1, or position 2, or both position 1 and position 2. Typically, the presence of the additional covalently bound polypeptide does not substantially reduce or otherwise diminish the function of selectively targeting and binding to infected tissue and/or microbial pathogens, such as Mycobacterium tuberculosis

In some forms, a multiplicity of molecules of the infected tissue and/or microbial pathogen binding peptides are combined to form peptide multimers, such as repeating units of one or more of SEQ ID NOs: 1-37 or functional variants thereof. For example, in some forms, multimers include a multiplicity of infected tissue and/or microbial pathogen binding peptides that independently have an amino acid sequence of any one of SEQ ID NOs: 1-37 or functional variants thereof. In some forms, the multimers include between two and one hundred copies of the infected tissue and/or microbial pathogen binding peptides.

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45652286 In some forms, the infected tissue and/or microbial pathogen-targeting peptide variants, or fusions, or multimers have a length of up to 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000 amino acid residues. In particular forms, an infected tissue and/or microbial pathogen-targeting peptide fusion or variant or multimer can have a length of at least 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further embodiments, an infected tissue and/or microbial pathogen-targeting peptide fusion or variant or multimer can have a length of 8 to 200 residues, 8 to 100 residues, 8 to 90 residues, 8 to 80 residues, 8 to 70 residues, 8 to 60 residues, 8 to 50 residues, 8 to 40 residues, 8 to 30 residues, 8 to 20 residues, 8 to 15 residues, 8 to 10 residues, 9 to 200 residues, 9 to 100 residues, 9 to 90 residues, 9 to 80 residues, 9 to 70 residues, 9 to 60 residues, 9 to 50 residues, 9 to 40 residues, 9 to 30 residues, 9 to 20 residues, 9 to 15 residues, 9 to 10 residues, 10 to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues, 10 to 20 residues, or 10 to 15 residues. As used herein, the term "residue" refers to an amino acid or amino acid analog.

The disclosed peptides can have a length of up to 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In particular embodiments, the disclosed peptides can have a length of at least 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further embodiments, the disclosed peptides can have a length of 7 to 200 residues, 7 to 100 residues, 7 to 90 residues, 7 to 80 residues, 7 to 70 residues, 7 to 60 residues, 7 to 50 residues, 7 to 40 residues, 7 to 30 residues, 7 to 20 residues, 7 to 15 residues, 7 to 10 residues, 8 to 200 residues, 8 to 100 residues, 8 to 90 residues, 8 to 80 residues, 8 to 70 residues, 8 to 60 residues, 8 to 50 residues, 8 to 40 residues, 8 to 30 residues, 8 to 20 residues, 8 to 15 residues, 8 to 10 residues, 9 to 200 residues, 9 to 100 residues, 9 to 90 residues, 9 to 80 residues, 9 to 70 residues, 9 to 60 residues, 9 to 50 residues, 9 to 40 residues, 9 to 30 residues, 9 to 20 residues, 9 to 15 residues, 9 to 10 residues, 10 to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues, 10 to 20 residues, 10 to 15 residues, 15 to 200 residues, 15 to 100 residues, 15 to 90 residues, 15 to 80 residues, 15 to 70 residues, 15 to 60 residues, 15 to 50 residues, 15 to 40 residues, 15 to 30 residues, 15 to 20 residues, 20 to 200 residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30 residues. As used herein, the term "residue" refers to an amino acid or amino acid analog.

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45652286 A protein or peptide containing an PL1 and/or PL3 and/or RPARPAR peptide can have a length of up to 50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In particular embodiments, the protein or peptide portion of an PL1 and/or PL3 and/or RPARPAR composition can have a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further embodiments, the protein or peptide containing an PL1 and/or PL3 and/or RPARPAR peptide can have a length of 7 to 200 residues, 7 to 100 residues, 7 to 90 residues, 7 to 80 residues, 7 to 70 residues, 7 to 60 residues, 7 to 50 residues, 7 to 40 residues, 7 to 30 residues, 7 to 20 residues, 7 to 15 residues, 7 to 10 residues, 8 to 200 residues, 8 to 100 residues, 8 to 90 residues, 8 to 80 residues, 8 to 70 residues, 8 to 60 residues, 8 to 50 residues, 8 to 40 residues, 8 to 30 residues, 8 to 20 residues, 8 to 15 residues, 8 to 10 residues, 9 to 200 residues, 9 to 100 residues, 9 to 90 residues, 9 to 80 residues, 9 to 70 residues, 9 to 60 residues, 9 to 50 residues, 9 to 40 residues, 9 to 30 residues, 9 to 20 residues, 9 to 15 residues, 9 to 10 residues, 10 to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues, 10 to 20 residues, 10 to 15 residues, 15 to 200 residues, 15 to 100 residues, 15 to 90 residues, 15 to 80 residues, 15 to 70 residues, 15 to 60 residues, 15 to 50 residues, 15 to 40 residues, 15 to 30 residues, 15 to 20 residues, 20 to 200 residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30 residues.

The disclosed conjugates can have a length of up to 50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In particular embodiments, the disclosed conjugates can have a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further embodiments, the disclosed conjugates can have a length of 10 to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues, 10 to 20 residues, 10 to 15 residues, 15 to 200 residues, 15 to 100 residues, 15 to 90 residues, 15 to 80 residues, 15 to 70 residues, 15 to 60 residues, 15 to 50 residues, 15 to 40 residues, 15 to 30 residues, 15 to 20 residues, 20 to 200 residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30 residues.

The protein or peptide portion of an PL1 and/or PL3 and/or RPARPAR composition can have a length of up to 50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In particular embodiments, the protein or peptide portion of an PL1 and/or PL3 and/or RPARPAR composition can have a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further embodiments, the protein or peptide portion of an

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45652286 PL1 and/or PL3 and/or RPARPAR composition can have a length of 7 to 200 residues, 7 to 100 residues, 7 to 90 residues, 7 to 80 residues, 7 to 70 residues, 7 to 60 residues, 7 to 50 residues, 7 to 40 residues, 7 to 30 residues, 7 to 20 residues, 7 to 15 residues, 7 to 10 residues, 8 to 200 residues, 8 to 100 residues, 8 to 90 residues, 8 to 80 residues, 8 to 70 residues, 8 to 60 residues, 8 to 50 residues, 8 to 40 residues, 8 to 30 residues, 8 to 20 residues, 8 to 15 residues, 8 to 10 residues, 9 to 200 residues, 9 to 100 residues, 9 to 90 residues, 9 to 80 residues, 9 to 70 residues, 9 to 60 residues, 9 to 50 residues, 9 to 40 residues, 9 to 30 residues, 9 to 20 residues, 9 to 15 residues, 9 to 10 residues, 10 to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues, 10 to 20 residues, 10 to 15 residues, 15 to 200 residues, 15 to 100 residues, 15 to 90 residues, 15 to 80 residues, 15 to 70 residues, 15 to 60 residues, 15 to 50 residues, 15 to 40 residues, 15 to 30 residues, 15 to 20 residues, 20 to 200 residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30 residues.

The disclosed compositions can have a length of up to 50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In particular embodiments, the disclosed compositions can have a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further embodiments, the disclosed compositions can have a length of 10 to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues, 10 to 20 residues, 10 to 15 residues, 15 to 200 residues, 15 to 100 residues, 15 to 90 residues, 15 to 80 residues, 15 to 70 residues, 15 to 60 residues, 15 to 50 residues, 15 to 40 residues, 15 to 30 residues, 15 to 20 residues, 20 to 200 residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30 residues.

F/T/F&T and other disclosed peptides can be stabilized against proteolysis. For example, the stability and activity of peptides can be increased by protecting some of the peptide bonds with N-methylation or C-methylation. Accessory peptides and homing peptides can also or similarly be stabilized against proteolysis.

The disclosed peptides can be made in the form of stabilized peptides and/or formulated as long-circulating forms. For example, a polyethylene glycol conjugate can be used. The disclosed peptides and/or cargos can also be administered over a period of time. For example, disclosed peptides and/or cargos can be delivered with an osmotic pump. This can extend the permeability of the target cells and tissues. Modified forms of the

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45652286 disclosed peptides can be used. For example, disclosed peptides can be methylated (which can stabilize the peptides against proteolysis). Stability against cleavage is desirable, except for bonds to be cleaved to activate a peptide. Modifications to the disclosed peptides generally should leave them functional. A peptide with a structural difference from naturally occurring forms of peptides can be considered a modified peptide.

It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed infected tissue and/or microbial pathogen-targeting peptide variants, or fusions, or multimers. For example, there are numerous D amino acids or amino acids which can be used. The opposite stereoisomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way (Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in Biotechnology, 3:348-354 (1992); Ibba, Biotechnology & Genetic Engineering Reviews 13: 197-216 (1995), Cahill et al., TIBS, 14(10): 400-403 (1989); Benner, TIB Tech, 12: 158-163 (1994); Ibba and Hennecke, Bio/technology, 12:678-682 (1994) all of which are herein incorporated by reference at least for material related to amino acid analogs).

In some forms, infected tissue and/or microbial pathogen- targeting peptide fusion, or variant, or multimer molecules are produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH2NH-, — CH2S-, — CH2-CH2 -, -CH=CH— (cis and trans), — COCH2 -, -CH(OH)CH2-, and -CHH2SO — (These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int I Pept Prot Res 14: 177-185 (1979) (-CH2NH-, CH2CH2-); Spatola et al. Life Sci 38: 1243-1249 (1986) (-CH H₂-S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (-CH-CH-, cis and trans); Almquist et al. J. Med. Chem. 23: 1392-1398 (1980) (— COCH2-); lennings-White et al. Tetrahedron Lett 23:2533 (1982) (-COCH2-); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (-CH(OH)CH₂-); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (-C(OH)CH₂-); and Hruby Life Sci 31 :189-199 (1982) (-CH2-S-); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is

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45652286 -CEpNH-. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g- aminobutyric acid, and the like. Amino acid analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others. D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D- amino acid of the same type e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference).

2. Target Receptors

Receptors that are selectively expressed on tissues infected with microbial pathogens in vivo, as well as at the surface of microbial pathogens themselves have been identified as potential targets for the described molecules that target infected tissue and/or microbial pathogens. In some forms, the target receptor is selectively expressed at the extracellular matrix of infected tissues, in vivo. In some forms, the target receptor is specifically bound by one or more peptide having an amino acid sequence of any one or more of SEQ ID NOs:l-37.

As set forth in the Examples, it has been established that peptide receptors in TB- infected pulmonary lesions include p32, NRP-1, Fn-EDB, CD206, Retinoid X receptor beta (RXR-beta), alpha- V-integrin, hyaluronan, heparin sulphate and TNC-C. In some forms, target receptors expressed in Mtb-infected tissues. In some forms, target receptors are upregulated in cellular and necrotic lesions in response to an increase in the number of Mtb bacilli. Therefore, in some forms the amount or number of target receptors in a tissue or organ is directly proportional to the amount or number of Mtb bacilli within or around an infected tissue in vivo.

In some forms, the target receptor is the protein known as p32, also known as HABP1 (hyaluronan-binding protein 1), gClqR (receptor for globular head domains complement Iq) or Clqbp (complement Iq-binding protein). The p32 protein is a doughnut-shaped trimer that has both mitochondrial and non-mitochondrial localization and functions, and has also been reported to be present at the cell surface and in the cell

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45652286 nucleus. Endogenous p32 protein is a mitochondrial protein in HeLa cells under control and stress conditions, and it has been shown that the overexpression of p32 increased mitochondrial fibrils. Therefore, in some forms, the target receptor is p32, and the described infected tissue binding molecules selectively bind p32 in vivo.

In some forms, the target receptor is the immunoregulatory receptor Neuropilin- 1 protein known as NRP- 1. Neuropilins (NRPs) are single-pass transmembrane, nontyrosine kinase surface glycoproteins found in all vertebrates and are highly conserved across species. Two homologous NRP isoforms are known to exist, namely NRP1 and NRP2, encoded by distinct neuropilin genes (Nrpl and Nrp2) which arose due to a gene duplication event. Both NRPs were originally discovered as neuronal adhesion molecules participating in semaphorin-mediated axonal guidance. They were later found to be fundamentally involved in vascular biology, with NRP1 required for normal embryonic vascular development and NRP2 involved in the formation of small lymphatic vessels and capillaries. Studies have revealed NRPs are multifunctional proteins participating in a variety of biological processes beyond nervous and vascular development, with NRP1 having a major role in immunity and tumorigenesis. NRP1 is also expressed by conventional DCs isolated from human peripheral blood, where it may promote early T cell priming by mediating the formation of immunological synapse between DCs and T cells via homotypic interactions. The NRP1 protein includes a long N-terminal extracellular domain, followed by a transmembrane region and a very short cytosolic tail of 43-44 amino acids. NRP-1 binds to the semaphorin group of ligands, as well as vascular endothelial growth factor (VEGF). Therefore, in some forms, the target receptor is NRP-1, and the described infected tissue binding molecules selectively bind NRP-1 in vivo.

In some forms, the target receptor is Extra domain B splice variant of fibronectin (Fn-EDB). Fn-EDB is an extracellular matrix protein (ECM) deposited by tumor- associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion Fn-EDB includes a 91 amino acid domain inserted into fibronectin 1 at the primary transcript level, is a non-internalizing, insoluble, ECM-associated protein, and is a marker of tissue remodeling and angiogenesis. Therefore, in some forms, the target receptor is Fn- EDB, and the described infected tissue binding molecules selectively bind Fn-EDB in vivo.

In some forms, the target receptor is Tenascin C (TNC-C). TNC-C is a glycoprotein that in humans is encoded by the TNC gene. It is expressed in the

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45652286 extracellular matrix of various tissues during development, disease or injury, and in restricted neurogenic areas of the central nervous system. Tenascin-C is the founding member of the tenascin protein family. In the embryo it is made by migrating cells like the neural crest; it is also abundant in developing tendons, bone and cartilage. Therefore, in some forms, the target receptor is TNC-C, and the described infected tissue binding molecules selectively bind TNC-C.

C. Active Agents/Cargo Molecules

Compositions including infected tissue and/or microbial pathogen-targeting molecules are described.

In some forms, the infected tissue and/or microbial pathogen-targeting molecules include one or more additional active agents, for example, to be targeted specifically to infected tissue, such as tissue infected by Mycobacterium tuberculosis bacteria. Typically, the compositions include one or more active agents that is delivered specifically to infected tissue, such as tissue infected by Mycobacterium tuberculosis bacteria in vivo. In some forms, the infected tissue and/or microbial pathogen-targeting molecules that bind one or more of p32, NRP-1, Fn-EDB, CD206, Retinoid X receptor beta (RXR-beta), alpha- V-integrin, hyaluronan (hyaluronic acid), heparin sulphate or TNC-C include one or more additional active agents. In exemplary forms, the infected tissue and/or microbial pathogen-targeting molecules include peptides having an amino acid sequence of any one or more of PPRRGLIKLKTS (“PL1”; SEQ ID NO:1); AGRGRLVR (“PL3”; SEQ ID NO:2); GRP ARP AR (“RPARPAR”; SEQ ID NO:3); CKRDLSRRC (“IP3”; SEQ ID NO:4); and AKRGARSTA (“LinTTl”; SEQ ID NO:5); CSPGAKVRC (SEQ ID NO:6); CSPGAK (SEQ ID NO:7); CRVLRSGSC (SEQ ID NO:8); YEQDPWGVKWWY (SEQ ID NO:9); KFRKAFKRFF (SEQ ID NOTO); CGNKRTR (SEQ ID NO: 11); CRGDKGPDC (SEQ ID NO: 12); CSGRRSSKC (SEQ ID NO: 13); CAQK (SEQ ID NO:14); TSKQNSR (SEQ ID NO:15); AGRGRLVRSAGGSVA (SEQ ID NO:16); CARSKNKDC (SEQ ID NO: 17); CREKA (SEQ ID NO: 18); CGLSGLGVA (SEQ ID NO: 19); CVGTNCY (SEQ ID NO:20); GGSRRVISRAKLAAAL (SEQ ID NO:21); TFFYGGSRGKRNNFKTEEY (SEQ ID NO:22); CAGALCY (SEQ ID NO:23); CGGGGGGGC (SEQ ID NO:24); SSVDKLAAALE (SEQ ID NO:25); NAVPNLRGDLQVLAQKVART (SEQ ID NO:26); TFATHGKHWAAP (SEQ ID NO:27); CNWMINKEC (SEQ ID NO:28); DRWVARDPASI (SEQ ID NO:29); GDGNSVLKPGNW (SEQ ID NQ:30); TFPDGFLWAVGSAAYQTEGGWRQHGKG (SEQ ID NO:31); CGLIIQKNEC (SEQ ID NO:32); CRNGRGPDC (SEQ ID NO:33);

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45652286 CGNSNPKSC (SEQ ID NO:34); CSSTRESAC (SEQ ID NO:35); CLHQSPHIC (SEQ ID NO:36); and CKRGARSTC (SEQ ID NO:37), and one or more additional active agents, for example, to be targeted specifically to infected tissue, such as tissue infected by Mycobacterium tuberculosis bacteria. Typically, the compositions include one or more active agents that is delivered specifically to infected tissue, such as tissue infected by Mycobacterium tuberculosis bacteria in vivo.

Compositions including one or more PL1 peptides and one or more PL3 peptides can be referred to as PL1-3 compositions. Compositions including one or more PL1 peptides and one or more RPARPAR peptides can be referred to as PL1R compositions. Compositions including one or more PL3 peptides and one or more RPARPAR peptides can be referred to as PL3R compositions. Compositions including one or more PL1 peptides, one or more PL3 peptides, and one or more RPARPAR peptides can be referred to as PL13R compositions.

Compositions including one or more PL1 peptides, one or more PL3 peptides, or both, can be referred to as PL1/3 compositions. Compositions including one or more PL1 peptides, one or more RPARPAR peptides, or both, can be referred to as PL1/R compositions. Compositions including one or more PL3 peptides, one or more RPARPAR peptides, or both can be referred to as PL3/R compositions. Compositions including one or more PL1 peptides, one or more PL3 peptides, one or more RPARPAR peptides, or combinations thereof, can be referred to as PL1/3/R compositions.

Disclosed are PL1 compositions, PL1 conjugates, PL1 molecules, PL1 proteins, and PL1 peptides. PL1 peptides are the basic feature of PL1 compositions, PL1 conjugates, PL1 molecules, PL1 proteins, and the PLlke. PL1 compositions are any composition, conglomeration, conjugate, molecule, protein, peptide, etc. that includes an PL1 peptide. PL1 conjugates are associations, whether covalent or non-covalent, of an PL1 peptide and one or more other elements, peptides, proteins, compounds, molecules, agents, compounds, etc. For example, an PL1 conjugate can include an PL1 peptide, PL1 protein, PL1 compound, PL1 molecule, etc. PL1 molecules are molecules that include an PL1 peptide. For example, an PL1 molecule can include an PL1 protein, PL1 peptide, etc. In general, PL1 peptides, PL1 proteins, PL1 molecules, and PL1 conjugates are all forms of PL1 compositions. PL1 compounds, PL1 peptides and PL1 proteins can be forms of PL1 molecules. Unless the context indicates otherwise, reference to an PL1 composition is intended to refer to PL1 compositions, PL1 molecules, PL1 proteins, PL1 peptides, and the like. An PL1 component is any molecule, peptide, protein, compound, conjugate,

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45652286 composition, etc. that includes an PL1 peptide. Examples of PL1 components include, for example, PL1 compositions, PL1 molecules, PL1 proteins, and PL1 peptides. PL1 components can include one or more PL1 peptides.

Disclosed are PL3 compositions, PL3 conjugates, PL3 molecules, PL3 proteins, and PL3 peptides. PL3 peptides are the basic feature of PL3 compositions, PL3 conjugates, PL3 molecules, PL3 proteins, and the like. PL3 compositions are any composition, conglomeration, conjugate, molecule, protein, peptide, etc. that includes an PL3 peptide. PL3 conjugates are associations, whether covalent or non-covalent, of an PL3 peptide and one or more other elements, peptides, proteins, compounds, molecules, agents, compounds, etc. For example, an PL3 conjugate can include an PL3 peptide, PL3 protein, PL3 compound, PL3 molecule, etc. PL3 molecules are molecules that include an PL3 peptide. For example, an PL3 molecule can include an PL3 protein, PL3 peptide, etc. In general, PL3 peptides, PL3 proteins, PL3 molecules, and PL3 conjugates are all forms of PL3 compositions. PL3 compounds, PL3 peptides and PL3 proteins can be forms of PL3 molecules. Unless the context indicates otherwise, reference to an PL3 composition is intended to refer to PL3 compositions, PL3 molecules, PL3 proteins, PL3 peptides, and the like. An PL3 component is any molecule, peptide, protein, compound, conjugate, composition, etc. that includes an PL3 peptide. Examples of PL3 components include, for example, PL3 compositions, PL3 molecules, PL3 proteins, and PL3 peptides. PL3 components can include one or more PL3 peptides.

Disclosed are RPARPAR compositions, RPARPAR conjugates, RPARPAR molecules, RPARPAR proteins, and RPARPAR peptides. RPARPAR peptides are the basic feature of RPARPAR compositions, RPARPAR conjugates, RPARPAR molecules, RPARPAR proteins, and the PL Ike. RPARPAR compositions are any composition, conglomeration, conjugate, molecule, protein, peptide, etc. that includes an RPARPAR peptide. RPARPAR conjugates are associations, whether covalent or non-covalent, of an RPARPAR peptide and one or more other elements, peptides, proteins, compounds, molecules, agents, compounds, etc. For example, an RPARPAR conjugate can include an RPARPAR peptide, RPARPAR protein, RPARPAR compound, RPARPAR molecule, etc. RPARPAR molecules are molecules that include an RPARPAR peptide. For example, an RPARPAR molecule can include an RPARPAR protein, RPARPAR peptide, etc. In general, RPARPAR peptides, RPARPAR proteins, RPARPAR molecules, and RPARPAR conjugates are all forms of RPARPAR compositions. RPARPAR compounds, RPARPAR peptides and RPARPAR proteins can be forms of RPARPAR molecules. Unless the

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45652286 context indicates otherwise, reference to an RPARPAR composition is intended to refer to RPARPAR compositions, RPARPAR molecules, RPARPAR proteins, RPARPAR peptides, and the PLlke. An RPARPAR component is any molecule, peptide, protein, compound, conjugate, composition, etc. that includes an RPARPAR peptide. Examples of RPARPAR components include, for example, RPARPAR compositions, RPARPAR molecules, RPARPAR proteins, and RPARPAR peptides. RPARPAR components can include one or more RPARPAR peptides.

The PL1, and/or PL3, and/or RPARPAR peptide can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of an amino acid sequence, a protein, or a peptide that includes the PL1, and/or PL3, and/or RPARPAR peptide. As another example, the accessory molecule can be covalently coupled or non- covalently associated with the PL1, and/or PL3, and/or RPARPAR peptide or an amino acid sequence, a protein, or a peptide that includes the PL1, and/or PL3, and/or RPARPAR peptide. The accessory molecule can be separate from or overlapping with the PL1, and/or PL3, and/or RPARPAR peptide. For example, some accessory molecules are amino acid sequences. This can allow the amino acid sequence including the PL1, and/or PL3, and/or RPARPAR peptide to overlap the amino acid sequence that includes the accessory amino acid sequence. Alternatively, the accessory peptide can be a separate entity that does not overlap with the PL1, and/or PL3, and/or RPARPAR peptide. In some forms, the accessory molecule can include a sequence in, for example, an PL1, and/or PL3, and/or RPARPAR peptide that binds to a specific receptor distinct from the receptor for the PL1 , and/or PL3, and/or RPARPAR peptide.

The amino acid sequence can include one or more accessory peptides. The protein or peptide can include one or more accessory peptides. In some forms, the cargo does not include an accessory molecule. The cargo can include one or more accessory molecules. In some forms, the cargo does not include an accessory peptide. The cargo can include one or more accessory peptides. The cargo can selectively home to cells and tissues having a microbial pathogen infection. In some forms, the cargo does not selectively home to cells and tissues having a microbial pathogen infection. In some forms, the cargo composition does not include an accessory molecule. The cargo composition can include one or more accessory molecules. In some forms, the cargo composition does not include an accessory peptide. The cargo composition can include one or more accessory peptides. The cargo composition can selectively home to cells and tissues having a microbial pathogen infection, such as Mtb infection. In some forms, the cargo composition does not

35

45652286 selectively home to cells and tissues having a microbial pathogen infection. The cargo composition can selectively home to cells and tissues having Mtb infection.

The peptide can be associated with one or more therapeutic agents. For example, a therapeutic agent can be a part of an amino acid sequence, a protein, or a peptide that includes the peptide. As another example, the therapeutic agent can be covalently coupled or non-covalently associated with the peptide or an amino acid sequence, a protein, or a peptide that includes the peptide. The therapeutic agent can be separate from or overlapping with the peptide. For example, some therapeutic agents are amino acid sequences. This can allow the amino acid sequence including the peptide to overlap the amino acid sequence that includes the therapeutic amino acid sequence. Alternatively, the therapeutic agent can be a separate entity that does not overlap with the peptide. In some forms, the therapeutic agent can include a sequence in, for example, a peptide that binds to a specific receptor distinct from the target for the peptide.

The disclosed peptides home to specific cells (cells and tissues having a microbial pathogen infection) and many homing molecules home to the vasculature of the target tissue. However, for the sake of convenience homing is referred to in some places herein as homing to the tissue associated with infection, or with the vasculature, to which the peptide or homing peptide may actually home. By including or associating a peptide or homing peptide with, for example, a protein, peptide, amino acid sequence, cargo, or cargo composition, the protein, peptide, amino acid sequence, cargo, or cargo composition can be targeted or can home to the target of the peptide or homing peptide. In this way, the protein, peptide, amino acid sequence, cargo, or cargo composition, or can be said to home to the target of the peptide or homing peptide. For convenience and unless otherwise indicated, reference to homing of a protein, peptide, amino acid sequence, cargo, cargo composition, etc. is intended to indicate that the protein, peptide, amino acid sequence, cargo, cargo composition, etc. includes or is associated with an appropriate peptide or homing peptide.

In some forms, the peptide and the cargo are not covalently coupled or directly non-covalently associated with each other. In some forms, the cargo does not include a peptide. The cargo can include one or more peptides. In some forms, the cargo does not include an PL1 peptide, an PL3 peptide, an RPARPAR peptide, or a homing peptide. The cargo can include one or more PL1 peptides, PL3 peptides, RPARPAR peptides, or homing peptides. The cargo can selectively home to cells and tissues having a microbial

36

45652286 pathogen infection. In some forms, the cargo does not selectively home to cells and tissues having a microbial pathogen infection.

In some forms, the peptide and the cargo composition are not covalently coupled or directly non-covalently associated with each other. In some forms, the cargo composition does not include a peptide. The cargo composition can include one or more peptides. In some forms, the cargo composition does not include an PL1 peptide, an PL3 peptide, an RPARPAR peptide, or a homing peptide. The cargo composition can include one or more PL1 peptides, PL3 peptides, RPARPAR peptides, or homing peptides. The cargo composition can selectively home to cells and tissues having a microbial pathogen infection. In some forms, the cargo composition does not selectively home to cells and tissues having microbial pathogen infection.

As used herein, reference to components (such as an PL1 , and/or PL3, and/or RPARPAR peptide and a cargo) as being “not covalently coupled” means that the components are not connected via covalent bonds (for example, that the PL1, and/or PL3, and/or RPARPAR peptide and the cargo are not connected via covalent bonds). That is, there is no continuous chain of covalent bonds between, for example, the PL1, and/or PL3, and/or RPARPAR peptide and the cargo. Conversely, reference to components (such as an PL1, and/or PL3, and/or RPARPAR peptide and a cargo composition) as being “covalently coupled” means that the components are connected via covalent bonds (for example, that the PL1 , and/or PL3, and/or RPARPAR peptide and the cargo composition are connected via covalent bonds). That is, there is a continuous chain of covalent bonds between, for example, the PL1, and/or PL3, and/or RPARPAR peptide and the cargo composition. Components can be covalently coupled either directly or indirectly. Direct covalent coupling refers to the presence of a covalent bond between atoms of each of the components. Indirect covalent coupling refers to the absence of a covalent bond between atoms of each of the components. That is, some other atom or atoms not belonging to either of the coupled components intervenes between atoms of the components. Both direct and indirect covalent coupling involve a continuous chain of covalent bonds.

Non-covalent association refers to association of components via non-covalent bonds and interactions. A non-covalent association can be either direct or indirect. A direct non-covalent association refers to a non-covalent bond involving atoms that are each respectively connected via a chain of covalent bonds to the components. Thus, in a direct non-covalent association, there is no other molecule intervening between the associated components. An indirect non-covalent association refers to any chain of molecules and

37

45652286 bonds linking the components where the components are not covalently coupled (that is, there is a least one separate molecule other than the components intervening between the components via non-covalent bonds).

Reference to components (such as an PL1, and/or PL3, and/or RPARPAR peptide and a cargo) as not being “non-covalently associated” means that there is no direct or indirect non-covalent association between the components. That is, for example, no atom covalently coupled to an PL1 , and/or PL3, and/or RPARPAR peptide is involved in a non- covalent bond with an atom covalently coupled to a cargo. Within this meaning, an PL1, and/or PL3, and/or RPARPAR peptide and a cargo can be together in a composition where they are indirectly associated via multiple intervening non-covalent bonds while not being non-covalently associated as that term is defined herein. For example, an PL1, and/or PL3, and/or RPARPAR peptide and a cargo can be mixed together in a carrier where they are not directly non-covalently associated. An PL1, and/or PL3, and/or RPARPAR peptide and a cargo that are referred to as not indirectly non-covalently associated cannot be mixed together in a continuous composition. Reference to components (such as an PL1, and/or PL3, and/or RPARPAR peptide and a cargo) as not being “directly non-covalently associated” means that there is no direct non-covalent association between the components (an indirect non-covalent association may be present). Reference to components (such as an PL1, and/or PL3, and/or RPARPAR peptide and a cargo) as not being “indirectly non- covalently associated” means that there is no direct or indirect non-covalent association between the components.

It is understood that components can be non-covalently associated via multiple chains and paths including both direct and indirect non-covalent associations. For the purposes of these definitions, the presence a single direct non-covalent association makes the association a direct non-covalent association even if there are also indirect non- covalent associations present. Similarly, the presence of a covalent connection between components means the components are covalently coupled even if there are also non- covalent associations present. It is also understood that covalently coupled components that happened to lack any non-covalent association with each other are not considered to fall under the definition of components that are not non-covalently associated.

In some forms, the cargo does not include a peptide. The cargo can include a peptide. In some forms, the cargo does not include a homing peptide. The cargo can include a homing peptide. The cargo can selectively home to cells and tissues having microbial pathogen infection, for example, cells and tissues having Mtb infection. In some

38

45652286 forms, the cargo does not selectively home to cells and tissues having microbial pathogen infection, for example, cells and tissues having Mtb infection. The cargo can selectively home to cells and tissues having microbial pathogen infection, for example, cells and tissues having Mtb infection. In some forms, the cargo does not include an accessory molecule. The cargo can include an accessory molecule. In some forms, the cargo does not include an accessory peptide. The cargo can include an accessory peptide. The cargo can selectively home to cells and tissues having Mtb infection, for example, cells and tissues having Mtb infection.

The PL1, and/or PL3, and/or RPARPAR peptide can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of an amino acid sequence, protein, peptide, conjugate, or composition that includes the PL1, and/or PL3, and/or RPARPAR peptide. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the PL1, and/or PL3, and/or RPARPAR peptide or an amino acid sequence, protein, peptide, conjugate, or composition that includes the PL I, and/or PL3, and/or RPARPAR peptide. Accessory molecules can be any molecule, compound, component, etc. that has a useful function and that can be used in combination with an PL1, and/or PL3, and/or RPARPAR composition, PL1, and/or PL3, and/or RPARPAR conjugate, PL1, and/or PL3, and/or RPARPAR molecule, PL1, and/or PL3, and/or RPARPAR protein, and/or PL1, and/or PL3, and/or RPARPAR peptide. Examples of useful accessory molecules include peptides, targeting molecules, affinity ligands, cell penetrating molecules, endosomal escape molecules, subcellular targeting molecules, nuclear targeting molecules. Different accessory molecules can have similar or different functions from each other. Accessory molecules having similar functions, different functions, or both, can be associated an PL1, and/or PL3, and/or RPARPAR composition, PL1 , and/or PL3, and/or RPARPAR conjugate, PL1, and/or PL3, and/or RPARPAR molecule, PL1, and/or PL3, and/or RPARPAR protein, and/or PL1, and/or PL3, and/or RPARPAR peptide.

The PL I, and/or PL3, and/or RPARPAR peptide can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of an amino acid sequence, protein, peptide, conjugate, or composition that includes the PL1, and/or PL3, and/or RPARPAR peptide. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the PL1, and/or PL3, and/or RPARPAR peptide or an amino acid sequence, protein, peptide, conjugate, or composition that includes the PL1, and/or PL3, and/or RPARPAR peptide. Accessory molecules can be

39

45652286 any molecule, compound, component, etc. that has a useful function and that can be used in combination with an PL1, and/or PL3, and/or RPARPAR composition, PL1, and/or PL3, and/or RPARPAR conjugate, PL1, and/or PL3, and/or RPARPAR molecule, PL1, and/or PL3, and/or RPARPAR protein, and/or PL1, and/or PL3, and/or RPARPAR peptide. Examples of useful accessory molecules include peptides, targeting molecules, affinity ligands, cell penetrating molecules, endosomal escape molecules, subcellular targeting molecules, nuclear targeting molecules. Different accessory molecules can have similar or different functions from each other. Accessory molecules having similar functions, different functions, or both, can be associated an PL1, and/or PL3, and/or RPARPAR composition, PL1, and/or PL3, and/or RPARPAR conjugate, PL1, and/or PL3, and/or RPARPAR molecule, PL1 , and/or PL3, and/or RPARPAR protein, and/or PL1 , and/or PL3, and/or RPARPAR peptide.

The accessory molecule can be separate from or overlapping with the PL1, and/or PL3, and/or RPARPAR peptide. For example, some accessory molecules are amino acid sequences. This can allow the amino acid sequence including the PL1, and/or PL3, and/or RPARPAR peptide to overlap the amino acid sequence that includes the accessory amino acid sequence. Alternatively the accessory molecule can be a separate entity that does not overlap with the PL1, and/or PL3, and/or RPARPAR peptide. In some forms, the accessory molecule can include a sequence in, for example, a peptide that binds to a specific receptor distinct from the receptor for the PL1, and/or PL3, and/or RPARPAR peptide.

The accessory molecule can be separate from or overlapping with the PL1, and/or PL3, and/or RPARPAR peptide. For example, some accessory molecules are amino acid sequences. This can allow the amino acid sequence including the PL1, and/or PL3, and/or RPARPAR peptide to overlap the amino acid sequence that includes the accessory amino acid sequence. Alternatively, the accessory molecule can be a separate entity that does not overlap with the PL1, and/or PL3, and/or RPARPAR peptide. In some forms, the accessory molecule can include a sequence in, for example, a peptide that binds to a specific receptor distinct from the receptor for the PL1, and/or PL3, and/or RPARPAR peptide.

The PL1, and/or PL3, and/or RPARPAR peptide can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of an amino acid sequence, protein, peptide, conjugate, or composition that includes the PL1, and/or PL3, and/or RPARPAR peptide. As another example, the accessory molecule can be

40

45652286 covalently coupled or non-covalently associated with the PL1, and/or PL3, and/or RPARPAR peptide or an amino acid sequence, protein, peptide, conjugate, or composition that includes the PL1, and/or PL3, and/or RPARPAR peptide. The PL1, and/or PL3, and/or RPARPAR conjugate can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of a conjugate or composition that includes the PL1, and/or PL3, and/or RPARPAR conjugate. As another example, the accessory molecule can he covalently coupled or non-covalently associated with the PL1 , and/or PL3, and/or RPARPAR conjugate or a conjugate or composition that includes the PL1, and/or PL3, and/or RPARPAR conjugate. The PL1, and/or PL3, and/or RPARPAR composition can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of a composition that includes the PL1, and/or PL3, and/or RPARPAR composition. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the PL1, and/or PL3, and/or RPARPAR composition or a composition that includes the PL1, and/or PL3, and/or RPARPAR composition.

The PL1, and/or PL3, and/or RPARPAR peptide can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of an amino acid sequence, protein, peptide, conjugate, or composition that includes the PL1, and/or PL3, and/or RPARPAR peptide. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the PL1, and/or PL3, and/or RPARPAR peptide or an amino acid sequence, protein, peptide, conjugate, or composition that includes the PL1, and/or PL3, and/or RPARPAR peptide. The PL1, and/or PL3, and/or RPARPAR conjugate can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of a conjugate or composition that includes the PL1, and/or PL3, and/or RPARPAR conjugate. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the PL1, and/or PL3, and/or RPARPAR conjugate or a conjugate or composition that includes the PL1, and/or PL3, and/or RPARPAR conjugate. The PL1, and/or PL3, and/or RPARPAR composition can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of a composition that includes the PL1, and/or PL3, and/or RPARPAR composition. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the PL1, and/or PL3, and/or RPARPAR composition or a composition that includes the PL1, and/or PL3, and/or RPARPAR composition.

41

45652286 The amino acid sequence can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of an amino acid sequence, protein, peptide, conjugate, or composition that includes the amino acid sequence. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the amino acid sequence or an amino acid sequence, protein, peptide, conjugate, or composition that includes the amino acid sequence. The protein or peptide can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of a protein, peptide, conjugate, or composition that includes the peptide. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the peptide or a protein, peptide, conjugate, or composition that includes the peptide. For example, an accessory molecule can be a part of a protein, conjugate, or composition that includes the protein. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the protein or a protein, conjugate, or composition that includes the protein. The conjugate can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of a conjugate or composition that includes the conjugate. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the conjugate or a conjugate or composition that includes the conjugate. The composition can be associated with one or more accessory molecules. For example, an accessory molecule can be a part of a composition that includes the composition. As another example, the accessory molecule can be covalently coupled or non-covalently associated with the composition or a composition that includes the composition.

The PL1, and/or PL3, and/or RPARPAR peptide can be associated with one or more peptides. For example, a peptide can be a part of an amino acid sequence, protein, peptide, conjugate, or composition that includes the PL1, and/or PL3, and/or RPARPAR peptide. As another example, the peptide can be covalently coupled or non-covalently associated with the PL1, and/or PL3, and/or RPARPAR peptide or an amino acid sequence, protein, peptide, conjugate, or composition that includes the PL1, and/or PL3, and/or RPARPAR peptide. The peptide can be separate from or overlapping with the PL1, and/or PL3, and/or RPARPAR peptide. For example, some peptides are amino acid sequences. This can allow the amino acid sequence including the PL1, and/or PL3, and/or RPARPAR peptide to overlap the amino acid sequence that includes the homing amino acid sequence. Alternatively the peptide can be a separate entity that does not overlap with the PL1, and/or PL3, and/or RPARPAR peptide. In some forms, the peptide can include a

42

45652286 sequence in, for example, an PL1, and/or PL3, and/or RPARPAR peptide that binds to a specific receptor distinct from the receptor for the PL1, and/or PL3, and/or RPARPAR peptide.

The amino acid sequence of any one or more of SEQ ID NOs:l-37 can be associated with one or more peptides. For example, a peptide can be a part of an amino acid sequence, protein, peptide, conjugate, or composition that includes the amino acid sequence. As another example, the peptide can be covalently coupled or non-covalently associated with the amino acid sequence or an amino acid sequence, protein, peptide, conjugate, or composition that includes the amino acid sequence. The protein or peptide can be associated with one or more peptides. For example, a peptide can be a part of a protein, peptide, conjugate, or composition that includes the peptide. As another example, the peptide can be covalently coupled or non-covalently associated with the peptide or a protein, peptide, conjugate, or composition that includes the peptide. For example, a peptide can be a part of a protein, conjugate, or composition that includes the protein. As another example, the peptide can be covalently coupled or non-covalently associated with the protein or a protein, conjugate, or composition that includes the protein. The conjugate can be associated with one or more peptides. For example, a peptide can be a part of a conjugate or composition that includes the conjugate. As another example, the peptide can be covalently coupled or non-covalently associated with the conjugate or a conjugate or composition that includes the conjugate. The composition can be associated with one or more peptides. For example, a peptide can be a part of a composition that includes the composition. As another example, the peptide can be covalently coupled or non-covalently associated with the composition or a composition that includes the composition.

As disclosed herein, the term “cargo composition” refers to any cargo that can be used in conjunction with the infected tissue-targeting molecules. One of skill in the art can determine what cargo can be coupled to an infected tissue-targeting molecule conjugate. The infected tissue and/or microbial pathogen-targeting peptide conjugates disclosed herein can include an infected tissue-targeting molecule coupled to or associated with the cargo composition. Examples of cargo compositions include, but are not limited to, an anti-microbial agent, diagnostic or labelling agents, therapeutic agents, pro-phylactic agents, anti-inflammatory agents, polypeptides, nucleic acid molecules, small molecules, nanoparticles, fluorophores, fluorescein, rhodamine, a radionuclide, indium- 111, technetium-99, carbon-11, carbon- 13, or a combination thereof. These cargo compositions associated with an infected tissue and/or microbial pathogen-targeting peptide in an

43

45652286 infected tissue-targeting molecule conjugate can be moieties. As used herein, the term "moiety" is used broadly to mean a physical, chemical, or biological material that generally imparts a biologically useful function to a linked cargo composition. A moiety can be any natural or nonnatural material including, without limitation, a biological material, such as a cell, phage or other virus; an organic chemical such as a small molecule; a nanoparticle, a radionuclide; a nucleic acid molecule or oligonucleotide; a polypeptide; or a peptide. For example, moieties that affect the target, such as moieties with therapeutic effect, or that facilitate detection, visualization or imaging of the target, such as fluorescent molecule or radionuclides.

Components of the disclosed infected tissue-targeting molecule conjugates can be combined, linked and/or coupled in any suitable manner. For example, moieties and homing molecules can be associated covalently or non-covalently, directly or indirectly, with or without a linker moiety.

In some forms, when the infected tissue -targeting peptides are included within, i.e.. incorporated into the external surface of a delivery vehicle, the delivery vehicle includes (e.g., encapsulates or is complexed with) one or more active agent, such as a therapeutic agent. In some forms, the therapeutic agent is an antimicrobial agent, such as an antibiotic.

Exemplary active agents are selected from therapeutic agents, diagnostic agents, nutraceutical agents and prophylactic agents. Typically, the active agents are selected from a protein, a nucleic acid, a carbohydrate, a polymer, a lipid, a small molecule, a cell, and a virus. Therefore, in some forms, nanoparticles or microparticles having attached thereto one or more infected tissue-targeting molecules encapsulate one or more active agents selected from proteins, nucleic acids, carbohydrates, polymers, lipids, small molecules, cells, and viruses, or combinations thereof.

In some forms, nanoparticles or microparticles having attached thereto one or more infected tissue- targeting molecules each independently having an amino acid sequence of any one or more of SEQ ID NOs: 1-37, or functional variants thereof, encapsulate active agents that are therapeutic agents. In other forms, nanoparticles or microparticles having attached thereto one or more infected tissue-targeting molecules each independently having an amino acid sequence of any one or more of SEQ ID NOs: 1-37, or functional variants thereof, encapsulate active agents that are diagnostic and/or labelling agents. In other forms, nanoparticles or microparticles having attached thereto one or more infected tissue and/or microbial pathogen-targeting peptides each independently having an amino

44

45652286 acid sequence of any one or more of SEQ ID NOs: 1-37, or functional variants thereof, encapsulate active agents that are diagnostic and/or labelling agents and also encapsulate active agents that are therapeutic agents. Therefore, in some forms, the particles encapsulate a multiplicity of therapeutic, diagnostic and/or labelling agents.

As used herein, "causing a cargo to be covalently coupled or directly non- covalently associated" with something else refers to any action that results in a cargo that is not covalently coupled or directly non-covalently associated with the something else becoming or coming into the state of being covalently coupled or directly non-covalently associated with the something else. More clearly, "causing a cargo to be covalently coupled or directly non-covalently associated" with something else refers to any action that results in a cargo and the something else becoming or coming into the state of being covalently coupled or directly non-covalently associated. As an example, covalently coupling a cargo to another cargo constitutes "causing a cargo to be covalently coupled or directly non-covalently associated" with the other cargo. As another example, a cargo that starts as a nonexistent concept and then is synthesized as part of a composition that includes the thing to which the cargo is to be coupled or directly associated constitutes "causing a cargo to be covalently coupled or directly non-covalently associated" with the thing.

As used herein, "causing a cargo composition to be covalently coupled or directly non-covalently associated" with something else refers to any action that results in a cargo composition that is not covalently coupled or directly non-covalently associated with the something else becoming or coming into the state of being covalently coupled or non- covalently associated with the something else. More clearly, "causing a cargo composition to be covalently coupled or directly non-covalently associated" with something else refers to any action that results in a cargo composition and the something else becoming or coming into the state of being covalently coupled or directly non-covalently associated. As an example, covalently coupling a cargo composition to another cargo composition constitutes "causing a cargo composition to be covalently coupled or directly non- covalently associated" with the other cargo composition. As another example, a cargo composition that starts as a nonexistent concept and then is synthesized as part of a composition that includes the thing to which the cargo composition is to be coupled or directly associated constitutes "causing a cargo composition to be covalently coupled or directly non-covalently associated" with the thing.

45

45652286 The cargo can be, for example, a molecule, or complex of molecules with therapeutic or diagnostic applications. Therapeutic cargos that can be targeted with the disclosed peptides include but are not limited to a molecule, a complex of molecules, an anti-bacterial agent, an anti-viral agent, or a combination of these. Therapeutic cargos that can be targeted with the disclosed peptides include but are not limited to a therapeutic protein, compound, or composition that is effective against microbial pathogens, such as an anti-bacterial agent, an antibody that targets microbial pathogens, a functional nucleic acid or a nucleic acid analog against one or more genes of microbial pathogens, a contrast agent, an imaging agent, a label, a labeling agent, or a combination thereof.

1. Therapeutic Agents

Exemplary therapeutic agents include, but are not limited to, antimicrobial agents, such as beta-lactam antibiotics (including penicillins such as ampicillin, cephalosporins selected in turn from cefuroxime, cefaclor, cephalexin, cephydroxil and cepfodoxime proxetil); tetracycline antibiotics (doxycycline and minocycline); macrolides antibiotics (azithromycin, erythromycin, rapamycin and clarithromycin); fluoroquinolones (moxifloxacin, ciprofloxacin, enrofloxacin, ofloxacin, gatifloxacin, levofloxacin) and norfloxacin and other inhibitors such as vancomycin, chloramphenicol, clindamyin, streptogramins, aminoglycosides, spectinomycin, spec tinamides, sulfonamides, trimethoprim, quinolones, diarylquinolines, such as bedaquiline, nitroimidazoles such as pretomanid and delamanid, oxazolidinones, such as sutezolid and linezolid, amphotericin B, flucytosine, azoles such as ketoconazole, itraconazole, fluconazole, clotrimazole, and miconazole, griseofulvin, terbinafine, and nystatin; kanamycin, neomycin, bacitracin, polymixin, mafenide acetate or silver sulfadiazine, or gentamicin sulfate; and carbapenem antibiotics, such as imipenem, meropenem, ertapenem, and doripenem. In some forms, the antimicrobial agent is a broad spectrum agent. Exemplary broad spectrum antimicrobial agents include, for example, a second, third, or fourth generation cephalosporin.

In some forms, the antibiotics are those associated clinically with treatment of an infection with a microbial pathogen. For example, in some forms, the antibiotics are those associated clinically with treatment of Mtb infection, such as first- line oral drugs (with typically adult daily dose) Isoniazid (300 mg), Rifampin (600 mg), Rifabutin (300 mg), Pyrazinamide (30 mg/kg), Ethambutol (15 - 25 mg/kg); and injectable drugs (with typically adult daily dose): Streptomycin (15 mg/kg), Amikacin (15 mg/kg), Kanamycin (15 mg/kg), Capreomycin (15 mg/kg).

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45652286 In some forms, the antibiotics are those associated clinically with treatment of Mtb infection, such as Second-line oral drugs (with typically adult daily dose): Ofloxacin (400 mg twice daily ), Levofloxacin (500 mg once daily), Moxifloxacin (400 mg qd), Gatifloxacin (400 mg once daily), Ethionamide (250 mg twice daily or three times daily), Aminosalicylic acid (3 g once daily), and Cycloserine (250 mg twice daily or three times daily), bedaquiline, pretomanid, and linezolid.

Therefore, in some forms, the antimicrobial agent is active against a wide range of bacteria in addition to Mycobacterium tuberculosis, including both gram positive and gram negative species. One of ordinary skill in the art will be able to select appropriate antimicrobial agents by considering factors such as the patient’s history (e.g., any history of an allergic reaction to such agents), and so forth. In preferred forms, the therapeutic agent is an antimicrobial that can kill or otherwise prevent viability of Mycobacterium tuberculosis. In an exemplary form, the therapeutic agent is bedaquiline.

In some forms, the nanoparticles or microparticles having attached thereto one or more peptides that selectively target and bind to Mycobacterium tuberculosis bacteria encapsulate vancomycin and optionally one or more therapeutic, diagnostic and/or labelling agents. a. Vancomycin

In some forms, the antimicrobial agent is vancomycin. Vancomycin is a glycopeptide antibiotic recommended intravenously as a treatment for complicated skin infections, bloodstream infections, endocarditis, bone and joint infections, and meningitis caused by methicillin-resistant bacteria. Blood levels may be measured to determine the correct dose. Common adverse drug reactions (>1% of patients) associated with IV vancomycin include: local pain, which may be severe, and thrombophlebitis. Damage to the kidneys (nephrotoxicity) and to the hearing (ototoxicity) were side effects of the early impure versions of vancomycin.

In some forms, vancomycin is combined for administered together with a second or further antimicrobial agent, such as Zosyn (piperacillin & tazobactam); Piperacillin; Timentin (Ticarcillin & clavulanate); Ticarcillin; Carbenicillin, or a 3^rd generation Cephalosporin, such as Ceftazidime.

2. Diagnostic agents

Exemplary diagnostic and/or labelling agents are selected from a fluorescent label, a radioactive label, an infrared label, a coloring agent, paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, X-ray imaging agents,

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45652286 and MRI contrast agents. Typically, the diagnostic agents are agents that can be visualized in the body of the subject, or in a sample obtained from the subject.

D. Delivery Vehicles

In some forms, the infected tissue-targeting molecules and/or conjugates thereof are associated with a vehicle for delivery of the peptide together with one or more active agents to infected tissues and bacteria such as Mycobacterium tuberculosis in vivo. Therefore, nanoparticles including infected tissue targeting peptides and one or more active agents are provided.

Exemplary delivery vehicles include substrates and particles, such as porous silicon particles, viral capsids, polymeric particles and liposomes. Exemplary particles include dendrimers and beads, liposomes, viral capsids and virus-like particles, metallic particles such as beads and nano worms/nano rods, and porous silicon materials. Typically, the delivery vehicles are nanoparticles or microparticles, sized for administration in vivo.

In some forms, the delivery vehicle includes up to 5, or 10, or 15, or 20, or 25, or 30, or 35, or 40, or 45, or more than 50 copies, such as 60, 70, 80, 90, 100, 200, 300, 400, 500 or more than 500, such as 1000 or more, such as 5,000, 10,000, 15,000, 20,000, 50,000 or 100,000 copies of an infected tissue targeting molecule. For example, in some forms, the delivery vehicles include between one and one hundred infected tissue-targeting molecule molecules, each independently having an amino acid sequence of any one or more of SEQ ID NOs:l-37 encapsulated within and/or associated with the delivery vehicle.

1. Particles

In some forms, the delivery vehicle conjugated or complexed with the infected tissue binding molecules is a particle suitable for administration to a subject in vivo.

Microparticles, microspheres, and microcapsules, referred to herein collectively as “microparticles”, are solid or semi-solid particles having a diameter of less than one millimeter, typically less than 100 microns, which can be formed of a variety of materials, including synthetic polymers, proteins, and polysaccharides. Nanoparticles, nanospheres, and nano-capsules, referred to herein collectively as “nanoparticles”, are solid or semisolid particles having a diameter of less than one micrometer, typically less than 999 nanometers, which can be formed of a variety of materials, including synthetic polymers, proteins, and polysaccharides.

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45652286 Nanoparticles and microparticles have been used in many different applications, primarily separations, diagnostics, and drug delivery. In the controlled drug delivery area, therapeutic molecules are encapsulated within nanoparticles or microparticles or incorporated into a monolithic matrix, for subsequent release. A number of different techniques are routinely used to make these nanoparticles or microparticles from synthetic polymers, natural polymers, proteins and polysaccharides, including phase separation, solvent evaporation, emulsification, and spray drying. Generally, the polymers form the supporting structure of these nanospheres and microspheres, and the therapeutic agent of interest is incorporated into the polymer structure.

“Mean particle size” as used herein, generally refers to the statistical mean particle size (diameter) of the particles in a population of particles. The diameter of an essentially spherical particle may refer to the physical or hydrodynamic diameter. The diameter of a non-spherical particle may refer preferentially to the hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as dynamic light scattering. “Monodisperse” and “homogeneous size distribution”, are used interchangeably herein and describe a population of nanoparticles or microparticles where all of the particles are the same or nearly the same size. As used herein, a monodisperse distribution refers to particle distributions in which 90% of the distribution lies within 15% of the median particle size, more preferably within 10% of the median particle size, most preferably within 5% of the median particle size.

Exemplary polymers used for the formation of nanospheres and microspheres that are conjugated or complexed with the infected tissue binding molecules include homopolymers and copolymers of lactic acid and glycolic acid (PLGA). Microspheres and nanospheres produced using polymers such as this exhibit a poor loading efficiency, however, and are often only able to incorporate a small percentage of the drug of interest into the polymer structure. Therefore, substantial quantities of microspheres and/or nanospheres often are administered to achieve a desired therapeutic effect.

In some forms, the particles are sized for uptake within the gut following oral administration, or for uptake via the mucosa of the lung following inhalation, or for administration into the bloodstream, i.e., by intravenous injection.

An exemplary way to measure the diameter of a particle is by using Dynamic light Scattering (DLS). The particle may have a diameter of between about 1,000 nm and about

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45652286 10 microns, inclusive, as measured by DLS, for example, between about 100 nm and about 1 micron, inclusive, between about 10 nm and about 500 nm, inclusive, between about 20 nm and about 500 nm, inclusive, or between about 25 nm and about 250 nm, inclusive. For example, the nanoparticle can have a diameter from 10 nm to 900 nm, from 10 nm to 800 nm, from 10 nm to 700 nm, from 10 nm to 600 nm, from 10 nm to 500 nm, from 20 nm from 500 nm, from 30 nm to 500 nm, from 40 nm to 500 nm, from 50 nm to 500 nm, from 60 nm to 400 nm, from 50 nm to 350 nm, from 50 nm to 300 nm, or from 50 nm to 200 nm. In preferred forms the nanoparticles can have a diameter less than 400 nm, less than 300 nm, or less than 200 nm. The preferred range for a nanoparticle is between 50 nm and 300 nm, or 25 nm and 250 nm, or 80 nm and 150 nm, inclusive. In particular forms a nanoparticle has a diameter of about 100 nm.

The particle or nanoparticle can have a zeta potential between -100 mV and +100 mV, inclusive, between -50 mV and +50 mV, inclusive, between -40 mV and +40 mV, inclusive, between -30 mV and +30 mV, inclusive, between -20 mV and +20 mV, inclusive, between -10 mV and +10mV, inclusive, or between -5mV and +5 mV, inclusive. The particle or nanoparticle can have a negative zeta potential. The particle can have a positive zeta potential. In some forms the particle has a substantially neutral zeta potential, i.e., the zeta potential is approximately 0 mV. In some forms, the particle has a zeta potential of approximately between -20 mV and +20 mV, inclusive, more preferably between -10 mV and +10 mV, inclusive. In some forms, the zeta potential is between 0 and +100, inclusive, e.g., between 0 mV and +40 mV, inclusive. a. Porous Silicon Particles

In some forms, the delivery vehicle that is conjugated or complexed with the infected tissue-targeting molecules or conjugates thereof is a particle or substrate formed from partly or entirely of a porous silicon particle. For example, in particular forms, the delivery vehicle is a porous silicon material loaded with a meltable composition. The term “porous silicon material” refers to a material that includes silicon, typically crystalline silicon, which has been treated by a process that has introduced a plurality of void spaces or pores into the material. For example, in some forms a “porous silicon material” includes some limited portion that is not silicon, such as carbon. A “porous silicon material” can include any shape or shapes. For example, a “porous silicon material” can include porous silicon films, porous silicon layers, porous silicon particles, etc. In some forms, the porous silicon material includes silicon oxide or silicon dioxide. Therefore, in some forms, the delivery vehicle is a particle formed partly or completely from a “porous silicon dioxide

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45652286 material” or a “porous silica material” that includes at least some silicon oxide or silicon dioxide. In specific forms, the porous silicon material has been oxidized so that one or more surfaces of the porous silicon material includes silicon dioxide or silica. The term, “porous silicon dioxide” or “porous silica” refers to porous silicon materials or particles that have been oxidized so that the materials or particles thereof include from about 1 % up to about 99% inclusive of silicon oxide. In other forms, the delivery vehicle is a particle formed partly or completely from a “porous silica material” that includes some portion that is not silica, e.g., silicon. In some forms, the delivery vehicle is a particle formed partly or completely from a porous silicon material prepared using a sol-gel process from appropriate soluble precursors. Where the porous silicon oxide or porous silicon dioxide is prepared using a sol-gel process, the resultant porous silicon material may be referred to as a silica gel.

In some forms, the delivery vehicle is a particle formed partly or completely from a material including silicon and optionally other elements, and which includes a plurality of void spaces or pores in the material. For example, a “porous silica material” as used herein, includes structures that have an internal “core” of silicon with an outer “sheath” of silica. In this context, a “porous silicon material” refers to a porous silicon-based material that contains a portion of silicon that is in its elemental form, that is, not oxidized, and a portion of silicon that has been oxidized so that one or more surfaces of the porous silicon material includes silicon dioxide. Additionally, and as mentioned above, a “porous silicon material” may further include some limited portion of which is not silicon, e.g., carbon. Typically, the void spaces or pores within a delivery vehicle that is a particle formed partly or completely from a material including silicon are between 1 nm to 100 pm in diameter or size. The dimensions of the void spaces or pores are generally tunable, i.e., the dimensions of the pores of the porous silicon material can be controlled to have a pore size dimension (e.g., diameter) of 1 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 70 nm, 80 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 pm, 10 pm, 50 pm, 100 pm, or any range of pore sizes that includes or is between any two of the foregoing percentages, including fractions thereof. In some forms, the size of a pore within the porous silicon material is varied in diameter e.g., one side of a porous silicon material may have substantially the same size pores while the opposite side of the porous silicon material may have pores that are substantially larger, by about 25% or greater). In some forms, the overall porosity density of the porous silicon material is user-defined, such that pore

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45652286 density and size is varied according to the structural requirements of a user. In certain forms, the delivery vehicle is a particle formed partly or completely from a porous silicon material having a porosity of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or any range of porosity that includes or is between any two of the foregoing percentages, including fractions thereof. The delivery vehicles formed partly or completely from a material including silicon porous silicon structures can easily withstand temperatures well in excess of what is required for wet or dry heat sterilization. For example, the delivery vehicles formed partly or completely from a material including silicon porous silicon materials readily tolerate heat sterilization without impacting their physical properties. Porous silicon materials have low toxicity profiles and excellent drug delivery properties. See, e.g., PCT International Publication Nos. W02006/050221 and W02009/009563. Under biological conditions, the primary degradation product of these materials is orthosilicic acid, a non-toxic water- soluble compound naturally found in human tissues that is readily cleared through the renal system.

Exemplary porous silicon materials for use as delivery vehicles are described in United States patent application publication number US/20200009053 Al ; US/20200397698 Al; and US/20210000744 Al, the contents of which are hereby incorporated by reference herein in their entirety.

Typically, delivery vehicles formed partly or completely from a porous silicon material exhibit a highly aligned nature of the pores, which results in anisotropic dissolution of an associated active agent/carrier matrix. For example, in some forms a carrier dissolves preferentially along the pore direction, guiding drug release into a zeroorder kinetic regime that result in a linear release profile. Therefore, the concentration of the free active agent is constant throughout the release period, and rapidly tapers at the end of the release period. Thus, in some forms, the delivery vehicle is a particle formed partly or completely from a porous silicon material that minimizes the initial burst release of an associated active agent. Therefore, a larger dose of an active agent can be administered without significant side effects than can be administered in the absence of the delivery vehicle formed partly or completely of a porous silicon material. In some forms, the delivery vehicle is a particle formed partly or completely from a porous silicon material configured to deliver a constant amount of an associated active agent to a subject in vivo over a set period of time, such as one day, two days, three days, four days, five days, six days, one week, ten days, two weeks, three weeks, four weeks, one month, two months,

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45652286 three months, one hundred days, six months, one year or more than one year. In exemplary forms, a porous silicon material has a porosity from about 15% to about 85%, inclusive, wherein the pores of the silicon material are loaded with a mixture including a thermally unstable therapeutic agent and a thermally unstable substance, wherein the thermally unstable therapeutic agent and the thermally unstable substance are not the same compound or molecule, and wherein the mixture has a lower melting point than the melting point of the thermally unstable therapeutic agent. Tn some forms, the porous silicon materials are porous silicon particles. In another form, the porous silicon particles of have an average diameter or length from about 10 nm to about 100 pm. In a further form, the porous silicon particles of the disclosure have an average diameter or length from about 10 nm to about 100 nm. In an alternate form, the porous silicon particles have an average diameter or length from about 100 nm to about 100 pm. In a certain form, the porous silicon material has a porosity from about 50% to about 80%. In another form, the porous silicon material has a porosity of about 75%. In yet another form, the porous silicon material includes pores that have average diameters from about 2 nm to about 250 nm.

Methods of preparing porous silicon nanoparticles (pSiNPs) are known in the art. In an exemplary method, pSiNPs are prepared by electrochemical etch of crystalline, (100) -oriented silicon wafers (p-type, boron-doped, ~ 1 m.Q cm resistivity). In brief, the silicon wafer is anodized in an electrolyte composed of 3:1 (v:v) of 48% aqueous HF:ethanol. The etching waveform included a square wave in which a lower current density of 50 mA cm-2 is applied for 1.8 s, followed by a higher current density pulse of 400 mA cm-2 for 0.36 s. The pulsed waveform is repeated for 140 cycles, and the resulting porous silicon nanostructure is lifted-off from the silicon substrate by application of a current density of 3.7 mA cm— 2 for 250 seconds in an electrolyte including 1:30 (v:v) of 48% aqueous HF:ethanol. The freestanding porous silicon films are then fractured into nanoparticles of mean diameter 170 nm by ultrasonication in deionized water overnight. b. Silver Particles (AgNP)

In some forms, the delivery vehicle that is conjugated or complexed with the infected tissue-targeting molecules or conjugates thereof is a particle or substrate formed from partly or entirely of a silver particle. For example, in particular forms, the delivery vehicle is a silver nanoparticle loaded with the protein and one or more therapeutic and/or diagnostic agents. Silver nanoparticles are known in the art (see, e.g., Lingasamy et al., 2020; and Tobi et al., 2021, the contents of which are incorporated herein in their entirety).

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45652286 In some forms, the silver particles are citric AgNP. Exemplary citric AgNP have an average core size of between about 5 nm and about 100 nm, inclusive such as between 10 nm and 90 nm, between 20 nm and 80 nm, or between 40 nm and 80 nm, such as 50 nm, 60 nm or 70 nm. In an exemplary form, the citric AgNP has an average core size of about 62 ± 20 nm. Exemplary citric AgNP have an average hydrodynamic size of between about 10 nm and about 200 nm, inclusive such as between 20 nm and 160 nm, between 40 nm and 150 nm, or between 60 nm and 140 nm, such as 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, or 140 nm. In an exemplary form, the citric AgNP have a hydrodynamic size of 103 ± 40 nm. In an exemplary form, the citric AgNP have an average core size of about 62 ± 20 nm, and hydrodynamic size of 103 ± 40 nm.

AgNPs have been developed as a model platform for targeted in vitro and in vivo delivery. The AgNPs have several unique features that make them useful for biodistribution studies including plasmonic enhancement emission from coupled fluorescent dyes to allow ultrasensitive imaging of single nanoparticles. An exemplary citric AgNP is depicted in Figure 3C.

In some forms, the particle includes a silver (Ag) core, covered with NeutrAvidin- PEG(5K)-thiols and Iipoic-PEG(1K)-NH2. For example, in some forms, the infected tissue-targeting molecules are attached to NeutrAvidin and the payload is conjugated to the free NH2 groups of the lipoic-PEG(lK)-linker. In some forms, isotopically pure silver nanoparticles (AgNPs) are synthesized and functionalized as previously described (Willmore et al., Nanoscale. 8:9096-9101 (2016)). For example, synthesis of wild type (wt) and isotopic (Agl07 and Agl09) AgNPs is carried out with biotinylated peptides being coated on NeutrAvidin (NA) on the surface of the AgNPs. Typically, transmission electron microscopy is used to image and DLS is used to assess the zeta potential, polydispersity and size of AgNPs. c. Viral Capsid Proteins

In some forms, the delivery vehicle is a viral capsid, or a virus-like particle formed from partly or entirely of a multiplicity of viral capsid proteins. Preferred virus capsids are stable toward thermal denaturation at temperatures up to 80-100°C, chaotropic agents, and to extremes of pH. Exemplary viral-like particles that are stable toward thermal denaturation at temperatures up to 80-100°C, chaotropic agents, and to extremes of pH include bacteriophage capsids and phage particles. In some forms, the delivery vehicle is a bacteriophage that infects Mycobacterium sp. In a preferred form, the delivery vehicle is a bacteriophage that infects Mycobacterium tuberculosis.

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45652286 i. Bacteriophage VLPs

In some forms, the delivery vehicle includes a viral-like particle (VLP), or vesicle, composed of a bacteriophage capsid protein.

In some forms, the bacteriophage is an RNA or DNA phage that infects a bacteria. In some forms, the bacteria is Mycobacterium tuberculosis.

The stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. The icosahedral capsid of the RNA bacteriophage PP7 is crosslinked by disulfide bonds between coat protein dimers at its 5-fold and quasi-6-fold symmetry axes, providing enhanced stability to VLPs formed from PP7 capsid proteins. In some forms, the PP7 capsid is a modified PP7, for example, modified by attachment of a peptide, carbohydrate, small molecule or nucleic acid to the viral capsid. Modified PP7 VLPs are described in Zhao, et al., ACS Nano. 2019 Apr 23; 13(4): 4443-4454, which is incorporated by reference herein in its entirety. Therefore, modified PP7 VLPs capable of encapsulating active agents are also described for use with the peptides. The structure of the coat protein is a five-stranded P-sheet with two a-helices and a hairpin. When the capsid is assembled, the helices and hairpin face the exterior of the particle, while the 3- sheet faces the interior. d. Polymeric Particles

In some forms, the delivery vehicle is or includes one or more polymers, such as polymeric nanoparticles or microparticles. Preferred polymers include biocompatible polymers. In some forms, the biocompatible polymer(s) is biodegradable or bioabsorbable. In other form, the polymer is non-degradable. In other forms, the particles are a mixture of degradable and non-degradable particles.

In some forms, the delivery vehicle is a particles that includes one or more biocompatible polymer(s) including, but not limited to, polyamino acids; cyclodextrin- containing polymers, in particular cationic cyclodextrin-containing polymers, such as those described in U.S. Patent No. 6,509,323; polymers prepared from lactones such as poly(caprolactone) (PCL); polyhydroxy acids and copolymers thereof such as poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co- glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co- glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L- lactide), and blends thereof, polyalkyl cyanoacralate, polyurethanes, poly(valeric acid), and poly-L-glutamic acid; hydroxypropyl methacrylate (HPMA); polyanhydrides; other

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45652286 polyesters; polyorthoesters; poly(ester amides); polyamides; poly(ester ethers); polycarbonates; polyalkylenes such as polyethylene and polypropylene; polyalkylene glycols such as poly(ethylene glycol) (PEG) and polyalkylene oxides (PEG), and block copolymers thereof such as polyoxyalkylene oxide (“PLURONICS®” or block copolymers containing PEG where PEG has a molecular weight of any values within the range of 300 Daltons to 1 MDa); polyalkylene terephthalates such as poly(ethylene terephthalate); ethylene vinyl acetate polymer (EVA); polyvinyl alcohols (PVA); polyvinyl ethers; polyvinyl esters such as poly(vinyl acetate); polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone; poly siloxanes; polystyrene (PS); and celluloses including alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, and carboxymethylcellulose; polymers of acrylic acids including poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as "polyacrylic acids"); polydioxanone and its copolymers; poly hydroxy alkanoates; polypropylene fumarate; polyoxymethylene; poloxamers; poly(butyric acid); trimethylene carbonate; and polyphosphazenes.

Examples of preferred natural polymers include proteins such as albumin, collagen, gelatin and prolamines, for example, zein, and polysaccharides such as alginate. Copolymers of the above, such as random, block, or graft copolymers, or blends of the polymers listed above can also be used.

Functional groups on the polymer can be capped to alter the properties of the polymer and/or modify (e.g., decrease or increase) the reactivity of the functional group. For example, the carboxyl termini of carboxylic acid contain polymers, such as lactide- and glycolide-containing polymers, may optionally be capped, e.g., by esterification, and the hydroxyl termini may optionally be capped, e.g. by etherification or esterification.

The weight average molecular weight can vary for a given polymer but is generally from about 1000 Daltons to 1,000,000 Daltons, 1000 Daltons to 500,000 Dalton, 1000 Daltons to 250,000 Daltons, 1000 Daltons to 100,000 Daltons, 5,000 Daltons to 100,000 Daltons, 5,000 Daltons to 75,000 Daltons, 5,000 Daltons to 50,000 Daltons, or 5,000 Daltons to 25,000 Daltons.

In some forms, the delivery vehicles are particles modified with one or more surfactants. Examples of surfactants include, but are not limited to, L-a-

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45652286 phosphatidylcholine (PC), 1 ,2-dipalmitoylphosphatidycholine (DPPC), oleic acid, sorbitan trioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, natural lecithin, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, block copolymers of oxyethylene and oxypropylene, synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, polyethylene glycol 400, cetyl pyridinium chloride, benzalkonium chloride, olive oil, glyceryl monolaurate, com oil, cotton seed oil, and sunflower seed oil, lecithin, oleic acid, and sorbitan trioleate.

In some forms where polyalkylene glycol (e.g., PEG) is used in a composition of polymers to modify the particles, PEG surface density may be controlled by varying the amount of PEG in the polymer composition or by mixing a blend of pegylated polymer component and non-pegylated polymer component. The density of PEG or polyalkylene glycol on the surface of formed particles may be evaluated using several techniques.

In some forms, the delivery vehicles are modified by the addition of one or more polymers to possess a specific ^-potential. For example, in some forms, the delivery vehicles are modified by the attachment of PEG and/or other polymers to the surface to possess a ^-potential of between about 20 mV and about -20 mV, preferably between about 10 mV and about -10 mV, more preferably between about 2 mV and about -2 mV. e. Liposomes, Micelles and Lipidic Particles

In some forms, the particles are lipidic particles, such as liposomes, or micelles. Lipidic particles include uni-lamellar phospholipid vesicles, liposomes, or lipoprotein particles. Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes, Chapter 10, 1979.

Formulations of liposomes and methods of making such formulations are well known to one of ordinary skill in the art. Liposomes are formed from commercially available phospholipids supplied by a variety of vendors including Av anti Polar Lipids, Inc. (Birmingham, Ala.). For example, in some forms, the liposome include the infected tissue binding molecules on the surface to direct the liposome to the infected tissue.

Suitable methods, materials and lipids for making liposomes are known in the art. Liposome delivery vehicles are commercially available from multiple sources. The liposome may be formed from a single lipid; however, in some embodiments, the

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45652286 liposome is formed from a combination of more than one lipid. The lipids can be neutral, anionic or cationic at physiologic pH. In some forms, the liposomes incorporate PEG, or PEGylated lipid derivatives. Incorporation of one or more PEGylated lipid derivatives can result in a liposome which displays polyethylene glycol chains on its surface. The resulting liposomes may possess increased stability and circulation time in vivo as compared to liposomes lacking PEG chains on their surfaces. Liposomes are formed from one or more lipids, which can be neutral, anionic, or cationic at physiologic pH. Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including, but limited to, 1 ,2-diacyl-glycero-3- phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin and sphingoglycolipids (also known as 1-ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; 1 ,2-diacyl-sn-glycero-3-phosphoethanolamine, including, but not limited to, 1 ,2-dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2-distearoylphosphatidylcholine (DSPC), 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), and 1 ,2-dimyristoylphosphatidylcholine (DMPC). The lipids can also include various natural (e.g., tissue derived L-a-phosphatidyl: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated l,2-diacyl-5n-glycero-3-phosphocholines, 1-acyl- 2-acyl-.s7?-glycero-3 -phosphocholines, l,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids. In some forms, the liposomes contain a phosphaditylcholine (PC) head group, and preferably sphingomyelin. In another form, the liposomes contain DPPC. In a further form, the liposomes contain a neutral lipid, preferably 1 ,2- dioleoylphosphatidylcholine (DOPC) .

In certain forms, the liposomes are generated from a single type of phospholipid. In such forms, preferably the phospholipid has a phosphaditylcholine head group, and, most preferably is sphingomyelin. The liposomes may include a sphingomyelin metabolite. Sphingomyelin metabolites used to formulate the liposomes include, without limitation, ceramide, sphingosine, or sphingosine 1 -phosphate. The concentration of the sphingomyelin metabolites included in the lipids used to formulate the liposomes can range from about 0.1 mol % to about 10 mol %. Preferably from about 2.0 mol % to about 5.0 mol %, and more preferably can be in a concentration of about 1.0 mol %.

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45652286 Suitable cationic lipids in the liposomes include, but are not limited to, N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, also references as TAP lipids, for example methylsulfate salt. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Suitable cationic lipids in the liposomes include, but are not limited to, dimethyldioctadecyl ammonium bromide (DDAB), 1 ,2-diacyloxy-3 -trimethylammonium propanes, N-[l-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine (DODAP), 1 ,2-diacyloxy-3- dimethylammonium propanes, N-[l -(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2-dialkyloxy-3-dimethylammonium propanes, dioctadecylamidoglycylspermine (DOGS), 3 -[N-(N',N'-dimethylamino- ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2-(sperminecarboxamido)- ethyl)-N,N-dimethyl-l-propanaminium trifluoro-acetate (DOSPA), |3-alanyl cholesterol, cetyl trimethyl ammonium bromide (CTAB), diCi4-amidine, N-ferf-butyl-N’-tetradecyl-3- tetradecylamino-propionamidine, N-(alpha-trimethylammonioacetyl)didodecyl-D- glutamate chloride (TMAG), ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride, l,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), and N, N, N', N'-tetramethyl-, N'-bis(2-hydroxylethyl)-2,3-dioleoyloxy- 1 ,4-butanediammonium iodide. In one form, the cationic lipids can be l-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2- hydroxyethyl)-imidazolinium chloride derivatives, for example, l-[2-(9(Z)- octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), and l-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2- hydroxyethyl)imidazolinium chloride (DPTIM). In one embodiment, the cationic lipids can be 2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine, for example, 1 ,2-dioleoyl-3-dimethyl- hydroxyethyl ammonium bromide (DORI), 1 ,2-dioleyloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DORIE), 1 ,2-dioleyloxypropyl-3-dimetyl- hydroxypropyl ammonium bromide (DORIE-HP), 1 ,2-dioleyl-oxy-propyl-3-dimethyl- hydroxybutyl ammonium bromide (DORIE-HB), 1 ,2-dioleyloxypropyl-3 -dimethylhydroxypentyl ammonium bromide (DORIE-Hpe), 1 ,2-dimyristyloxypropyl-3-dimethyl- hydroxylethyl ammonium bromide (DMRIE), 1 ,2-dipalmityloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DPRIE), and 1 ,2-disteryloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DSRIE).

The lipids may be formed from a combination of more than one lipid, for example, a charged lipid may be combined with a lipid that is non-ionic or uncharged at

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45652286 physiological pH. Non-ionic lipids include, but are not limited to, cholesterol and DOPE ( 1 ,2-dioleolylgly ceryl phosphatidylethanolamine), with cholesterol being most preferred. The molar ratio of a first phospholipid, such as sphingomyelin, to second lipid can range from about 5:1 to about 1:1 or 3:1 to about 1: 1, more preferably from about 1.5:1 to about 1:1, and most preferably, the molar ratio is about 1:1.

E. Linking Moieties

Tn some forms, the compositions of infected tissue-targeting molecules or conjugates thereof include a linking chemical moiety that serves to connect, directly or indirectly, one or more moieties to the infected tissue-targeting molecules.

Linking chemical moieties can be any organic, inorganic, or organometallic moiety which is polyvalent, so as to provide more than two points of attachment. The linking chemical moiety can be an organic molecule that contains multiple functional groups, or an organic moiety such as a substituted alkyl, unsubstituted alkyl, substituted alkylene, unsubstituted alkylene, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aryl, unsubstituted aryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocyclyl, unsubstituted heterocyclyl, substituted arylalkyl, or unsubstituted arylalkyl.

The functional groups can be any atom or group of atoms that contains at least one atom that is neither carbon nor hydrogen, with the proviso that the groups are capable of reacting with a nucleophile or an electrophile. Suitable functional groups include halogens (bromine, chlorine, and iodine); oxygen-containing functional groups such as a hydroxyls, epoxides, carbonyls, aldehydes, ester, carboxyls, and acid chlorides; nitrogen-containing functional groups such as amines and azides; and sulfur-containing groups such as thiols. The functional group may also be a hydrocarbon moiety which contains one or more nonaromatic pi-bonds, such as an alkyne, alkene, or diene. The linking chemical moiety can contain at least two different types of functional groups (e.g., one or more amines and one or more hydroxyls, one or more hydroxyls and one or more carboxyls, or one or more halides and one or more hydroxyls). In such cases, the different functional groups present on the linking chemical moiety can be independently addressed synthetically, permitting the covalent attachment of the rest of the compound and the one or more modulators in controlled stoichiometric ratios.

Following reaction with functional groups on the linking chemical moiety, one or more moieties will be covalently joined to the linking chemical moiety via bonds, and/or

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45652286 to the nanoparticle. The identity of these bonds will be determined by the identity of the functional group on the linking chemical moiety, the reactive loci of the rest of the compound and that of the functional group that attaches the modulator (directly or indirectly) to the linking chemical moiety. Examples of suitable bonds that connect the portions of the compound to the linking chemical moiety include -C(O)NH-, -C(O)NR’-, - NR’C(O)-, -C(O)O-, -OC(O)-, -C(0)0CH2-, -SO2NR’-, -CH2R’-, -O-, -NR’H-, -NR’-, - OCONH-, -NHCOO-, -OCONR’-, -NRCOO-, -NHCONH-, -NR’CONH-, -NHCONR’-, - NR’CONR’-, -CHOH-, -CROH-, unsubstituted alkyl (such as unsubstituted C1-C12 alkyl), substituted alkyl (such as substituted C1-C12 alkyl), wherein R’ is hydrogen, halogen (F, Cl, Br, I), hydroxyl, unsubstituted alkyl (such as unsubstituted C1-C12 alkyl), substituted alkyl (such as substituted C1-C12 alkyl), substituted alkylene (such as substituted Cl -Cl 2 alkylene), unsubstituted alkylene (such as unsubstituted Cl -Cl 2 alkylene), an aryl group, or a heterocyclic group.

In some forms, the linking moiety is or includes polyethylene glycol (PEG). Exemplary PEGs include PEG having a molecular weight of from about 100 DA to about 20,000 Da, inclusive. In some forms, the PEG has a molecular weight of between about 1,000 Da and about 10,000 Da. In some forms, the PEG has a molecular weight of about 5,000 Da.

F. Formulations

Formulations of infected tissue-targeting molecules or conjugates thereof, such as compositions including delivery vehicles and active agents are also provided. The compositions can be formulated for administration to a subject, for example, as a pharmaceutical formulation. Therefore, pharmaceutical formulations including a plurality of functional infected tissue-targeting molecules, each independently having an amino acid sequence of any one or more of SEQ ID NOs: 1-37, or a functional variant thereof are described. Exemplary formulations include a solution, a dry powder, a tablet, micelles, colloids, nanodroplets, nano- structured hydrogel, nanocrystals, and a nanosuspension. Typically, the formulation includes a determined amount of infected tissue binding molecules, in a form appropriate for a desired route of administration. Exemplary formulations of infected tissue-targeting molecules including delivery vehicles and active agents include liquids and dry powders. In some forms, where the delivery vehicle is a nanoparticle, the compositions include infected tissue-targeting molecules in an amount from about 0.1% to about 50%, by weight, inclusive, from about 1% to about 40%, from about 1% to about 50%, preferably from about 1% to about 30% by weight, more

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45652286 preferably from about 1 % to about 20% by weight, most preferably from about 1 % to about 10% by weight. The ranges above are inclusive of all values from 1% to 99%. In some forms, the infected tissue-targeting molecules to be delivered may be encapsulated within and/or associated with the surface of the particle. For those forms where the infected tissue binding peptide is associated with the surface of the particle, the percent loading may be higher since the amount of is not limited by the methods of encapsulation.

1. Liquid Formulations

In some forms, the compositions of infected tissue-targeting molecules or conjugates thereof, e.g., including nanoparticles are formulated as a liquid. Suitable liquid carriers include, but are not limited to, distilled water, de-ionized water, pure or ultrapure water, saline, and other physiologically acceptable aqueous solutions containing salts and/or buffers, such as phosphate buffered saline (PBS), Ringer's solution, and isotonic sodium chloride, or any other aqueous solution acceptable for administration to an animal or human.

Liquid formulations may include one or more suspending agents, such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, gum tragacanth, or lecithin. Liquid formulations may also include one or more preservatives, such as ethyl or n-propyl p- hydroxybenzoate.

Formulations may be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof. Liquid formulations may also contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, "minor amounts" means no excipients are present that might adversely affect the delivery of the nanoparticle compositions to organs or tissues, e.g., through circulation.

2. Dry Powder Formulations and Kit

In some forms, the compositions of infected tissue-targeting molecules or conjugates thereof, e.g., including nanoparticles are formulated in dry powder forms as finely divided solid formulations. The dry powder components can be stored in separate containers or mixed at specific ratios and stored. In some forms, suitable aqueous and organic solvents are included in additional containers. In other forms, dry powder components, one or more solvents, and instructions on procedures to mix and prepare assembled nanostructures are included in a kit. Alternatively, stabilized, assembled particles, nanoparticles or bulk gel thereof are dried via vacuum-drying or freeze-drying,

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45652286 and suitable pharmaceutical liquid carrier can be added to rehydrate and suspend the assembled nanostructures or gel compositions upon use.

Dry powder formulations are typically prepared by blending one or more gelators, stabilizing agents, or active agents with one or more pharmaceutically acceptable carriers. Pharmaceutical carrier may include one or more dispersing agents. The pharmaceutical carrier may also include one or more pH adjusters or buffers. Suitable buffers include organic salts prepared from organic acids and bases, such as sodium citrate or sodium ascorbate. The pharmaceutical carrier may also include one or more salts, such as sodium chloride or potassium chloride. The dry powder formulations can be suspended in the liquid formulations to form nanoparticle solutions, and administered systemically or regionally using methods known in the art for the delivery of liquid formulations.

3. Injectable Formulations

In some forms, the compositions of infected tissue-targeting molecules or conjugates thereof, e.g., including nanoparticles are formulated for parenteral delivery, such as injection or infusion, in the form of a solution or suspension. The formulation is preferably administered into the blood stream or, alternatively, directly to an organ or tissue in a subject. Formulations can be prepared as aqueous compositions using techniques is known in the art. Typically, such compositions can be prepared as injectable or infusible formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection.

In some forms, the compositions of infected tissue-targeting molecules or conjugates thereof are formulated in a suitable carrier. A carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, com oil, sesame oil, etc.), and combinations thereof.

In some forms, the compositions of infected tissue-targeting molecules or conjugates thereof are formulated to contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s). Preservatives can be used to prevent the growth of fungi and microorganisms. Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride,

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45652286 benzyl peroxide, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.

In some forms, the compositions of infected tissue-targeting molecules or conjugates thereof are formulated to be buffered to a pH, for example, pH 2, 3, 4, 5, 6, 7, 8, 9 or pH 10. In an exemplary form, the formulation is typically buffered to a pH of 3-8 for parenteral administration. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

In some forms, the compositions of infected tissue -targeting molecules or conjugates thereof are formulated to include one or more water soluble polymers. Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

In some forms, the compositions of infected tissue-targeting molecules or conjugates thereof are formulated to be a sterile injectable solution. Sterile injectable solutions can be prepared by incorporating the nanoparticles in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized gelators, stabilizing agents, and/or active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Formulations including compositions of infected tissue-targeting molecules or conjugates thereof may be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington - The science and practice of pharmacy,” 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems,” 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

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45652286 G. Exemplary Formulations of Targeted Drug Delivery Particles

An exemplary formulation of infected tissue-targeting molecules or conjugates thereof is a drug-loaded porous silicon particle having infected tissue-targeting molecules attached to the surface and containing one or more antibiotic agents. In exemplary forms, the drug-loaded particles exhibit a temporal and linear drug release profile of an optimized formulation in vivo for an extended period of time are provided. In some forms, the targeted drug-loaded porous silicon particles release the entire amount of loaded drug in vivo over a period of from about two days to about 90 days, or from between about 90 days to about one year, e.g., with an overall estimated release duration in vivo of greater than 6 months. In some forms, the release profile showed a rapid taper at the end of the release. Thus, the performance of the targeted drug delivery formulations is superior to “free” drug. Preferably, the targeted drug delivery formulations have reproducible syringability and exhibit a low toxicity in vivo.

In exemplary forms, the targeted drug delivery formulations include a porous silicon particle loaded with a meltable composition, whereby the meltable composition includes a therapeutic agent and a melting point suppression agent; whereby the meltable composition has a melting temperature; whereby the therapeutic agent has a melting temperature and a decomposition temperature; and whereby the melting temperature of the meltable composition is lower than the melting temperature and the decomposition temperature of the therapeutic agent.

In an exemplary form, the porous silicon nanoparticles are loaded with vancomycin. In an exemplary method, vancomycin is loaded and trapped in the pSiNPs. Briefly, an aqueous dispersion of pSiNPs (1 mg ml-1 in deionized water) is added to an aqueous solution of vancomycin (0.5 mg ml-1) in a 1 :1 (v:v) ratio, and ultrasonicated for 10 min. An equal volume of aqueous calcium chloride solution (2 M) is then added to the mixture, which is agitated (< 5 min) and ultrasonicated in an ice bath for 2 h. Drug-loaded nanoparticles are purified by centrifugation and washing steps with deionized water, 70% ethanol and 100% ethanol, sequentially. To analyze the vancomycin loading capacity, the supernatants from centrifugation were collected and assayed for free vancomycin by measuring absorbance at 280 nm. The mass loading is verified by spectrophotometric quantification (/.max = 280 nm) of vancomycin released from the nanoparticles.

In an exemplary form, the infected tissue-targeting molecule including the amino acid sequence of any one of PPRRGLIKLKTS (SEQ ID NO: 1), AGRGRLVR (SEQ ID NO:2) and GRPARPAR (SEQ ID NO:3), or a functional variant thereof having at least

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45652286 80% sequence identity to any one of SEQ ID NOs: l-37, is coupled to the vancomycin- loaded porous silicon nanoparticles. In an exemplary method, aliquots (1 ml) of pSiNPs (1 mg ml-1 in ethanol) are treated with 20 p 1 of 3-(ethoxydimethyl)-propylamine silane by vortexing overnight at room temperature. The amine-terminated nanoparticles are rinsed with ethanol and water, and then further functionalized by either MAL-PEG-SCM (maleimidepolyethylene glycol-succinimidyl carboxy methyl ester, MW 5000) or mPEG- SCM (methoxy PEG-succinimidyl carboxy methyl ester, MW 5000). The maleimide activated nanoparticles are then mixed with the peptide solution (0.5 mg ml-1 in water, 1 ml) and vortexed for 2 h to conjugate the peptide via the free cysteine residue at the terminal group of the peptide.

In some forms, peptide coupling is quantified and confirmed by measuring the fluorescence of a FAM label of the peptide. Characterization of the porous silicon nanoparticles is carried out by detecting the hydrodynamic size of the nanoparticles as measured by dynamic light scattering (DLS, e.g., Zetasizer ZS90, Malvern Instruments). Transmission electron microscopy (TEM) images are obtained with a JEOL-1200 EX II. Photoluminescence spectra (lex = 365 nm) were monitored using a spectrometer (QE Pro, Ocean Optics) with a 460 nm long-pass filter. Quantification of vancomycin is conducted by measuring absorbance at 280 nm with a spectrophotometer (NanoDrop 2000, Thermo Scientific) based on the standard curve of vancomycin.

III. Methods of Use

Methods of using infected tissue targeting peptides or conjugates and compositions thereof are provided. Typically, the methods administer infected tissue targeting peptides or conjugates thereof associated with one or more delivery vehicles including a therapeutic antimicrobial agent to reduce or remove a pathogenic infection, such as an infection of Mycobacterium tuberculosis in a human subject.

It has been established that infected tissue targeting peptides are useful in improving the treatment of antibiotic resistant infections. Such infections are typically treated with “last-resort” antibiotics that are typically toxic to the recipient. The improvement in efficacy made possible by the described infected tissue targeting peptides reduces the unwanted side effects of such antibiotics. Alternatively, the increase in efficacy may make it possible to overcome partial resistance of bacteria to an antibiotic.

Methods of using infected tissue targeting peptides combine targeting with the use of nanoparticles as a delivery vector for potentially toxic antibiotics to enhance specificity of drug delivery, thereby reducing dosages and side effects. The described infected tissue

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45652286 binding molecules have a property that may make them particularly effective in targeting infections; they not only bind to the bacteria in the infected area, but also accumulate in places, where there do not appear to be any intact bacteria. It may be that the described infected tissue binding molecules bind to a bacterial component that is released from disintegrated bacteria or shed from intact ones and that binds to tissue in the infected area. This property increases the amount of antibiotic that can be brought to the site of infection.

In some forms, the methods treat diseases and disorders associated with microbial infections in a subject.

Methods of treatment for an infection by a microbial pathogen in a subject include administering to the subject a pharmaceutical formulation including

(a) a composition including an infected tissue-targeting molecule having an amino acid sequence of any of SEQ ID NOs:l-37, or a functional variant thereof;

(b) a nanoparticle;

(c) an antimicrobial agent; and

(d) a pharmaceutically acceptable excipient for administration in vivo, wherein the composition is in an amount effective to kill or otherwise mitigate the microbial pathogen in the subject.

In some forms, compositions of infected tissue targeting peptides are provided for use in treating or preventing one or more symptoms of an infection by a microbial pathogenic in a subject in need thereof, for example, including administering to the subject a composition including:

(a) an infected tissue targeting peptide having an amino acid sequence of any of SEQ ID NOs:l-37, or a functional variant thereof;

(b) a nanoparticle;

(c) an antimicrobial agent; and

(d) a pharmaceutically acceptable excipient for administration in vivo, wherein the composition is in an amount effective to kill or otherwise mitigate the microbial pathogenic Mycobacterium sp. in the subject.

A. Treating Diseases and Disorders

Methods of treating or preventing infections, such as bacterial infections in a subject are provided.

The methods typically include administering to the subject an effective amount of a pharmaceutical formulation of nanoparticles including antibiotics and having one or more infected tissue-binding peptides each independently having an amino acid sequence of any

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45652286 of SEQ ID NOs:l-37, or a functional variant thereof attached thereto as a targeting agent, to treat or prevent microbial infection, such as an infection with Mycobacterium tuberculosis in the subject.

In preferred forms the methods treat or prevent one or more diseases or disorders associated with pathogenic bacterial infection. An exemplary pathogenic bacterial infection is a Mycobacterium tuberculosis infection. In some forms, the methods administer pharmaceutical formulations including the infected tissue binding molecules to a subject in need thereof via a route selected from oral administration, intramuscular injection, intravenous injection, sub-cutaneous injection, and intra-articular injection. In some forms the formulation is administered in an amount effective to reduce a microbial pathogen, such as Mycobacterium tuberculosis in the blood of the recipient to a sub- infectious amount, or amount less than or equal to between 1 and 10 cells of viable microbial pathogen per dL in blood. In some forms the formulation is administered in an amount effective to reduce Mycobacterium sp., in the blood of the recipient to a sub- infectious amount, or amount less than or equal to between 1 and 10 cells of viable Mycobacterium sp. cells per dL in blood. For example, in some forms, the compositions and methods typically treat or prevent infection of a subject by one or more pathogenic species of microorganisms. In an exemplary form, the methods enhance the efficacy of antimicrobial agents such as antibiotics for the treatment of an infection with a pathogenic microorganism by targeting the antibiotic directly to the site of the infection following systemic administration of the composition to the subject.

1. Infectious Diseases to be Treated

In general, the described compositions and methods of treatment thereof are useful in the context of treating and preventing infection with a microbial pathogen, and/or latent or active infectious diseases in a subject. For example, in some forms, the compositions and methods treat or prevent infection of a subject by one or more pathogenic strains of Mycobacterium sp. In exemplary forms, the compositions and methods treat or prevent infection of a subject by one or more pathogenic strains of Mycobacterium tuberculosis.

In some embodiments, the methods treat or prevent an infection by a microbial pathogen that is a viral, fungal, bacterial or other microbial pathogen.

Exemplary viral pathogens include any virus including, but not limited to, a virus from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus,

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45652286 Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (EBOV) (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., Human herpesvirus 1, 3, 4, 5, and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenza virus A, such as H1N1 strain, and B and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (for example, rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (for example, rubella virus, dengue virus, etc.), and Totiviridae. Suitable viral antigens also include all or part of Dengue protein M, Dengue protein E, Dengue D INS 1 , Dengue D1NS2, and Dengue D1NS3. Viral antigens may be derived from a particular strain such as a papilloma virus, a herpes virus, i.e. herpes simplex 1 and 2; a hepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-borne encephalitis viruses; parainfluenza, varicella-zoster, cytomegalovirus, Epstein- Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomeningitis, viruses from the Orthomyxovirus family, for example, the Influenza virus A (e.g., Influenza A virus subtypes including H1N 1, H1N2, H3N2, H3N1, H5N1, H2N2, and H7N7), Influenza virus B, Influenza virus C, Isavirus, Thogoto virus and Quaranjavirus, Ebolavirus, for example, the Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Tai Forest ebolavirus (TAFV), Reston ebolavirus (RESTV), and Bundibugyo ebolavirus (BDBV) and Flavivirus, for example, the Zika virus (ZIKV).

In some forms, the pathogen is a bacterial pathogen. Exemplary bacterial pathogens can be any pathogenic bacteria including, but not limited to bacterial strains of Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Helicobacter, Haemophilus, Haemophilus influenza type B (HIB), Hyphomicrobium,

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45652286 Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Mycobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, and Yersinia.

In other forms, the microbial pathogen is a protozoan pathogen. Exemplary protozoan pathogens include but are not limited to, Cryptococcus neoformans, Histoplasma capsulation, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.

In some forms, the microbial pathogen is an antibiotic-resistant microbial pathogen. Antimicrobial resistance (AMR) is a significant problem in some clinical strains. AMR can occur by (a) acquisition of resistance genes via horizontal gene transfer; or (b) mutations in genes already present in the genome.

In some forms the antibiotic-resistant strain of pathogen is a multi-drug resistant (MDR) pathogen, responsible for multi-drug resistant disease, as well as extensively drug- resistant (XDR) strains. In some forms, the described infected tissue-binding molecules can effectively deliver drugs to treat an infection by an AMR pathogen that is resistant to one or more classes of antibiotics and therapeutic agents in a subject in need thereof. In some forms, the described compositions including the described infected tissue-binding molecules can effectively deliver a therapeutic agent in an amount effective to treat an infection by an AMR pathogen that is resistant to treatment with the same amount of the same therapeutic agent in the absence of the infected tissue homing molecules in a subject in need thereof. a. Infection by Mycobacterium sp.

In some forms the methods treat or prevent one or more symptoms of infection with Mycobacterium Sp.

In some forms the methods treat or prevent one or more symptoms of infection with one or more Mycobacterium selected from M. tuberculosis, M. leprae, M. avium, M. ulcerans, M. xenopi, M. kansasii, M. abscessus, M. chelonae, M. fortuitum, M. africanum, M. canetti, M. bovis, M. caprae, M. microti, M. pinnipedii, M. mungi, and M. orygis.

In exemplary forms the methods treat or prevent one or more symptoms of infection with Mycobacterium tuberculosis, and/or Mycobacterium leprae in a subject in

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45652286 need thereof. For example, in some forms the methods treat or prevent one or more symptoms of tuberculosis and/or leprosy in a subject in need thereof.

In other forms, the methods treat or prevent treat or prevent one or more symptoms of infection with a non-tuberculosis strain of mycobacterium (“NTM”; also referred to as atypical mycobacteria, mycobacteria other than tuberculosis (MOTT), or environmental mycobacteria).

NTM are a large diverse cause of opportunistic lung disease that are hard to treat. NTM are common in the US where there is higher potential for more expensive treatment options. The most common type of NTM bacteria in the U.S. is Mycobacterium avium complex (MAC). The great majority of NTM lung disease in the U.S. is caused by MAC.

Therefore, in some forms the methods treat or prevent one or more symptoms of Mycobacterium avium complex (MAC) in a subject in need thereof.

Two of the other more common NTM species that infect the lungs are M. abscessus and M. kansasii. Each type of NTM affects the body differently. The severity of disease, how it is treated, and the likelihood of recovery can vary widely from person to person. Some of the factors that impact the course of someone’s NTM lung disease are the virulence of the organism, the amount of exposure they’ve gotten, and their overall health. Therefore, in some forms the methods treat or prevent one or more symptoms of infection with M. abscessus and/or M. kansasii. in a subject in need thereof.

Mycobacterium tuberculosis(Mtb) bacteria usually attack the lungs, but TB bacteria can attack any part of the body such as the kidney, spine, and brain. Not everyone infected with TB bacteria becomes sick. For example, in some forms, the methods enhance the efficacy of antimicrobial agents such as antibiotics for the treatment of an infection with a pathogenic Mycobacterium tuberculosis by targeting the antibiotic directly to the site of Mycobacterium following systemic administration of the composition to the subject.

The compositions can also be used for treatment and/or prevention of other diseases and disorders including those associated with bacterial infection, such as secondary infections. For example, in some forms, the methods treat and/or prevent a secondary infection by one or more of a virus, a bacteria, a fungus or a protozoan.

In some forms, the subject to be treated is a human. All the methods described can include the step of identifying and selecting a subject in need of treatment, or a subject who would benefit from administration with the described compositions.

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45652286 Primary Mtb infection is spread by air droplets from a cough or sneeze from a person infected with the bacterium and can affect anyone, but the clinical disease is limited except in people with weakened immune systems, as well as infants and the elderly. Most people recover from primary Mtb infection, but the infection stays inactive (latent, dormant) for years.

Secondary TB is the reactivation of a dormant infection with Mtb, usually but not always in a person with a weakened immune system. i. M. tuberculosis infection

The methods treat or prevent an infection of Mycobacterium tuberculosis (Mtb) in a subject in need thereof.

Mtb, also known as Koch's bacillus, is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. First discovered in 1882 by Robert Koch, M. tuberculosis has an unusual, waxy coating on its cell surface primarily due to the presence of mycolic acid. The physiology of Mtb is highly aerobic and requires high levels of oxygen. Primarily a pathogen of the mammalian respiratory system, it infects the lungs. The most frequently used diagnostic methods for tuberculosis are the tuberculin skin test, acid-fast stain, culture, and polymerase chain reaction. Mtb requires oxygen to grow, and is nonmotile. Mtb divides every 18-24 hours, which is extremely slow compared with other bacteria, which tend to have division times measured in minutes (Escherichia coli can divide roughly every 20 minutes). Mtb is a small bacillus that can withstand weak disinfectants and can survive in a dry state for weeks. Its unusual cell wall is rich in lipids such as mycolic acid and cord factor glycolipid, is likely responsible for its resistance to desiccation and is a key virulence factor.

Mtb is part of a genetically related complex group of Mycobacterium species, known as Mycobacterium tuberculosis complex, including: M. tuberculosis sensu stricto; M. africanum M. canetti; M. bovis M. caprae; M. microti; M. pinnipedii; M. mungi; and M. orygis.

In some forms, the methods treat or prevent an infection by Mycobacterium tuberculosis complex in a subject. Therefore, the methods treat and prevent infection by any one or more of M. tuberculosis sensu stricto; M. africanum; M. canetti; M. bovis; M. caprae; M. microti; M. pinnipedii; M. mungi; and M. orygis in a subject.

The syndemic of human immunodeficiency virus (HIV)/tuberculosis co-infection has grown as a result of the considerable sociogeographic overlaps between the two

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45652286 epidemics. TB is the leading killer of HIV-positive people, causing 25% of all HIV-related deaths. ii. Antibiotic-Resistant M. tuberculosis infection

In some forms, the pathogenic Mtb is an antibiotic-resistant strain of Mtb. Antimicrobial resistance (AMR) is a significant problem in some clinical strains. AMR in M. tuberculosis typically occurs by mutations in genes already present in the genome.

In some forms the antibiotic-resistant strain of Mtb is a multi-drug resistant Mtb (MDR-Mtb), responsible for multi-drug resistant disease (MDR-TB), as well as extensively drug -resistant (XDR-) TB.

Globally in 2013, an estimated 480,000 people developed MDR-TB, accounting for around 5% of all estimated TB cases. Among 1278 patients consecutive adults with locally confirmed pulmonary MDR tuberculosis started on second-line drug treatment in eight countries (Estonia, Latvia, Peru, Philippines, Russia, South Africa, South Korea, and Thailand), 43-7% showed resistance to at least one second-line drug, 20-0% to at least one second-line injectable drug, 12-9% to at least one fluoroquinolone, 6-7% (range 0-8- 15-2% ) of patients had XDR tuberculosis, and previous treatment with second-line drugs was consistently the strongest risk factor for resistance to second-line drugs. Notwithstanding that, most cases of MDR-TB and XDR-TB resulted from primary transmission in some countries with high TB drug resistance burden.

Therefore, in some forms, the pathogenic M. tuberculosis is resistant to one or more types of antibiotics. In some forms, the pathogenic M. tuberculosis is a multi-drug resistant (MDR) strain, that is resistant to multiple types of antibiotics, or an extensively drug resistant (XDR) strain, that is resistant to all types of antibiotics.

AMR Mtb that are resistant to many classes of antibiotics and therapeutic agents are problematic during infection as it can be difficult to treat. iii. Symptoms of Tuberculosis

In some forms, the methods treat or prevent one or more symptoms of Tuberculosis (TB). For example, in some forms, the methods treat or prevent one or more symptoms of pulmonary TB, CNS TB, pericardial TB, pleural TB, Lymphadenitis, Abdominal TB, skeletal TB, genitourinary TB or miliary TB in a subject in need thereof.

Tuberculosis can develop through progression of recently acquired infection (primary disease), reactivation of latent infection, or exogenous reinfection. In immunocompetent individuals, approximately 3-10% of those with tuberculous infection

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45652286 will develop tuberculosis in the first 1-2 years after infection and another 5% will develop tuberculosis during their lifetime.

Therefore, in some forms the methods treat or prevent active TB in a subject in need thereof. In other form, the methods treat or prevent latent TB in a subject in need thereof.

Most tuberculosis occurs as pulmonary disease with 17% occurring at an extrapulmonary site, only. However, as much as 70% of HIV- 1 infected patients will have evidence of extrapulmonary disease or mycobacteremia once the CD4 count is below 100 cells pl/ml. Co-infected persons are more likely to present atypically, potentially delaying the diagnosis of tuberculosis. iv. Pulmonary Tuberculosis

In some forms the methods treat or prevent one or more symptoms of pulmonary TB in a subject in need thereof. Symptoms of active pulmonary TB may occur during the primary infection (and they may be relatively mild in an otherwise healthy person) or a secondary reactivation of MTB infection. These typically include: Chest pain, Wheezing or difficulty breathing, Cough (usually with mucus, sometimes with blood), Fever, sometimes with night sweats, Fatigue, and unintentional weight loss. Therefore, in some forms, the methods treat or prevent one or more symptom including Chest pain, Wheezing or difficulty breathing, Cough (usually with mucus, sometimes with blood), Fever, sometimes with night sweats, Fatigue, and unintentional weight loss in a subject having pulmonary TB. v. Central Nervous System (CNS) TB

In some forms the methods treat or prevent one or more symptoms of CNS TB in a subject in need thereof. Tuberculosis of the central nervous system can present as tuberculous meningitis, tuberculomas, or tuberculous spinal meningitis. The clinical spectrum of tuberculous meningitis ranges from chronic headache and subtle mental status changes to sudden, severe meningitis progressing to coma. A prodrome of malaise, intermittent headache, and low grade fever can be followed by protracted headache, vomiting, confusion, meningismus, and focal neurologic signs within 2 to 3 weeks. If untreated stupor, coma, seizures, and hemiparesis and death can occur within five to eight weeks after the onset of illness. Fever is not always present, and the peripheral white blood cell count is usually normal. Patients may have mild anemia or hyponatremia due to inappropriate antidiuretic hormone secretion.

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45652286 Therefore, in some forms, the methods treat or prevent one or more symptom including malaise, intermittent headache, fever, protracted headache, vomiting, confusion, meningismus, stupor, coma, seizures, and hemiparesis in a subject having CNS TB.

In some forms, the methods treat or prevent one or more symptom of TB meningitis. Tuberculomas are space-occupying lesions in the brain. They are usually multiple but can be single. Patients may present with seizures or other focal neurologic symptoms without evidence of systemic illness or meningeal inflammation. The meninges can become involved with encasement of the spinal cord by a gelatinous or fibrous exudates in advanced cases. Patients may have bladder or rectal sphincter weakness, hypesthesia, anesthesia, paresthesias in the distribution of a nerve root, or paralysis and pain resulting from nerve root or cord compression. Therefore, in some forms, the methods treat or prevent one or more symptom including bladder or rectal sphincter weakness, hypesthesia, anesthesia, paresthesias in the distribution of a nerve root, or paralysis and pain resulting from nerve root or cord compression in a subject having TB meningitis. vi. Pleural Tuberculosis

In some forms the methods treat or prevent one or more symptoms of Tuberculous pleurisy in a subject in need thereof. Tuberculous pleurisy can occur within weeks to months after primary infection (early postprimary pleurisy), complicate chronic pulmonary tuberculosis, or develop concurrently in 10%-30% of cases with military tuberculosis. Early postprimary pleurisy usually affects adolescents and young adults. The effusion can resolve within several months in as many as 90% of cases; however, without treatment, 65% will relapse with chronic organ tuberculosis within 5 years. Elderly patients with chronic pulmonary tuberculosis may have cirrhosis or congestive heart failure; so tuberculous pleurisy may be easily mistakenly attributed to underlying comorbidities. The clinical course of tuberculous pleurisy may be low grade and subtle or abrupt and severe and can be confused with acute bacterial pneumonia. Patients usually have cough, pleuritic chest pain, and occasionally high fever. Night sweats, chills, weakness, dyspnea, and weight loss can also occur. The effusion is usually minimal to moderate in volume and almost always unilateral (unless military tuberculosis exists concurrently). Empyema can occur when a cavity ruptures into the pleural space; empyema is associated with bronchopleural fistula formation and frank pus. It is rapidly fatal if antituberculosis therapy is not given expeditiously.

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45652286 Therefore, in some forms, the methods treat or prevent one or more symptom including cough, pleuritic chest pain, and occasionally high fever. Night sweats, chills, weakness, dyspnea, and weight loss in a subject infected with Mtb. vii. Lymphadenitis

In some forms the methods treat or prevent one or more symptoms of Lymphadenitis in a subject in need thereof. Lymphadenitis is the most common form of extrapulmonary tuberculosis. In HIV-negative persons, it is usually unilateral and located in the cervical or supraclavicular area. The most common site is the upper border of the sternocleidomastoid muscle. Patients usually present with a painless, red, firm mass without systemic symptoms. It is most often seen in young adult females. Children often have an ongoing primary infection, but other age groups seldom have concurrent extranodal tuberculosis. Mediastinal adenopathy is often seen in children with primary infection, but it is uncommon in young adults and elderly persons. Differential diagnosis of mediastinal adenopathy includes histoplasmosis, lymphoma, and cardinoma. Less commonly, tuberculosis can also cause fibrosing mediastinitis, and patients can present with dyspnea on exertion due to compression of pulmonary veins and arteries or superior vena cava syndrome. In individuals with AIDS, peripheral lymph node tuberculosis is always multifocal and associated with systemic symptoms, such as fever and weight loss. Mediastinal lymphadenopathy is frequent, and CT scan reveals multiple coalescing mediastinal masses with low-density centers, peripheral contrast enhancement, and no calcification. Abdominal lymphadenopathy in the intra- abdominal cavity is also common in AIDS patients. Lymph nodes can obstruct the biliary tract, ureters, or bowel. Abscesses in the liver, spleen, pancreas, or kidney can exist concurrently. Therefore, in some forms the methods treat or prevent obstruction of the biliary tract, ureters, or bowel, and/or formation of abscesses in the liver, spleen, pancreas, or kidney in a subject with an Mtb infection, optionally also having HIV/AIDS. viii. Pericardial Tuberculosis

In some forms the methods treat or prevent one or more symptoms of Pericardial tuberculosis in a subject in need thereof.

Pericardial tuberculosis is usually caused by extension from a contiguous focus of infection, such as mediastinal or hilar nodes, the lung, spine, or sternum. Dissemination to the pericardium can occur with military tuberculosis. The onset may be abrupt or insidious. Patients may present with dyspnea, orthopnea, dull retrosternal pain, a pericardial friction rub, or symptoms and signs of cardiac tamponade. Fever, weight loss

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45652286 and night sweats usually occur before cardiopulmonary complaints. A few patients present with findings of chronic constrictive pericarditis. A pleural effusion can be found in as many as 39% of cases with pericardial tuberculosis, and radiographic evidence of concurrent pulmonary tuberculosis in 32%-72% of cases.

Therefore, in some forms the methods treat or prevent one or more of dyspnea, orthopnea, dull retrosternal pain, a pericardial friction rub, or symptoms and signs of cardiac tamponade, fever, weight loss and night sweats in a subject having Pericardial tuberculosis. ix. Bone and Joint Tuberculosis

In some forms the methods treat or prevent one or more symptoms of Bone and Joint tuberculosis in a subject in need thereof.

One third of cases with skeletal tuberculosis involve the spine (Pott’s disease or tuberculous spondylitis). The disease can occur via hematogenous, contiguous, or lymphatic spread. In the early stage, only back pain or stiffness is present. Diagnosis may be delayed with the sequelae of paralysis, deformity, or sinus formation. The earliest manifestation of tuberculous arthritis is pain, and the symptom may precede signs of inflammation and radiographic changes by weeks or months. Tenosynovitis of the hand, arthritis of the wrist, and carpal tunnel syndrome can also be caused by tuberculosis. Tuberculous osteomyelitis can affect all bones including the ribs, skull, phalanx, pelvis, and long bones. Tuberculosis is the most common infectious cause for single of multiple osteomyelitic rib lesions since other causes of osteomyelitis of the rib are rare.

Therefore, in some forms the methods treat or prevent one or more of tuberculous arthritis orthopnea, tuberculous osteomyelitis, back pain or stiffness, paralysis, deformity, tenosynovitis of the hand, arthritis of the wrist, and carpal tunnel syndrome in a subject having skeletal tuberculosis. x. Disseminated (Miliary) Tuberculosis

In some forms the methods treat or prevent one or more symptoms of miliary tuberculosis in a subject in need thereof.

The term, miliary tuberculosis, initially was used to describe the resemblance of the pathologic lesions to millet seeds. Now this term denotes any progressive disseminated tuberculosis spread hematogenously. It can be divided into acute miliary tuberculosis, cryptic miliary tuberculosis, and nonreactive tuberculosis.

Acute miliary tuberculosis is associated with a brisk and histologically typical tissue reaction. Children have acute or subacute onset, high intermittent fevers, night

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45652286 sweats, and occasional rigors. Pleural effusion, peritonitis, or meningitis occurs in as many as two thirds of cases. The clinical course of young adults is usually more chronic and initially less severe. However, now older individuals are affected by miliary tuberculosis more frequently, and their underlying illnesses may obscure the diagnosis. Patients usually have nonspecific constitutional symptoms, such as fever, anorexia, weakness, and weight loss. They may have headache due to meningitis, abdominal pain resulting from peritonitis, or pleural pain caused by pleuritis. Patients may have normal white cell count, anemia, hyponatremia, elevation of alkaline phosphatase and transaminases. Fulminant miliary tuberculosis may be associated with severe refractory hypoxemia (adult respiratory distress syndrome) and disseminated intravascular coagulation.

Cryptic miliary tuberculosis occurs in older patients with miliary tuberculosis; chest X-rays are normal and tuberculin test results are negative. Patients have a chronic clinical course characterized by mild intermittent fever, anemia, and, ultimately, meningeal involvement preceding death.

Nonreactive tuberculosis is very rare and characterized by massive hematogenous dissemination of tubercle bacilli, "non-granulomatous" ("nonreactive") tissue lesions, and often a septic presentation. Patients present with overwhelming sepsis, splenomegaly and subtle diffuse mottling on the chest X-ray. Hematologic abnormalities include leukopenia, thrombocytopenia, anemia, pancytopenia, leukemoid reactions, myelofibrosis, or polycythemia. Disseminated tuberculosis should be considered when pancytopenia is associated with fever and weight loss.

Miliary tuberculosis developed in 10% of AIDS patients with tuberculosis and 38% of AIDS patients with extrapulmonary tuberculosis. Patients can also have abscesses of various soft tissue and organs, including the liver, spleen, pancreas, psoas muscle, mediastinum, neck, chest wall, abdominal wall, and prostate.

Therefore, in some forms the methods treat or prevent one or more symptoms of acute miliary tuberculosis, cryptic miliary tuberculosis, or nonreactive tuberculosis in a subject having miliary tuberculosis. xi. Genitourinary Tuberculosis

In some forms the methods treat or prevent one or more symptoms of Genitourinary tuberculosis in a subject in need thereof.

Genitourinary tuberculosis is mostly a disease of middle-aged adults, and the onset is usually insidious. Asymptomatic renal cortical foci may occur in all forms of tuberculosis. In normal hosts, the interval between infection and active renal disease is

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45652286 usually years and sometimes decades. Genitourinary symptoms of dysuria and gross hematuria are most common. Constitutional symptoms and Pyuria, albuminuria, and hematuria were the most common laboratory abnormalities. Renal tuberculosis may spread to the prostate, seminal vesicles, epididymis, and testis in that order. The usual clinical manifestations are a tender scrotal mass associated with a draining sinus or oligospermia unresponsive to treatment. Genital foci can also result from lymphatic or hematogenous spread and present as a painful testicular or scrotal mass. The presence of epididymal or prostatic calcification suggests the diagnosis, but nontuberculous chronic prostatitis may also have a similar presentation. Female genital tuberculosis starts with a hematogenous focus in the endosalpinx, and may spread to the endometrium, ovaries, cervix, and vagina. A granulomatous ulcerating mass in the cervix may resemble carcinoma. Abdominal pain, menstrual disorders, or infertility are common complaints. Patients may present with the pictures of pelvic inflammatory disease with unresponsiveness to therapy. It is uncommon for patients to have systemic symptoms, and signs of old tuberculosis are often absent. Pregnancies are often ectopic in the presence of pelvic tuberculosis. xii. Abdominal Tuberculosis

In some forms the methods treat or prevent one or more symptoms of abdominal tuberculosis in a subject in need thereof.

Abdominal tuberculosis can affect the gastrointestinal tract, the peritoneum, the liver, and the pancreas. Tuberculosis can involve any gastrointestinal site from the oropharynx to the anus. Patients can present with nonhealing ulcers of the tongue or oropharynx, or nonhealing sockets after tooth extraction. An adjacent caseous node might result in esophageal stricture with obstruction, tracheoesophageal fistula formation, and rare fatal hematemesis from an aortoesophageal fistula. Patients might have ulcerative or hyperplastic lesions in the stomach or gastric outlet obstruction. Duodenal involvement may lead to symptoms of peptic ulcer or obstruction. Perforation, obstruction, enteroenteric and enterocutaneous fistula, massive hemorrhage, and severe malabsorption may follow small bowel involvement. The most typical site of enteric tuberculosis is the ileocecal area producing symptoms of pain, anorexia, diarrhea, obstruction, hemorrhage, and a palpable mass. Patients with anal tuberculosis might have ulcers, perianal warty growths, and fistulas. Tuberculous peritonitis results from either spread of adjacent tuberculous disease or military tuberculosis. The clinical picture has two types: plastic and serous. The plastic type is less common; characterized by tender abdominal masses and a "doughy abdomen". The serous type presents with ascites often without signs of

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45652286 peritonitis. Patients usually have symptoms of fever, abdominal pain, and weight loss. The onset may be insidious or acute. Some cases were diagnosed at routine hernia repair, or during surgery for an unknown mass or an acute abdomen. Tuberculous peritonitis was often undiagnosed in patients having cirrhosis with ascites. Peritoneal dialysis patients may present with the clinical pictures of bacterial peritonitis unresponsive to routine antibiotics.

Tuberculosis is a frequent cause of granulomatous hepatitis with elevated alkaline phosphatase and gamma-glutamyl transpeptidase levels that are out of proportion to bilirubin levels with normal or mildly elevated transaminase levels. Very rarely, tuberculosis granulomatous hepatitis causes jaundice without evidence of extrahepatic tuberculosis (primary tuberculosis of the liver). Focal hepatic tuberculosis describes single or multiple tuberculous abscesses occurring in patients with little natural immunity to tuberculosis and in children. Pancreatic tuberculosis may present with an abscess or a mass involving local nodes and resembling carcinoma. Abdominal lymph nodes may obstruct the biliary tract causing tuberculous ascending cholangitis. b. Infectious Diseases leading to Granulomas

In some forms, the described compositions and methods treat or prevent formation of granulomas in a subject in need thereof.

Granulomas form in response to chronic inflammation. Accordingly, the most common cause of granulomas are infections. Caseating granulomas are formed by infections, such as tuberculosis and fungal infections. Noncaseating granulomas may be formed by an inflammatory condition (e.g., sarcoidosis and Crohn disease), vasculitis, and exposure to foreign objects.

Formation of granulomas is characteristic of certain diseases. Most commonly, in chronic granulomatous disease (CGD), an inherited genetic mutation reduces the ability of white blood cells to kill certain bacteria and fungi, like Staphylococcus aureus and Aspergillus. Individuals with CGD are highly susceptible to infections that lead to granuloma development throughout the body. Similarly, granuloma annulare is a chronic skin disorder characterized by granulomas appearing as small red or yellow bumps in a ring shape on the skin. Finally, granulomatosis with polyangiitis (GPA) is a rare autoimmune-induced vasculitis characterized by granuloma formation, causing inflammation of the blood vessels (primarily small-sized arteries) and, ultimately, affecting blood flow.

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45652286 Two factors contribute to the pathogenesis of the granulomatous process, including when the inducer agent is a pathogen/invader or foreign body the intrinsic toxicity of the agents can damage the tissues; and when the vigorous immune-inflammatory T cell- mediated response evoked by the pathogen recruits macrophages and other cells that during the activated stage and phagocytosis secrete tissue-damaging substances. Examples for the former are found in M. tuberculosis that has a waxy lipid cell wall constituent the cord factor (trehalose 6, 6’-dimycolate) which regulates in vitro the rope-like growth of the bacteria. An additional component is muramyl dipeptide that is also granulomagenic. Both components trigger the innate immune response with cytokine (TNFa, IL- la, IL-6, IL- 10, IFNy and chemokine CCL2 production. Mycobacterium leprae the causative organism of leprosy lives intracellularly in the skin, nasal mucosa and Schwann cells of the peripheral nerves. As a consequence, there is gross thickening of the facial skin, hypopigmentation and loss of sensation to heat, cold and pain. The Gram -ve bacillus Brucella rapidly multiplies in the lymph node and causes probably by the Lipid A component of its endotoxin destruction of the lymphoreticular organs. When it colonizes the heart tissue it can cause fatal endocarditis. The Gram+ve Listeria secretes the exotoxin Listeriolysin O, a P hemolysin that destroys red cells, neutrophils and monocytes. The fungus Histoplasma has tropism for the mucous membrane in the mouth where it causes lesions. All the quoted pathogens can sustain their intra-macrophage survival by subverting the killing machinery of the cells (arrest of the phagolysosomal fusion, disruption of signaling pathways) thereby assuring the chronicity of the infection. It should be pointed out that because of the intimate relationship between host and invading pathogen it is difficult to clearly isolate the pathogen or host-derived factors in the pathogenesis of the diseases. Therefore, methods of using compositions of the described infected tissue targeting peptides to deliver one or more active agents, such as therapeutic agents, to the site of a granuloma in an amount effective to treat or prevent one or more symptoms of the granuloma in a subject are provided.

2. Subjects

All the methods described can also include the step of identifying and selecting a subject in need of treatment, or a subject who would benefit from administration with the described compositions. Tuberculosis (TB) is a chronic disease of animals caused by a Mycobacterium sp. For example, Mycobacterium bovis can infect practically all mammals, causing a general state of illness, coughing and eventual death. Therefore, in some forms, the subject is a fish, a mammal or an avian. In some forms, the subject is a shellfish. In

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45652286 some forms, the subject is a poultry bird, such as a chicken, a turkey, a duck or other fowl raised for human consumption. In some forms, the subject is a farm animal, such as a cow, pig, sheep, goat or rabbit. In preferred forms, the subject is a primate, such as a human. Therefore, in some forms, the subject is a human patient. hi some forms, the subject has been medically diagnosed as having an infection with a microorganism, or a disease or disorder associated with an infection with a microorganism by exhibiting clinical (e.g., physical) symptoms of a disease. For example, in some forms, the methods administer the compositions including infected tissuetargeting molecules to a subject having one or more symptoms of an infection, disease or disorder associated with Mtb.

In immunocompetent individuals, approximately 3-10% of those with tuberculous infection will develop tuberculosis in the first 1-2 years after infection and another 5% will develop tuberculosis during their lifetime. The risk of Mtb infection is modified by the age of acquiring infection (e.g. being lowest in the age range of 5 to 9 years) and many other host factors. Exogenous reinfection is thought to be uncommon in immunocompetent persons residing in areas with a low prevalence of tuberculosis, but life-style- related factors and chronic diseases, such as active or passive smoking, nutritional status and diabetes mellitus may significantly affect the risk of reactivation of endogenous infection. In the setting of HIV-1 infection, the risk of progressing rapidly to infection once infected with M. tuberculosis, the risk of reactivation, and the risk of exogenous reinfection are all increased compared to HIV-1 seronegative persons.

Therefore, in some forms, the subject is identified as having an increased risk of an infection with Mtb, and/or developing a disease associated with Mtb. For example, in some forms, the subject has been medically diagnosed as having a latent Mtb infection, but has no symptoms. In other forms, the subject has a predisposition to Mtb infection, or to be at risk of a disease or disorder associated with Mtb infection by exhibiting clinical (e.g., physical) symptoms, which are indicative of an increased risk or likelihood of developing Mtb infection, or a disease or disorder associated with Mtb infection. In some forms, the subject is an immunocompromised subject, or subject having one or more factors associated with increased risk of Mtb infection or active tuberculosis, such as active or passive smoking, having poor or minimal nutritional status, and/or having diabetes mellitus.

Therefore, in some forms, the methods administer pharmaceutical formulations including the infected tissue-targeting molecules or conjugates thereof to a subject prior to

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45652286 a clinical diagnosis of bacterial infection, or a disease or disorder associated with bacterial infection. In preferred forms, the methods administer pharmaceutical formulations including the Mycobacterium tuberculosis binding peptides to a subject via oral administration, or via sub-cutaneous or intravenous injection. In some forms, the subject is a patient with an underlying disease or disorder that reduces the efficacy of the immune system for preventing Mtb infection and/or onset of disease caused by Mtb in the subject. In some forms, the subject has previously received therapy for TB. In some forms, the subject has resurgent TB, for example, following prior treatment. In some forms, the subject has HIV. In some forms, the subject has diabetes mellitus. In some forms, the subject is an active or passive smoker.

B. Detection of microbial pathogen infection

Any of the methods can include one or more steps of diagnosis of a microbial infection, such as a bacterial infection. In an exemplary form, the methods detect and diagnose a Mycobacterium infection/detection of the presence of Mycobacterium cells in a subject or sample. For example, in some forms, the compositions of infected tissuetargeting molecules, each independently having an amino acid sequence of any of SEQ ID NOs: 1-37, or a functional variant thereof, are attached to a dye, or other imaging agent, to label and identify Mycobacterium bacteria and/or tissues infected therewith. In some forms, methods of using the labelled compositions to detect and diagnose subjects as having Mycobacterium infection include administering a composition to the subject, or to a sample from the subject and detecting the label or imaging agent in an organ of the subject, or in the sample. The methods can include one or more additional tests known in the art for detecting and typing Mycobacterium bacteria.

C. Treatment Regimens

Treatment regimens are also described. A treatment regimen can include one or multiple administrations of a pharmaceutical formulation including active agents such as antibiotics and having infected tissue-targeting molecules each independently having an amino acid sequence of any of SEQ ID NOs: 1-37, or a functional variant thereof attached thereto as a targeting agent, and formulations thereof for achieving a desired physiological change, including administering to an animal, such as a bird or mammal, especially a human being, an effective amount of the compositions to treat the disease or symptom thereof, or to produce the physiological change. In preferred forms, the desired physiological change is the reduction in the amount of pathogenic Mycobacterium bacteria

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45652286 in the body (i.e., in a bodily location or site, such as the blood and/or tissues and organs of the subject).

Generally, the treatment regimens include administering the compositions to treat a disease or symptom associated with infection by pathogenic Mycobacterium bacteria in the subject. In some forms, the methods are administered in a regimen together with other treatments or procedures, for example, to treat and/or prevent pneumonia in the subject.

1. Dosage and Effective Amounts

A therapeutically effective amount of compositions including active agents such as antibiotics and having infected tissue-targeting molecules each independently having an amino acid sequence of any of SEQ ID NOs: 1-37, or a functional variant thereof attached thereto as a targeting agent are typically sufficient to reduce or alleviate one or more symptoms associated with pathogenic Mycobacterium infection in a subject are provided. Symptoms of diseases and disorders associated with pathogenic Mycobacterium infection may be physical, such as fever, or biological such as increased amounts of one or more toxin(s) in the blood.

In exemplary forms, the compositions include nanoparticulate compositions, such as porous silicon nanoparticles loaded with antibiotics and having infected tissue-targeting molecules each independently having an amino acid sequence of any of SEQ ID NOs: 1- 37, or a functional variant thereof attached thereto as a targeting agent. In some forms, the amount of nanoparticles can be effective to, for example, treat or prevent one or more symptoms of a pathogenic Mycobacterium infection in a subject. Preferably the nanoparticles are delivered systemically, for example, orally or via sub-cutaneous or intravenous injection, or by inhalation. Generally the nanoparticulate compositions are non-toxic in vivo and do not target or otherwise modulate non-infected tissue or metabolic products. In some forms, the nanoparticles are administered in an amount effective to reduce one or more symptoms associated with the pathogenic Mycobacterium infection in the subject. In some forms, the nanoparticle composition is administered in an effective amount to prevent Mycobacterium infection in the subject, or to prevent one or more diseases or disorders associated with pathogenic Mycobacterium infection in a subject at risk of severe disease resulting from pathogenic Mycobacterium infection.

The therapeutic result of the nanoparticles can be compared to a control. Suitable controls are known in the art. A typical control is a comparison of a condition or symptom of a subject prior to and after administration of the nanoparticle composition. The condition or symptom can be a biochemical, molecular, physiological, or pathological

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45652286 readout. For example, the effect of the nanoparticle compositions on a particular symptom, pharmacologic, or physiologic indicator can be compared to an untreated subject, or the condition of the subject prior to treatment. In some forms, the symptom, pharmacologic, or physiologic indicator is measured in an infected subject prior to treatment, and again one or more times after treatment is initiated. In some forms, the control is a reference level, or average determined based on measuring the symptom, pharmacologic, or physiologic indicator in one or more subjects that do not have the disease or infection to be treated (e.g., healthy subjects). In some forms, the effect of the treatment is compared to a conventional treatment that is known the art.

The actual effective amounts of the nanoparticle composition can vary according to factors including the specific nanoparticles administered, the particular composition formulated, the mode of administration, and the age, weight, condition of the subject being treated, as well as the route of administration and the disease or disorder. Generally, the active agent that is administered is an antibiotic. Since some antibiotics, such as vancomycin, are to some extent toxic to a subject when administered systemically at therapeutic amounts, the described compositions including infected tissue-targeting molecules reduce the toxicity of the therapeutically effective dose of the antibiotics by selectively targeting the antibiotics to the site of infection. Therefore, in some forms, the methods reduce the toxicity of the antibiotics required to treat and/or prevent infection with Mtb. Generally, when the compositions include porous silicon nanoparticles having vancomycin loaded within the pores of the nanoparticle, the amount of the antibiotics required to treat the infection in the subject is less than the amount of the same antibiotic that is required when administered to a subject alone to treat a corresponding infection. Therefore, in some forms, the amount of the compositions that is required can be compared to a control treated with the same antibiotic and/or same nanoparticles in the absence of the targeting peptide(s). Typically, the amount of the antibiotic that is required when loaded into a targeted nanoparticle having Mycobacterium tuberculosis binding peptides each independently having an amino acid sequence of any of SEQ ID NOs:l-37, or a functional variant thereof attached thereto as a targeting agent is about 1/100, 1/50, 1/10, 1/9, 1/8, 1/7, 1/6, 1/5, 14, 1/3, 14 or less than 14 the amount of the same antibiotic that is required to treat the infection when administered in the absence of the nanoparticles.

Therefore, in some forms, the composition increases the efficacy of the antibiotic by more than 1%, such as by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 200%, 300%, 400% or 500%, or more than 500%. In some forms, the composition reduces the

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45652286 dosage of the antibiotic that is required to treat or prevent the infection by up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 200%, 300%, 400% or 500%, or more than 500%.

In some forms, the effective amount of nanoparticle compositions causes little or no killing of the cells of the subject, and preferably little or no inhibition of metabolism in the cells. It is particularly preferred that the composition have little or no effect on the normal bacterial flora of the subject outside of the site of the bacterial infection.

In some forms, dosages of nanoparticles are administered once, twice, or three times daily, or every other day, two days, three days, four days, five days, or six days to a human. In some forms, dosages of nanoparticle compositions are administered about once or twice every week, every two weeks, every three weeks, or every four weeks. In some forms, dosages are administered about once or twice every month, every two months, every three months, every four months, every five months, or every six months.

In some forms, the regimen includes one or more cycles of a round of therapy with the nanoparticle compositions followed by a drug holiday (e.g., no compositions conjugated or complexed with the infected tissue-targeting molecules nanoparticles). The round of the therapy can be, for example, any of the administrations discussed above. Likewise, the drug holiday can be 1, 2, 3, 4, 5, 6, or 7 days; or 1, 2, 3, 4 weeks, or 1, 2, 3, 4, 5, or 6 months.

In particular forms, the subject is administered a dosage of between about 0.1 mg/kg body weight and 100 mg/kg body weight, inclusive, of the composition, such as nanoparticles having infected tissue binding molecules each independently having an amino acid sequence of any of SEQ ID NOs: 1-37, or a functional variant thereof attached thereto as a targeting agent and having loaded therein an antibiotic such as vancomycin. In some forms, the subject is administered a dosage of between about 0.1 mg/kg body weight and 1,000 mg/kg body weight, inclusive, of the composition. In some forms, the subject is administered a dosage of between about 0.2 mg/kg body weight and 10 mg/kg body weight, inclusive, of the composition. In some forms, the subject is administered a dosage of between about 0.3 mg/kg body weight and 1,000 mg/kg body weight, inclusive, of drug-filled nanoparticles that are conjugated or complexed with the Mycobacterium tuberculosis binding peptides. In some forms, the subject is administered a dosage of between about 0.4 mg/kg body weight and 500 mg/kg body weight, inclusive, of the composition. In some forms, the subject is administered a dosage of between about 0.5 mg/kg body weight and 100 mg/kg body weight, inclusive, of the composition. In some

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45652286 forms, the subject is administered a dosage of between about 1.0 mg/kg body weight and 100 mg/kg body weight, inclusive, of drug-filled nanoparticles that are conjugated or complexed with the infected tissue-targeting molecules. In some forms, the subject is administered a dosage of between about 1.0 mg/kg body weight and 5 mg/kg body weight, inclusive, of the composition. Particular dosage regimens include, for example, one or more cycles in which the subject is administered the of the composition on each of two, three, four, five, six or seven days, weeks or months in a row, followed by a one, two, three, four, five, six or seven-day, week, or month drug holiday.

In the most preferred forms, methods of using the described compositions lead to direct or indirect reduction in the number of viable cells of pathogenic Mycobacterium bacteria in the body (e.g., in the blood or tissue or organs) and/or direct or indirect inhibition of the production of Mycobacterium toxins or equivalents, or the direct or indirect reduction of one or more symptoms of a disease or disorder associated with pathogenic Mtb bacteria in the subject. In some forms, the effective amount of compositions is suitable for achieving a specific target amount of antibiotic in the body, such as a specific concentration at the site of infection. In an exemplary form, the composition delivers an effective amount of an antibiotic for achieving therapeutic efficacy against a strain of pathogenic Mycobacterium that is resistant to the antibiotic when administered at a pharmaceutically acceptable dosage in the absence of the composition.

In some forms, the methods administer antimicrobial agents conjugated or complexed with one or more infected tissue-targeting molecules each independently having an amino acid sequence of any one or more of SEQ ID NOs: 1-37, or functional variants thereof to reduce side effects associated with the anti-microbial agents in the subject, for example, by reducing the systemic exposure to the drug and/or dosage of the therapeutically effective amount. In some forms, the methods administer vancomycin conjugated or complexed with one or more infected tissue-targeting molecules each independently having an amino acid sequence of any one or more of SEQ ID NOs: 1-37, or functional variants thereof to reduce side effects associated with vancomycin, such as local pain, thrombophlebitis, damage to the kidneys (nephrotoxicity) and/or to the hearing (ototoxicity).

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45652286 D. Combination Therapies and Procedures

In some forms, the compositions are administered alone, or in combination with one or more conventional therapies, for example, a conventional antibiotic or other therapy.

Typically, tuberculosis is treated with at least two drugs to which the isolate is susceptible. The duration of the treatment regimen, expected drug toxicities, and overall effectiveness will depend on the drugs used. Currently, there are 1 1 drugs approved by the United States Food and Drug Administration (FDA) for treating tuberculosis. The fluoroquinolones and rifabutin, drugs that are commonly used to treat drug-resistant tuberculosis and HIV-related tuberculosis, respectively, do not have FDA approval for the treatment of tuberculosis. In some forms, the antibiotics are two or more of those associated clinically with treatment of Mtb infection, such as first- line oral drugs (with typically adult daily dose) Isoniazid (300 mg), Rifampin (600 mg), Rifabutin (300 mg), Pyrazinamide (30 mg/kg), Ethambutol (15 - 25 mg/kg); and injectable drugs (with typically adult daily dose): Streptomycin (15 mg/kg), Amikacin (15 mg/kg), Kanamycin (15 mg/kg), Capreomycin (15 mg/kg). In other forms, the antibiotics two or more of those associated clinically with treatment of Mtb infection, such as Second-line oral drugs (with typically adult daily dose): Bedaquiline (400 qd or 200 mg q.i.d.), Pretomanid (200 mg qd), Linezolid (600 or 1,200 mg qd), Ofloxacin (400 mg b.i.d.), Levofloxacin (500 mg qd), Moxifloxacin (400 mg qd), Gatifloxacin (400 mg qd), Ethionamide (250 mg b.i.d. or t.i.d.) , Aminosalicylic acid (3 g q.i.d.), and Cycloserine (250 mg b.i.d. or t.i.d.).

In some forms, the antibiotics are two or more of Isoniazid (300 mg), Rifampin (600 mg), Rifabutin (300 mg), Pyrazinamide (30 mg/kg), Ethambutol (15 - 25 mg/kg); and injectable drugs (with typically adult daily dose) Streptomycin (15 mg/kg), Amikacin (15 mg/kg), Kanamycin (15 mg/kg), Capreomycin (15 mg/kg), Ofloxacin (400 mg b.i.d.), Levofloxacin (500 mg qd), Moxifloxacin (400 mg qd), Gatifloxacin (400 mg qd), Ethionamide (250 mg b.i.d. or t.i.d.), Aminosalicylic acid (3 g q.i.d.), and Cycloserine (250 mg b.i.d. or t.i.d.).

In some forms, the methods include administration of nanoparticles having infected tissue-targeting molecules each independently having an amino acid sequence of any of SEQ ID NOs:l-37, or a functional variant thereof attached thereto as a targeting agent and having loaded therein an antibiotic such as vancomycin in combination with one or more additional active agents. The combination therapies can include administration of the described compositions and other active agents together in the same admixture, or in

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45652286 separate admixtures. Therefore, in some forms, the pharmaceutical composition includes drug-filled nanoparticles that are conjugated or complexed with the infected tissuetargeting molecules and one, two, three, or more additional active agents. The additional active agent(s) can have the same, or different mechanisms of action. In some forms, the combination results in an additive effect on the treatment of pathogenic Mycobacterium infection. In some forms, the combinations result in a more than additive effect on the treatment of the disease or disorder.

The additional therapy or procedure can be simultaneous or sequential with the combination therapy. In some forms the additional therapy is performed between drug cycles or during a drug holiday that is part of the compositions dosage regime. For example, in some forms, the additional therapy or procedure is surgery.

Additional therapeutic agents include conventional therapeutics such as nonsteroidal anti-inflammatory drugs (NSAID), pain relief and antimicrobial drugs. Therefore, in some forms, the additional therapeutic agents include antibacterial antibiotics, bacteriophage and immunotherapies. In some forms, the additional active agent is another drug directly or indirectly targeting pathogenic Mtb, or other microorganisms present or suspected to be present at the site of infection.

In some forms, the methods administer two or more antimicrobial agents that are encapsulated alone or together within particles conjugated or complexed with one or more infected tissue-targeting molecules each independently having an amino acid sequence of any one or more of SEQ ID NOs: 1-52, or functional variants thereof. For example, in some forms, the methods administer vancomycin together with Ceftazidime encapsulated alone or together within particles conjugated or complexed with one or more Mycobacterium tuberculosis binding peptides each independently having an amino acid sequence of any one or more of SEQ ID NOs: 1-52, or functional variants thereof.

The description will be better understood by reference to the following numbered paragraphs.

1. A method for treatment or prevention of an infection by a microbial pathogen in a subject in need thereof, the method including administering to the subject a pharmaceutical formulation including:

(a) an infected tissue homing peptide; and

(b) an antimicrobial agent, wherein the antimicrobial agent is conjugated or complexed with the infected tissue homing peptide, and

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45652286 wherein the antimicrobial agent is in an amount effective to treat or prevent one or more symptoms of the infection in the subject.

2. A method for identification of an infection by a microbial pathogen in a subject, including administering to the subject a pharmaceutical formulation including:

(a) an infected tissue homing peptide; and

(b) an imaging or diagnostic agent, wherein the imaging or diagnostic agent is in an amount effective to bind to infected tissues and/or microbial pathogens, and label or otherwise identify the infection in the subject.

3. The method of paragraph 1 or 2, wherein the infected tissue homing peptide includes any one or more of SEQ ID NOs:l-52, or a functional variant thereof having at least 70% sequence identity to any one of SEQ ID NOs:l-37.

4. The method of paragraph 3, wherein the infected tissue homing peptide includes PPRRGLIKLKTS (SEQ ID NO:1).

5. The method of paragraph 3, wherein the infected tissue homing peptide includes a functional variant having at least 70% sequence identity to PPRRGLIKLKTS (SEQ ID NO:1).

6. The method of paragraph 3, wherein the infected tissue homing peptide consists of PPRRGLIKLKTS (SEQ ID NO: 1).

7. The method of paragraph 3, wherein the infected tissue homing peptide includes AGRGRLVR (SEQ ID NO:2).

8. The method of paragraph 3, wherein the infected tissue homing peptide includes a functional variant having at least 70% sequence identity to AGRGRLVR (SEQ ID NO:2).

9. The method of paragraph 3, wherein the infected tissue homing peptide consists of AGRGRLVR (SEQ ID NO:2).

10. The method of paragraph 3, wherein the infected tissue homing peptide includes GRPARPAR (SEQ ID NO:3).

11. The method of paragraph 3, wherein the infected tissue homing peptide includes a functional variant having at least 70% sequence identity to GRPARPAR (SEQ ID NO:3).

12. The method of paragraph 3, wherein the infected tissue homing peptide consists of GRPARPAR (SEQ ID NO:3).

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45652286 13. The method of paragraph 3, wherein the infected tissue homing peptide includes CKRDLSRRC (SEQ ID NO:4).

14. The method of paragraph 3, wherein the infected tissue homing peptide includes a functional variant having at least 70% sequence identity to GRP ARP AR (SEQ ID NO:3).

15. The method of paragraph 3, wherein the infected tissue homing peptide consists of CKRDLSRRC (SEQ ID NO:4).

16. The method of paragraph 3, wherein the infected tissue homing peptide includes AKRGARSTA (SEQ ID NO:5).

17. The method of paragraph 3, wherein the infected tissue homing peptide includes a functional variant having at least 70% sequence identity to AKRGARSTA (SEQ ID NO:5).

18. The method of paragraph 3, wherein the infected tissue homing peptide consists of AKRGARSTA (SEQ ID NO:5).

19. The method of any one of paragraphs 1-18, wherein the formulation further includes one or more polypeptides or other amino acid sequences, wherein the polypeptides or other amino acid sequences are contiguous with the amino (NH) or carboxyl (COOH) terminus of the infected tissue homing peptide.

20. The method of any one of paragraphs 1-19, wherein the formulation further includes one or more additional molecules selected from the group including a carbohydrate, a lipid, a small organic molecule, a polymer, and a nucleic acid.

21. The method of any one of paragraphs 1-20, wherein the antimicrobial agent is selected from the group including bedaquiline, pretomanid, sutezolid, delamanid, isoniazid, vancomycin, rifampin, rifabutin, pyrazinamide, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, ethionamide, aminosalicylic acid, and cycloserine.

22. The method of paragraph 21, wherein the antimicrobial is vancomycin.

23. The method of any one of paragraphs 21 to 22, wherein the antimicrobial agent is attached to the peptide via a linker.

24. The method of paragraph 23, wherein the linker is a cleavable linker.

25. The method of any one of paragraphs 1 to 24, wherein the formulation further includes a delivery vehicle.

26. The method of paragraph 25, wherein the delivery vehicle is a particle, and wherein the particle is a microparticle or a nanoparticle.

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45652286 27. The method of paragraph 25 or 26, wherein the delivery vehicle is selected from the group including a citric acid silver nanoparticle, a liposome, a micelle, a viral capsid, a polymeric particle, a dendrimer, a porous silicon particle, a metal particle, and an iron oxide nanoparticle.

28. The method of any one of paragraphs 25 to 27, wherein the delivery vehicle is a citric acid nanoparticle.

29. The method of any one of paragraphs 25 to 28, wherein the infected tissue homing peptide is conjugated to, or dispersed within the outer surface of the particle.

30. The method of paragraph 29, wherein the infected tissue homing peptide is attached to the surface of the particle via a linker.

31. The method of paragraph 30, wherein the linker is a cleavable linker.

32. The method of paragraph 31, wherein the infected tissue-targeting peptide is attached to the surface of the particle via a polyethylene glycol (PEG) linker.

33. The method of paragraph 32, wherein the PEG has a molecular weight of between about 1,000 Da and about 10,000 Da, inclusive, optionally wherein the PEG has a molecular weight of about 5,000 Da.

34. The method of any one of paragraphs 26 to 33, wherein the antimicrobial agent is encapsulated or embedded within one or more cavities and/or pores within the particle.

35. The method of any one of paragraphs 26 to 34, wherein the particle has a mean hydrodynamic diameter of between about 40 nm and about 80 nm, inclusive, optionally wherein the particle has a mean diameter of about 60 nm.

36. The method of any one of paragraphs 26 to 35, wherein the particle does not include another targeting or homing agent.

37. The method of any one of paragraphs 26 to 36, wherein the particle is conjugated to between one and about 10,000 molecules of the infected tissue homing peptide.

38. The method of any one of paragraphs 26 to 37, wherein the formulation is in a form selected from the group including a solution, a dry powder, micelles, colloids, nanodroplets, nano-structured hydrogel, nanocrystals, and a nanosuspension.

39. The method of any one of paragraphs 1 to 38, wherein the subject has been diagnosed as having, or as being at risk of having an infection with one or more Mycobacterium sp. selected from the group including M. tuberculosis, M. leprae, M. avium, M. ulcerans, M. xenopi, M. kansasii, M. abscessus, M. chelonae, M. fortuitum, M.

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45652286 africanum, M. canelti, M. bovis, M. caprae, M. microti, M. pinnipedii, M. mungi, and M. orygis.

40. The method of any one of paragraphs 1 to 38, wherein the subject has been diagnosed as having, or as being at risk of having an infection with M. tuberculosis.

41. The method of paragraph 39 or 40, wherein the Mycobacterium sp. is an antibiotic resistant strain that is resistant to one or more antibiotics when administered in vivo alone at a safe dose.

42. The method of paragraph 41 , wherein the Mycobacterium sp. is resistant to one or more antibiotics selected from the group including bedaquiline, pretomanid, sutezolid, delamanid, isoniazid, vancomycin, rifampin, rifabutin, pyrazinamide, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, ethionamide, aminosalicylic acid, and cycloserine.

43. The method of paragraph 42, wherein the antibiotic resistant strain is susceptible to the same antibiotic when administered conjugated with the infected tissue homing peptide.

44. The method of any one of paragraphs 40 to 43, wherein the subject has been diagnosed as having, or as being at risk of having tuberculosis (TB) selected from the group including pulmonary TB, CNS TB, pericardial TB, pleural TB, Lymphadenitis, Abdominal TB, skeletal TB, genitourinary TB and miliary TB.

45. The method of any one of paragraphs 40 to 44, wherein the method is effective to reduce, eliminate, or prevent one or more symptoms of M. tuberculosis (Mtb) infection selected from the group including fever, chest pain, wheezing or difficulty breathing, cough, fatigue, and unintentional weight loss.

46. The method of any one of paragraphs 1 to 45, wherein the pharmaceutical formulation is administered to the subject via a route selected from the group including oral administration, intramuscular injection, intravenous injection, sub-cutaneous injection, and intra-articular injection.

47. The method of any one of paragraphs 1 to 46, wherein the effective amount of the antimicrobial agent administered in the formulation is less than the amount of the same therapeutic agent that is required to be effective when administered alone.

48. A dosage form for treatment of an infection by Mycobacterium tuberculosis (Mtb) in a subject in need thereof, including

(a) a infected tissue homing peptide including an amino acid sequence selected from SEQ ID NOs:l-52; and

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45652286 (b) an antimicrobial agent, wherein the antimicrobial agent is in an amount effective to treat or prevent one or more symptoms of the infection in the subject.

The description will be better understood by reference to the following Examples.

Examples

Example 1: M. tuberculosis-binding peptides that penetrate the cellular rim and/or the caseous core of TB lesions identified by peptide library screening

Peptides that penetrate the cellular rim and/or the caseous core of TB lesions with significantly improved selectivity and plasma-to-lesion penetration coefficient relative to control peptide, following systemic injection were sought.

Materials and Methods

To identify potential peptides, peptide receptors in M. tuberculosis-infected lung vs control lung tissue were stained. The readout is not quantitative, but highly specific staining of Mtb-infected tissues with antibodies to known peptide receptors can identify key receptors for further studies.

Immunostaining of tissue sections from TB-infected mice was carried out with antibodies against the known target molecules (receptors) for the candidate peptides from other disease indications. Slides with paraffin-embedded TB-infected C3HeB/FeJ lung sections were used. Fluorescent immunostaining was performed on the tissue sections carrying Type I caseous necrotic and Type III cellular lesions with peptide receptor antibodies listed in Table 1.

Antibodies against receptors p32, NRP-1, Fn-EDB and TNC-C were made inhouse, the rest of the required antibodies were ordered from commercial companies. Fluorescent immunostainings of both necrotic and cellular lesions were imaged (at least 3 animals and 7 images per tissue) with Olympus Fluoview FV1200 MPE confocal microscope.

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45652286 Table 1: List of the antibodies against peptide receptors used in the immunostaining analysis.

Results Multiple peptides showing highly specific binding to Mtb-infected tissues were identified and characterized. Altogether 13 antibodies were used to detect the presence of peptide receptors in TB -infected pulmonary lesions and 10 of 13 showed specific staining on tissue sections. Three antibodies (CD206, CD47 and heparin sulfate proteoglycan) did not show binding to the receptor proteins on tissue sections. Most likely these antibodies were not suitable for paraffin-embedded tissue sections. One antibody (TNC-C) did not work well with the standard staining protocol but gave suitable results after optimization

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45652286 of the staining conditions. The results of the staining assay are depicted in Figures 1A-1T, with the stained tissues in cellular lesion depicted in Figs. 1A-1J, and the stained tissues in necrotic lesions depicted in Figs. 1K-1T; regions in white indicate positive staining for each of the antibodies tested, meaning that the receptors are expressed in Mtb-infected C3HeB/FeJ mouse tissues and as expected, were found to be upregulated in cellular and necrotic lesions where the majority of Mtb bacilli are located. Peptides that home at least 3x over the control peptides have a high probability of much higher homing potential.

Example 2: In vivo playoff auditioning of the candidate peptides for targeting the TB lesions

In vivo playoff auditioning is useful for head-to-head comparative evaluation of multiple peptide phages in the same animal. The most important advantage of in vivo playoff studies - that all the peptide phages are auditioned in parallel under identical conditions - avoids inter-animal variability that may arise due to differences in physiological state of the animal and/or disease status heterogeneity and subtle variations in experimental conditions. In addition, inclusion of positive and negative phages provides quality control for homing experiments.

Some peptides that have been shown in the literature to specifically home and penetrate tumors may also recognize infected tissue and could potentially be used in targeted delivery of treatments to bacterial infections. The aim was to test such already available peptides to identify ones that accumulate in TB-infected tissues, and that therefore can potentially be used to deliver therapeutic agents to the sites of infection. A significant advantage of testing these peptides first is that they are already at hand. In addition to potentially directing more drug into the TB granulomas in general, selectively concentrating an antibiotic in macrophages may increase the efficacy of bacterial eradication because macrophages are an important reservoir of intracellular bacilli.

It was proposed to use the highly validated phage library screening in vivo play off platform to identify peptides that recognize the bacilli rather than tissue changes brought about by the infection. Such peptides can take a drug payload all the way to the bacilli, not just into the infected area in general. These screens will include an in vivo screening step, and may also yield peptides that recognize new tissue changes caused by the infection, similar in principle to the targets recognized by existing peptides, but distinct from them. Granuloma tissue in particular is likely to upregulate molecules that are not expressed in normal tissues at significant levels and may present new targets detectable by phage display.

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45652286 Materials and Methods

A pool of equally represented phages displaying candidate and control peptides was administered to live mice (Mtb-infected and healthy uninfected), and the representation of each phage in target and control organs was determined by sequencing (Sanger sequencing for low complexity pools and HTS for high complexity pools).

A phage play-off pool with 23 peptide-phages was prepared (encompassing the candidate homing peptides listed in Table 2). This required cloning of some of the peptides, sequencing of all the peptides with Sanger method, amplification, purification and determining the titer of the samples. TB-infected and non-infected mice were dosed with the phage pool and DNA samples of lungs and livers were sent for high throughput sequencing and data analysis.

Results

First, representation of targeting peptides over the control peptides was estimated in both infected and non-infected tissue (Figure 2A) and second, the over-representation of targeting peptides in the infected tissue was studied, focusing on peptides that homed at least 3 times more to the infected lung than to the normal lung (Figure 2B).

Five peptides - PL1, PL3, RPARPAR, IP3 and linTTl - met the criteria and were proposed as potential targeting peptides of TB-infected pulmonary lesions. Importantly, each of the candidates identified by play-off genetics was shown to also bind fixed Mtb- infected lung tissues by immunofluorescence, as indicated in Figures 1A-1T.

Each of the candidate homing peptides, originally identified in other disease indications, recognize different cell surface and extracellular molecules upregulated in reactive tissues as summarized below:

1. PL 1 peptide (PPRRGLIKLKTS ; SEQ ID NO : 1 ) showed the highest difference between infected and non-infected lung tissue (Figure 2B) and highest fold over the control peptide in infected lungs (Figure 2A); PL1 interacts with receptors Fn- EDB and TNC-C that are both present in the solid tumor extracellular matrix, and absent in normal adult tissues (Lingasamy et al., 2019). PL 1 has been shown to target also endometriotic lesions (Simon-Gracia et al., 2021).

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45652286 Table 2: Components of in vivo play-off screen and peptides used in the phage pool dosed in TB mice. over the control peptide in both infected and uninfected lungs in the play-off screen

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45652286 (Figures 2A, 2B). PL3 interacts with TNC-C receptor and NRP-1 in solid tumors (Lingasamy et al., 2020).

3. RPARPAR peptide (GRPARPAR; SEQ ID NOG) showed high presence in infected and normal lung tissue (Figure 2A). RPARPAR, a prototypic C-end rule (CendR) peptide interacts with NRP1 protein and accumulates in the lung parenchyma when injected intravenously to mice (Teesalu et al., 2009).

4. TP3 peptide (CKRDLSRRC; SEQ ID NO:4) showed 5 times higher fold increase in infected lungs over normal lung (Figure 2B). IP3 peptide contains a hyaluronan-binding motif that homes to macrophage-rich regions in peritoneal tumors (gastric and colon tumors), including poorly vascularized micro-tumors (Ikemoto et al., 2017).

5. LinTTl peptide (AKRGARSTA; SEQ ID NOG) had 3 times higher fold in infected lungs over the non-infected lungs (Figure 2B). LinTTl is targeting activated macrophages in the tumors and inflammatory lesions (Sharma et al., 2017).

Example 3: Homing studies using peptide-functionalized silver nanoparticles

Peptides identified from the screens were tested for their ability to deliver drugs to the sites of infection at concentrations and depth of penetration not achieved in conventional therapy. The delivery may be best accomplished with a drug-filled nanoparticle coated with a TB homing peptide for infection targeting. Nanoparticles can be designed to release the drug slowly for a long-lasting therapeutics effect, which would reduce the frequency of treatments. Nanoparticles (or small dendrimers) have significant advantages over simple peptide-drug conjugates as drug delivery vehicles, such as increasing the efficacy of the peptide. The presentation of the peptide at the surface of a nanoparticle allows for multivalent binding that makes up for the fact that peptides generally do not have high affinities for their target (Ruoslahti, 2012). Moreover, a major limitation of affinity-based drug targeting is low amount of available target molecule in the target tissue. Nanoparticles can be designed to have a favorable drug load to target molecule ratio. Finally, peptide-coated nanoparticles have already been used in bacterial targeting and can readily be redesigned for TB targeting. Once a proof of concept has been established with nanoparticles, it will be possible to convert the peptide-based targeting to other chemistries, such as a conjugate to an antibody against the same target the peptide uses (multivalent presentation is not important with antibodies because they are naturally divalent and generally have higher affinities than peptides). However, they may not

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45652286 reproduce the tissue-penetrating and cell-penetrating properties of the peptides, and the drug payload to target molecule ratio is less favorable than with nanoparticles.

Methods

Homing of 5 peptides chosen based on the play-off screen (PL1, PL3, RPARPAR, IP3 and linTTl) was tested in TB -infected mice in vivo. Synthetic fluorescein-labelled peptides were ordered from TAG Copenhagen (Denmark) and peptide-functionalized silver nanoparticles (AgNP) were prepared and sent for homing studies.

Results

Citric AgNP have an average core size of 62 ± 20 nm and hydrodynamic size of 103 ± 40 nm (Figures 3A-3C) (Lingasamy et al., 2020).

AgNPs were developed as a model platform for targeted in vitro and in vivo delivery (Tobi et al., 2021). The AgNPs have several unique features that make them useful for biodistribution studies including plasmonic enhancement emission from coupled fluorescent dyes to allow ultrasensitive imaging of single nanoparticles.

Example 4: Evaluation of priority peptides that were identified in an initial in vivo biopanning

Methods

To evaluate priority peptides that were identified in an initial in vivo biopanning study and confirmed by immunofluorescent staining of target receptors using PFA-fixed M. tuberculosis C3HeB/FeJ lung tissues (see Figures 1A-1T and 2A-2B). C3HeB/FeJ TB Infection (“Kramnik” mouse) model

The drawback of the standard mouse infection models for tuberculosis is their lack of advanced lung lesion types as the progression of disease rarely reaches stages of extensive necrosis and calcification in lungs. The C3HeB/FeJ mouse strain with a recessive allele was studied first by Igor Kramnik, et al (2000). Developed under the ‘Kramnik model’ development grant funded by the BMGF, the C3HeB/FeJ TB efficacy model has been optimized for several years now at CSU in terms of achieving consistent infections and reproducibly showing caseous necrotic lesion types at treatment start. The model uses a low dose aerosol infection with M. tuberculosis Erdman (50-120 CFU/ mouse), with treatment start at 8 weeks post aerosol. The lung pathology in C3HeB/FeJ mice is heterogeneous, generating diverse lesion types which present unique microenvironments each harboring metabolically distinct bacterial subpopulations (Driver et al., 2012; Irwin et al., 2015). The C3HeB/FeJ mouse infection model is characterized by developing hypoxic, caseous necrotic lesions in lungs upon an M. tuberculosis infection.

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45652286 Necrosis causes a proportion of the bacteria to become extracellular within the necrotic debris and caseum, which is hypoxic.

Three distinct lesion types have been characterized, including highly encapsulated caseous necrotic granulomas (type I), fulminant neutrophilic alveolitis (type II), and cellular non-necrotizing lesions (type III), with only the latter lesion type being present in BALB/c mice. These different lesion types exhibit highly different microenvironmental conditions that vary in certain parameters such as oxygen tension, pH, nutrient supply, and cellular composition and are more closely aligned with the heterogamous pathology found in human TB patients (Lenaerts et al., 2015). The presence of caseum with a higher ratio of extracellular to intracellular bacilli, and the altered microenvironments that arise, are known to have a profound impact on drug efficacy. Drug penetration across the caseous necrotic lesions into the caseum can also be hampered resulting in limited drug exposure at the site where the majority of difficult to eradicate bacilli are located. For some drugs, this manifests as a bimodal treatment response composed of drug responsive and non- responsive animals, a major challenge also observed in the treatment of human TB, for which the peptide-targeting drug embedded NP approaches should prove invaluable. The C3HeB/FeJ mouse is an ideal model to establish this initial proof of concept that peptide targeting drug-loaded NPs can overcome lesion PK limitations of conventional therapies allowing more effective drug treatments of the bacterial phenotypes found within TB- infected tissues or lesion PD.

Peptides and Particles

Synthetic fluorescein-labelled peptides and peptide-functionalized silver nanoparticles loaded with CF555 (AgNP) (see Table 3 a) were administered to Mtb Erdman infected C3HeB/FeJ mice as a single intravenous (IV) or intraperitoneal (IP) injection and allowed to circulate as indicated prior to a full body perfusion to remove unbound material (Figure 4).

Lung and liver tissues were recovered and fixed prior to embedding in paraffin. Blocks were subsequently sectioned for image analysis which included hematoxylin and eosin (H&E, histology), fluorescence and DAPI staining (peptide/ Ag-NP localization and eukaryotic DNA), and Sybr Gold (stains Mtb bacilli). All images were captured at CSU using a Vectra Polaris Multispectral Imaging and Whole Slide Scanning system.

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45652286 Table 3: Synthetic fluorescein-labelled peptides and peptide-functionalized silver nanoparticles loaded with CF555 dose groups.

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45652286 Results

Altogether 129 whole slide scans and corresponding H&Es plus SYBR gold staining were used to map and detect peptide and peptide-functionalized silver nanoparticles interactions across lungs and livers and in TB-infected pulmonary lesions (Table 3). All of peptides that were genetically-enriched from the in vivo play-off screen showed strong evidence of highly specific staining on tissue sections when administered by a single dose injection to infected mice, whereas, control constructs showed non-specific interactions only (See Figures 5A-5B).

To further demonstrate specificity of peptide interactions in vivo, adjacent slide sections from the study underwent secondary fluorescent immunostaining with their corresponding peptide receptor antibody listed in Table 1. Spatial biology analysis and quantification confirmed co-localization of receptors targeted by peptide-AgNP and also by antibody specific immunofluorescence. Excellent co-localization was observed in all cases, especially for receptors identified in vascularized Type III cellular lesions, with a somewhat lower frequency of co-localization with receptors identified by fluorescent immunostaining in non-vascularized necrotic Type I lesions (not shown). This observation is not unexpected given that these initial studies examined lesion penetration by fluorescent staining after only a single administration and a relatively short circulation time (i.e., 30 to 180 mins), where past experience using other nanoparticle delivery systems indicate that full lesion penetration may require dosing to steady state.

Summary

Firstly, a panel of existing homing peptides was tested for ability to home to TB infected tissues using a series of experiments of increasing labor intensity. In vivo playoff was carried out using a set of ~20 phages displaying homing peptides with targeting specificity towards activated macrophages, angiogenic sites, and activated extracellular matrix. TB infected mice were injected with equimolar mixture of different candidate and control peptide phages, and their representation in TB lesions and control tissues will be studied by high-throughput sequencing. The expression of receptors for the peptides showing systemic selectivity towards TB lesions was studied by immunostaining to confirm the playoff data and to develop better understanding of the peptide target sites. Finally, systemic homing experiments with selected fluorophore-labeled synthetic peptides were performed in TB infected mice, and peptide bio-distribution tested by confocal immunofluorescence imaging.

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45652286 Peptide receptor immunostaining of tissue sections from TB-infected tissue with antibodies against the known target molecules (receptors) identified the lead peptides. In parallel, tissue sections stained with markers of vascular endothelial cells and other cell types in the lesion with existing materials and with no exposure to M. tuberculosis bacteria provided information on the presence and spatial distribution of peptide receptors to guide decisions as to which peptides to test further.

In vivo playoff auditioning with multiplexed injection of up to 20 peptides per mouse, using the C3HeB/FeJ mouse model enabled representation of each peptide phage to be assessed by high-throughput sequencing of peptide-encoding region of the phage genome. The internally controlled playoff approach established peptides with systemic homing ability to the TB lesions.

Peptides that penetrate the cellular rim and/or the caseous core of TB lesions, with significantly improved selectivity and plasma-to-lesion penetration coefficient relative to control peptide, following systemic injection were identified.

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45652286

Claims

CLAIMS We claim:

1. A method for treatment or prevention of an infection by a microbial pathogen in a subject in need thereof, the method comprising administering to the subject a pharmaceutical formulation comprising:

(a) an infected tissue homing molecule; and

(b) an antimicrobial agent, wherein the antimicrobial agent is conjugated or complexed with the infected tissue homing molecule, and wherein the antimicrobial agent is in an amount effective to treat or prevent one or more symptoms of the infection in the subject.

2. A method for identification of an infection by a microbial pathogen in a subject, comprising administering to the subject a pharmaceutical formulation comprising:

(a) an infected tissue homing molecule; and

(b) an imaging or diagnostic agent, wherein the imaging or diagnostic agent is in an amount effective to bind to infected tissues and/or microbial pathogens, and label or otherwise identify the infection in the subject.

3. The method of claim 1 or 2, wherein the infected tissue homing molecule comprises a peptide comprising any one or more of SEQ ID NOs:l-37 or 40, or a functional variant thereof having at least 70% sequence identity to any one of SEQ ID NOs:l-37 or 40.

4. The method of claim 3, wherein the infected tissue homing peptide comprises PPRRGLIKLKTS (SEQ ID NO: 1).

5. The method of claim 3, wherein the infected tissue homing peptide comprises a functional variant having at least 70% sequence identity to PPRRGLIKLKTS (SEQ ID NO:1).

6. The method of claim 3, wherein the infected tissue homing peptide consists of PPRRGLIKLKTS (SEQ ID NO:1).

7. The method of claim 3, wherein the infected tissue homing peptide comprises AGRGRLVR (SEQ ID NO:2).

8. The method of claim 3, wherein the infected tissue homing peptide comprises a functional variant having at least 70% sequence identity to AGRGRLVR (SEQ ID NO:2).

9. The method of claim 3, wherein the infected tissue homing peptide consists of AGRGRLVR (SEQ ID NO:2).

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10. The method of claim 3, wherein the infected tissue homing peptide comprises GRPARPAR (SEQ ID NO:3).

11. The method of claim 3, wherein the infected tissue homing peptide comprises a functional variant having at least 70% sequence identity to GRPARPAR (SEQ ID NO:3).

12. The method of claim 3, wherein the infected tissue homing peptide consists of GRPARPAR (SEQ ID NO:3).

13. The method of claim 3, wherein the infected tissue homing peptide comprises CKRDLSRRC (SEQ ID NO:4).

14. The method of claim 3, wherein the infected tissue homing peptide comprises a functional variant having at least 70% sequence identity to GRPARPAR (SEQ ID NO:3).

15. The method of claim 3, wherein the infected tissue homing peptide consists of CKRDLSRRC (SEQ ID NO:4).

16. The method of claim 3, wherein the infected tissue homing peptide comprises AKRGARSTA (SEQ ID NO:5).

17. The method of claim 3, wherein the infected tissue homing peptide comprises a functional variant having at least 70% sequence identity to AKRGARSTA (SEQ ID NO:5).

18. The method of claim 3, wherein the infected tissue homing peptide consists of AKRGARSTA (SEQ ID NO:5).

19. The method of any one of claims 1-18, wherein the formulation further comprises one or more polypeptides or other amino acid sequences, wherein the polypeptides or other amino acid sequences are contiguous with the amino (NH) or carboxyl (COOH) terminus of the infected tissue homing peptide.

20. The method of any one of claims 1-19, wherein the formulation further comprises one or more additional molecules selected from the group consisting of a carbohydrate, a lipid, a small organic molecule, a polymer, and a nucleic acid.

21. The method of any one of claims 1-20, wherein the antimicrobial agent is selected from the group consisting of bedaquiline, pretomanid, sutezolid, delamanid, isoniazid, vancomycin, rifampin, rifabutin, pyrazinamide, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, ethionamide, aminosalicylic acid, and cycloserine.

22. The method of claim 21, wherein the antimicrobial is vancomycin.

23. The method of any one of claims 21 to 22, wherein the antimicrobial agent is attached to the peptide via a linker.

24. The method of claim 23, wherein the linker is a cleavable linker.

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25. The method of any one of claims 1 to 24, wherein the formulation further comprises a delivery vehicle.

26. The method of claim 25, wherein the delivery vehicle is a particle, and wherein the particle is a microparticle or a nanoparticle.

27. The method of claim 25 or 26, wherein the delivery vehicle is selected from the group consisting of a citric acid silver nanoparticle, a liposome, a micelle, a viral capsid, a polymeric particle, a dendrimer, a porous silicon particle, a metal particle, and an iron oxide nanoparticle.

28. The method of any one of claims 25 to 27, wherein the delivery vehicle is a citric acid nanoparticle.

29. The method of any one of claims 25 to 28, wherein the infected tissue homing peptide is conjugated to, or dispersed within the outer surface of the particle.

30. The method of claim 29, wherein the infected tissue homing peptide is attached to the surface of the particle via a linker.

31. The method of claim 30, wherein the linker is a cleavable linker.

32. The method of claim 31, wherein the infected tissue-targeting peptide is attached to the surface of the particle via a polyethylene glycol (PEG) linker.

33. The method of claim 32, wherein the PEG has a molecular weight of between about 1,000 Da and about 10,000 Da, inclusive, optionally wherein the PEG has a molecular weight of about 5,000 Da.

34. The method of any one of claims 26 to 33, wherein the antimicrobial agent is encapsulated or embedded within one or more cavities and/or pores within the particle.

35. The method of any one of claims 26 to 34, wherein the particle has a mean hydrodynamic diameter of between about 40 nm and about 80 nm, inclusive, optionally wherein the particle has a mean diameter of about 60 nm.

36. The method of any one of claims 26 to 35, wherein the particle does not include another targeting or homing agent.

37. The method of any one of claims 26 to 36, wherein the particle is conjugated to between one and about 10,000 molecules of the infected tissue homing peptide.

38. The method of any one of claims 26 to 37, wherein the formulation is in a form selected from the group consisting of a solution, a dry powder, micelles, colloids, nanodroplets, nano-structured hydrogel, nanocrystals, and a nanosuspension.

39. The method of any one of claims 1 to 38, wherein the subject has been diagnosed as having, or as being at risk of having an infection with one or more

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40. The method of any one of claims 1 to 38, wherein the subject has been diagnosed as having, or as being at risk of having an infection with M. tuberculosis.

41. The method of claim 39 or 40, wherein the Mycobacterium sp. is an antibiotic resistant strain that is resistant to one or more antibiotics when administered in vivo alone at a safe dose.

42. The method of claim 41, wherein the Mycobacterium sp. is resistant to one or more antibiotics selected from the group consisting of bedaquiline, pretomanid, sutezolid, delamanid, isoniazid, vancomycin, rifampin, rifabutin, pyrazinamide, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, ethionamide, aminosalicylic acid, and cycloserine.

43. The method of claim 42, wherein the antibiotic resistant strain is susceptible to the same antibiotic when administered conjugated with the infected tissue homing peptide.

44. The method of any one of claims 40 to 43, wherein the subject has been diagnosed as having, or as being at risk of having tuberculosis (TB) selected from the group consisting of pulmonary TB, CNS TB, pericardial TB, pleural TB, Lymphadenitis, Abdominal TB, skeletal TB, genitourinary TB and miliary TB.

45. The method of any one of claims 40 to 44, wherein the method is effective to reduce, eliminate, or prevent one or more symptoms of M. tuberculosis (Mtb) infection selected from the group consisting of fever, chest pain, wheezing or difficulty breathing, cough, fatigue, and unintentional weight loss.

46. The method of any one of claims 1 to 45, wherein the pharmaceutical formulation is administered to the subject via a route selected from the group consisting of oral administration, intramuscular injection, intravenous injection, sub-cutaneous injection, and intra- articular injection.

47. The method of any one of claims 1 to 46, wherein the effective amount of the antimicrobial agent administered in the formulation is less than the amount of the same therapeutic agent that is required to be effective when administered alone.

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48. A dosage form for treatment of an infection by Mycobacterium tuberculosis (Mtb) in a subject in need thereof, comprising

(a) a infected tissue homing peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-37 or 40; and

(b) an antimicrobial agent, wherein the antimicrobial agent is in an amount effective to treat or prevent one or more symptoms of the infection in the subject.

49. A method for treatment or prevention of an infection by a microbial pathogen in a subject in need thereof, the method comprising administering to the subject a pharmaceutical formulation comprising:

(a) an infected tissue homing molecule that targets one or more receptor selected from the group consisting of p32, NRP-1, Fn-EDB, CD206, Retinoid X receptor beta (RXR-beta), alpha- V-integrin, hyaluronin, heparin sulphate and TNC-C; and

(b) an antimicrobial agent, wherein the antimicrobial agent is conjugated or complexed with the infected tissue homing molecule, and wherein the antimicrobial agent is in an amount effective to treat or prevent one or more symptoms of the infection in the subject.

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