(12) United States Patent (10) Patent No.: US 9,593,160 B2 Ingber Et Al

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(12) United States Patent (10) Patent No.: US 9,593,160 B2 Ingber et al. (45) Date of Patent: Mar. 14, 2017

(54) ENGINEERED MICROBE-TARGETING (56) References Cited MOLECULES AND USES THEREOF U.S. PATENT DOCUMENTS (75) Inventors: Donald E. Ingber, Boston, MA (US); 5,137,810 A 8, 1992 Sizemore et al. Michael Super, Lexington, MA (US); 5,270,199 A 12/1993 Ezekowitz Jeffrey Charles Way, Cambridge, MA 5,545,820 A 8, 1996 Gatehouse et al. 5,951,976 A 9/1999 Segal (US); Mark J. Cartwright, West 6,117,977 A 9/2000 Lasky et al. Newton, MA (US); Julia B. Berthet, 6,225,046 B1 5/2001 Vesey et al. Framingham, MA (US); Dinah R. 6,503,761 B1 1/2003 Koenig et al. Super, Lexington, MA (US); Martin 6,528,624 B1 3/2003 Idusogie et al. 6,562,784 B1 5/2003 Thiel et al. M. Rottman, Cambridge, MA (US); 6,733,753 B2 5, 2004 Boone et al. Alexander Watters, Melrose, MA (US) 6,846,649 B1 1/2005 Thiel et al. 6,900,292 B2 5/2005 Sun et al. (73) Assignee: PRESIDENT AND FELLOWS OF 7,202,207 B2 4/2007 Thiel et al. 7,211,396 B2 5/2007 Uttenthal HARVARD COLLEGE, Cambridge, 7,226,429 B2 6, 2007 Tullis MA (US) (Continued) (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 FOREIGN PATENT DOCUMENTS U.S.C. 154(b) by 0 days. EP O375,736 B1 5, 1998 EP O861667 A2 9, 1998 (21) Appl. No.: 14/233,553 (Continued)

(22) PCT Filed: Jul. 18, 2012 OTHER PUBLICATIONS (86). PCT No.: Azevedo et al. 2003; Horseradish peroxidase: a valuable tool in biotechnology. Biotechnology Annual Review, 9: 199-247.* S 371 (c)(1), Functional MBL Elisa kit (ligand elisa); created Feb. 28, 2006 on (2), (4) Date: Apr. 18, 2014 the web at mastgrp.biz Sanquin Glossies/MBL%20flyerO6.pdf.* Brooks et al., “Expression and secretion of ficolin beta by porcine (87) PCT Pub. No.: WO2013/012924 neutrophils'. Biochim. Biophys. Acta 1624 (1-3):36-45 (2003). Fox et al., “Single amino acid substitutions on the Surface of PCT Pub. Date: Jan. 24, 2013 Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins”. Protein Sci. 10(3):622 (65) Prior Publication Data 630 (2001). Grogl et al., “Leishmania braziliensis: protein, carbohydrate, and US 2014/0227723 A1 Aug. 14, 2014 antigen differences between log phase and stationary phase promastigotes in vitro”. Exp. Parasitol. 63(3):352-359 (1987). Related U.S. Application Data (Continued) (60) Provisional application No. 61/508,957, filed on Jul. Primary Examiner — Karen Cochrane Carlson 18, 2011, provisional application No. 61/605,052, (74) Attorney, Agent, or Firm — Nixon Peabody LLP filed on Feb. 29, 2012, provisional application No. 61/605,081, filed on Feb. 29, 2012. (57) ABSTRACT Described herein are engineered microbe-targeting or (51) Int. Cl. microbe-binding molecules, kits comprising the same and C07K I4/42 (2006.01) uses thereof. Some particular embodiments of the microbe C07K 6/12 (2006.01) targeting or microbe-binding molecules comprise a carbo A6 IK 47/48 (2006.01) hydrate recognition domain of mannose-binding lectin, or a GOIN 33/543 (2006.01) fragment thereof, linked to a portion of a Fc region. In some GOIN 33/569 (2006.01) embodiments, the microbe-targeting molecules or microbe (52) U.S. Cl. binding molecules can be conjugated to a Substrate, e.g., a CPC ...... C07K 16/12 (2013.01); A61K 47/4843 magnetic microbead, forming a microbe-targeting Substrate (2013.01); A61K 47/48369 (2013.01); C07K (e.g., a microbe-targeting magnetic microbead). Such 14/42 (2013.01); G0IN 33/54353 (2013.01); microbe-targeting molecules and/or Substrates and the kits G0IN 33/569 II (2013.01); G0IN 33/569 16 comprising the same can bind and/or capture of a microbe (2013.01); G0IN 33/56938 (2013.01); G0IN and/or microbial matter thereof, and can thus be used in 33/56961 (2013.01); C07K 2319/33 (2013.01); various applications, e.g., diagnosis and/or treatment of an G0IN 2333/42 (2013.01); G0IN 2333/4724 infection caused by microbes such as sepsis in a Subject or (2013.01) any environmental Surface. Microbe-targeting molecules (58) Field of Classification Search and/or Substrates can be regenerated after use by washing CPC C07K 2319/30; C07K 16/2866; C07K 14/42: with a low pH buffer or buffer in which calcium is insoluble. A61K 39/395 See application file for complete search history. 18 Claims, 40 Drawing Sheets US 9,593,160 B2 Page 2

(56) References Cited Jarva et al., “Streptococcus pneumoniae evades complement attack and opsonophagocytosis by expressing the pspC locus-encoded Hic U.S. PATENT DOCUMENTS protein that binds to short consensus repeats 8-11 of factor H'. J. Immunol. 168(4): 1886-1894 (2002). 7,439,224 B2 10/2008 Thiel et al. Kehres, “A kinetic model for binding protein-mediated arabinose 7.462,596 B2 12/2008 Larsen et al. transport', Protein Sci. 1 (12): 1661-1665 (1992). 7,695,937 B2 4/2010 Baum Ye et al., "Surface display of a glucose binding protein'. Journal of 7,763,436 B2 7, 2010 Das et al. Molecular Catalysis B: Enzymatic 28(4-6):201-206 (2004). 8,080.245 B2 12/2011 Visintin et al. Bangs Laboratories, Inc., “Protein Coated Microspheres'. Tech. 8,084.275 B2 12/2011 Hirai et al. Note #51 (1997). 8,088,596 B2 1/2012 Zeng et al. Brouwer et al., “Mannose-Binding Lectin (MBL) Facilitates 8,415, 118 B2 4/2013 Huang et al. 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(56) References Cited Arakawa et al., “Elution of antibodies from a protein-A column by aqueous arginine solutions.” Protein Expression and Purification OTHER PUBLICATIONS 36:244-248 (2004). Armour et al., “Recombinant human IgG molecules lacking Nisnevitch et al., J. Biochem. Biophys. Methods, 49:467-480 Ficgamma receptor I binding and monocyte triggering activities.” (2001). “The Solid Phase in Affinity Chromatography: Strategies for Antibody Attachment.” Eur, J. Immunol. 29:2613-2624 (1999). Terai et al., Eur, J. Immunol. 33:2755-2763 (2003). “Relationship Ashkenazi et al., “Immunoadhesins as research tools and therapeu between Gene Polymorphisms of Mannose-Binding Lectin (MBL) tic agents.” Curr Opin Immunol. 9(2):195-200 (1997). and Two Molecular Forms of MBL.” Chamow et al., “Immunoadhesins: principles and applications.” Witus et al., J. Am. Chem. Soc., 132: 16812-16817 (2010). “Iden Trends Biotechnol. 14(2):52-60 (1996). tification of Highly Reactive Sequences for PLP-Mediated Chang et al., “Crystallization and preliminary X-ray analysis of a Bioconjugation Using a Combinatorial Peptide Library.” trimeric form of human mannose binding protein.” J. Mol. Biol. Nadesalingam et al., “Mannose-binding lectin recognizes 241:125-127 (1994). peptidoglycan via the N-acetyl glucosamine moiety, and inhibits Foster “Immune evasion by Staphylococci.” Nature 3:948-958 ligand-induced proinflammatory effect and promotes chemokine (2005). production by macrophages.” Journal of Immunology 175: 1785 Hinton et al., “Engineered human IgG antibodies with longer serum 1794 (2005). half-lives in primates.” The Journal of Biological Chemistry Nakamura et al., “Characterization of the interaction between serum 279(8):6213-6216 (2004). mannan-binding protein and nucleic acid ligands.” Journal of Leu Idusogie et al., “Engineered antibodies with increased activity to kocyte Biology 86:737-748 (2009). recruit complement.” The Journal of Immunology 166 (4):2571-5 Ogden et al., “C1q and mannose binding lectin engagement of cell (2001). Surface calreticulin and CD91 initiates macropinocytosis and uptake Keen et al., “Interrelationship between pH and surface growth of of apoptotic cells.” J. Exp. Med. 194:781-795 (2001). nitrobacter.” Soil Biol. Biochem. 19(6):665-672 (1987). Stuart et al., “Mannose-binding lectin-deficient mice display defec Schmidt, "Fusion proteins as biopharmaceuticals—applications and tive apoptotic cell clearance but no autoimmune phenotype.” Jour challenges.” Current Opinion in Drug Discovery & Development nal of Immunology 174:3220-3226 (2005). Warwicket al., “Use of quantitative 16S ribosomal DNA detection 12(2):284-295 (2009). for diagnosis of central vascular catheter-associated bacterial infec Shields et al., “High resolution mapping of the binding site on tion.” Journal of Clinical Microbiology 42:1402-1408 (2004). human IgG1 for Fc gamma RI. Fc gamma RII, Fc gamma RIII, and Bossola et al., “Circulating bacterial-derived DNA fragments and FcRn and design of IgG1 variants with improved binding to the Fc markers of inflammation in chronic hemodialysis patients.” Clin J. gamma R.” The Journal of Biological Chemistry 276(9):6591-6604 Am. Soc. Nephrol. 4:379-385 (2009). (2001). Bayston et al., "Bacterial endotoxin and current concepts in the Steurer et al., “Ex vivo coating of islet cell allografts with murine diagnosis and treatment of endotoxaemia.” J. Med. Microbiol CTLA4/Fc promotes graft tolerance.” The Journal of Immunology 31:73-83 (1990). 155(3): 1165-1174 (1995). Michelow et al., “A novel L-ficolin?mannose-binding lectin chime Vaccaro et al., “Engineering the Fc region of immunoglobulin G to ric molecule with enhanced activity against Ebola virus.” Journal of modulate in vivo antibody levels.” Nature Biotechnology Biological Chemistry 285(32):24729-24739 (2010). 23(1): 1283-1288 (2005). Takahashi et al., “Mannose-binding lectin and its associated Wong et al., “Bioinspired self-repairing slippery Surfaces with proteases (MASPs) mediate coagulation and its deficiency is a risk pressure—stable omniphobicity.” Nature 477:443-447 (2011). factor in developing complications from infection, including dis Gouin et al., “Multimeric Lactoside “Click Clusters' as Tools to Seminated intravascular coagulation.” Immunobiology 216(1-2):96 Investigate the Effect of Linker Length in Specific Interactions with 102 (2011). Peanut Lectin, Galectin-1, and -3', ChemEBioChem 11(10): 1430 Invivo Gen Insight, “IgG-Fc Engineering for Therapeutic Use.” 1442 (2010). Apr./May 2006. Sibille et al., "Comparison of serological tests for the diagnosis of Product Data Sheet: Human Mannan Binding Lectin peptide (237 feline immunodeficiency virus infection of cats'. Veterinary Micro 248) (Carboxyterminal end) ab45655. biology 45(2-3): 259-267 1995). Loosdrecht et al., “Influence of interfaces on microbial activity.” Microbiological Reviews 54(1):75-87 (1990). * cited by examiner U.S. Patent Mar. 14, 2017 Sheet 1 of 40 US 9,593,160 B2

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s as s s a. & w w s s s: as: ex xces ex as se: s 3. & w sw: -w (.333i US 9,593,160 B2 1. 2 ENGINEERED MICROBE-TARGETING cal means (stain, culture, antigenemia or antigenuria, nucleic MOLECULES AND USES THEREOF acid detection) or pathognomonic signs of infection obtained by imaging or clinical examination. The infection can affect CROSS REFERENCE TO RELATED any organ system, but the more severe cases present as APPLICATIONS 5 septicemia (i.e., organisms, their metabolic end-products or toxins in the blood stream), bacteremia (i.e., bacteria in the This Application is a 35 U.S.C. S371 National Phase blood), toxemia (i.e., toxins in the blood), endotoxemia (i.e., Entry Application of International Application No. PCT/ endotoxin in the blood). Sepsis can also result from US2012/047201 filed Jul. 18, 2012, which designates the fungemia (i.e., fungi in the blood), Viremia (i.e., viruses or U.S., and which claims the benefit under 35 U.S.C S119(e) 10 virus particles in the blood), and parasitemia (i.e., helminthic of U.S. Provisional Application No. 61/508,957 filed Jul.18, or protozoan parasites in the blood). Thus, septicemia and 2011: 61/605,081 filed Feb. 29, 2012; and 61/605,052 filed septic shock (acute circulatory failure resulting from Septi Feb. 29, 2012, the contents of each of which are herein cemia often associated with multiple organ failure and a high incorporated by reference in their entirety. 15 mortality rate) may be caused by various microorganisms. There are three major types of sepsis characterized by the GOVERNMENT SUPPORT type of infecting organism. For example, gram-negative sepsis is the most frequently isolated (with a case fatality This invention was made with Government support under rate of about 35%). The majority of these infections are grant no. N66001-11-1-4180 awarded by DARPA. The caused by Escherichia coli, Klebsiella pneumoniae and Government has certain rights in the invention. 2O Pseudomonas aeruginosa. Gram-positive pathogens such as SEQUENCE LISTING the Staphylococci and Streptococci are the second major cause of sepsis. The third major group includes fungi, with The instant application contains a Sequence Listing which fungal infections causing a relatively small percentage of has been Submitted in ASCII format via EFS-Web and is 25 sepsis cases, but with a high mortality rate; these types of hereby incorporated by reference in its entirety. Said ASCII infections also have a higher incidence in immunocomprised copy, created on Jan. 15, 2014, is named 28671233.txt and patients. is 22,557 bytes in size. Some of these infections can be acquired in a hospital setting and can result from certain types of Surgery (e.g., TECHNICAL FIELD 30 abdominal procedures), immune Suppression due to cancer or transplantation therapy, immune deficiency diseases, and Described herein relates generally to molecules, products, exposure through intravenous catheters. Sepsis is also com kits and methods for detecting and/or removing microbes in monly caused by trauma, difficult newborn deliveries, and a sample or a target area, including bodily fluids such as intestinal torsion (especially in dogs and horses). Infections blood and tissues of a subject, food, water, and environmen- 35 in the lungs (pneumonia), bladder and kidneys (urinary tract tal Surfaces. infections), skin (cellulitis), abdomen (such as appendicitis), bone (osteomyelitis) and joints (arthritis) and other areas BACKGROUND (such as meningitis) can spread and also lead to sepsis. In Some circumstances, ingestion of microbe-contaminated Sepsis is a major cause of morbidity and mortality in 40 water, fluid or food, or contact with microbe-covered envi humans and other animals. In the United States, sepsis is the ronmental Surfaces can cause infections that lead to sepsis, second leading cause of death in intensive care units among and infection with food-borne and water-borne pathogens patients with non-traumatic illnesses. It is also the leading Such as Shigella spp., or certain serotypes of Escherichichia cause of death in young livestock, affecting 7.5-29% of coli (such as O157H7), Salmonella spp including Salmo neonatal calves, and is a common medical problem in 45 nella enterica serovar typhi or Listeria monocytogenes can neonatal foals. Despite the major advances of the past also lead to sepsis. several decades in the treatment of serious infections, the Many patients with septicemia or Suspected septicemia incidence and mortality due to sepsis continues to rise. exhibit a rapid decline over a 24-48 hour period. It has been Sepsis results from the systemic invasion of microorgan reported that patients with septic shock require adapted isms into blood and can present two distinct problems. First, 50 treatment in less than 6 hours in order to benefit from the growth of the microorganisms can directly damage antimicrobial therapy. Thus, rapid and reliable diagnostic tissues, organs, and vascular function. Second, toxic com and treatment methods are essential for effective patient ponents of the microorganisms can lead to rapid systemic care. Unfortunately, a confirmed diagnosis as to the type of inflammatory responses that can quickly damage vital infection, e.g., sepsis, traditionally requires microbiological organs and lead to circulatory collapse (i.e., septic shock) 55 analysis involving inoculation of blood cultures, incubation and, often times, death. for 18-24 hours, plating the causative microorganism on Sepsis is a systemic reaction defined by the American Solid media, another incubation period, and final identifica College of Chest Physicians and the Society of Critical Care tion 1-2 days later. Even with immediate and aggressive Medicine by a systemic inflammatory response (SIRS) in treatment, some patients can develop multiple organ dys response to a confirmed infectious process. SIRS is defined 60 function syndrome and eventually death. Hence, there by the presence of two or more of the following: altered remains a strong need for improved techniques for diagnosis body temperature (<36° C. or >38° C.), tachycardia (heart and treatment of patients with infectious diseases, blood rate-90/min), tachypnea (respiratory rate-20/min) or borne infections, sepsis, or systemic inflammatory response hypocapnia (PCO less than 4.3 kPa), leucopenia (white syndrome. The ability to rapidly detect infectious pathogens blood cells (WBCs)<4000 cells/mm or leucocytosis 65 in food, water, and/or environmental Surfaces would also (>12000 WBC/mm) or >10% band forms. The confirma have great value for preventing infections and sepsis in the tion of the infectious process is confirmed by microbiologi population. US 9,593,160 B2 3 4 SUMMARY for orienting the CRD away from the substrate; and (c) at least one linker between the CRD and the substrate-binding Embodiments described herein are based on, at least in domain. part, engineering a microbe-targeting molecule or a In some embodiments, the microbe-Surface binding microbe-binding molecule. For example, in one embodi domain comprises the full amino acid sequence of a carbo ment, a microbe-targeting molecule is engineered by fusing hydrate-binding protein. In some embodiments, the amino the carbohydrate recognition domain and neck region of a acid sequence of the carbohydrate-binding protein does not carbohydrate-binding protein (e.g., mannose-binding lectin) include a complement region. In some embodiments, the to the C-terminal of a Fc fragment of human IgG1. Such amino acid sequence of the carbohydrate-binding protein microbe-targeting molecules can be also modified to reduce 10 does not include a coagulation activation region. the complement activation and coagulation side effects In some embodiments of any aspects described herein, the which are present in the wild-type mannose-binding lectin, linker can comprise a portion of a Fc region of an immu and can complicate binding and detection. Further, the noglobulin, e.g., IgG1. In Such embodiments, the portion of microbe-targeting molecules described herein can be engi the Fc region can be linked, directly or indirectly, to N-ter neered, e.g., by inserting an AKT tripeptide to the N-termi 15 minal of the carbohydrate recognition domain. In some nal of the Fc fragment for site-specific biotinylation, such embodiments, the portion of the Fc region can be genetically that their carbohydrate recognition domains orient away modified, e.g., to increase half-life of the engineered mol from a substrate to which they attach, thus increasing the ecules, or modulate an immune response (e.g., antibody microbe-binding capacity. The microbe-targeting molecules dependent cell-mediated cytotoxicity and complement-de can be attached to various Substrates, e.g., a magnetic pendent cytotoxicity). microbead, in a multivalent oriented manner to form a In some embodiments of any aspects described herein, the microbe-targeting substrate. The term “microbead' as used Substrate-binding domain can comprise at least one oligo herein generally refers to a bead or a particle of any material peptide comprising an amino acid sequence of AKT. In other having a size of about 0.001 um to about 1000 um or about embodiments, the Substrate-binding domain can comprise a 0.001 um to about 100 um, or about 0.01 um to about 10 um. 25 biotin molecule. Depending on various applications, e.g., for In one embodiment, the microbead is a nanobead. The term use as a soluble protein in pharmaceutical compositions, the “nanobead” as used herein generally refers to a bead or Substrate-binding domain can become non-essential in some particle having a size ranging from about 1 nm to about 1000 embodiments of the engineered microbe-targeting mol nm, from about 10 nm to about 500 nm, from about 25 nm. ecules. Otherwise, the engineered microbe-targeting mol to about 300 nm, from about 40 nm to about 250 nm, or from 30 ecules can be used to coat various Substrates for a wide about 50 nm to about 200 nm. variety of applications. In some embodiments, the Substrate In some embodiments, the microbe-targeting molecules is a magnetic microbead, resulting in formation of a can be modified, e.g., to facilitate attachment of the microbe microbe-targeting magnetic microbead or opsonin. In some targeting molecules to a Substrate. For example, in one embodiments, the microbe-targeting magnetic microbead or embodiment, the microbe-targeting molecules can be bioti 35 opsonin can encompass a microbe-targeting nanobead. nylated, e.g., for attachment to an avidin- or avidin-like Not only can the microbe-targeting magnetic microbeads coated Substrate. Thus, the engineered microbe-targeting be used to remove microbes or pathogens in a sample, e.g., molecules described herein provide a valuable building blood and tissues, they can also be used to develop assays for block for various applications, e.g., diagnosis and/or treat detecting the presence or absence of, and/or differentiating ment of diseases caused by microbes or pathogens, removal 40 between, different microbes or pathogens. Accordingly, kits of microbes or pathogens from a sample, including bodily and assays for detecting the presence or absence of fluids and tissues of a Subject, foods, water, or an environ microbes, and/or differentiating between, different microbes mental Surface; and development of targeted drug delivery or pathogens in a test sample are also provided herein. In devices. Some embodiments, the kits comprise microbe-targeting Accordingly, provided herein is directed to an engineered 45 Substrates (e.g., but not limited to, one or more containers microbe-targeting molecule comprising: (a) at least one each containing a population of magnetic microbeads coated microbe Surface-binding domain; (b) a Substrate-binding with a plurality of the engineered microbe-targeting mol domain adapted for orienting the microbe Surface-binding ecules); and at least one reagent. In some embodiments, the domain away from the Substrate; and (c) at least one linker kits can further comprise one or more containers each between the microbe surface-binding domain and the sub 50 containing a population of detectable labels, wherein each of strate-binding domain. the detectable labels is conjugated to a molecule that binds In some embodiments, the microbe-Surface binding to the microbes or pathogens. Such kits can be used for domain can comprise a carbohydrate recognition domain analysis, e.g., by an enzyme-linked immunosorbent assay (CRD) or a fragment thereof. In some embodiments, the (ELISA), fluorescent linked immunosorbent assay (FLISA), microbe-Surface binding domain can further comprise a non 55 immunofluorescent microscopy, fluorescence in situ hybrid carbohydrate recognition domain or fragment thereof from ization (FISH), or any other radiological, chemical, enzy the carbohydrate-binding protein, e.g., a neck region of the matic or optical detection assays. In some embodiments, the carbohydrate-binding protein. As used herein, the term “non kits and assays described herein can be adapted for antibiotic carbohydrate recognition domain refers to the portion or Susceptibility tests, e.g., to determine Susceptibility of a fragment of a carbohydrate-binding protein that does not 60 microbe in a test sample to one or more antibiotics, regard directly bind with the microbe surface. less of whether the identity of the microbe is known or not. In some embodiments, the CRD or the carbohydrate Without limitations, in some embodiments, the engi binding protein can be derived from, e.g., mannose-binding neered microbe-targeting molecules can be formulated as an lectin. Hence, another aspect provided herein is directed to antibiotic or antiseptic for use in various applications, e.g., an engineered mannose-binding lectin molecule comprising: 65 wound dressings, alone or in combination with other wound (a) at least one carbohydrate recognition domain (CRD) or dressing protocols, e.g., silver nanoparticles and other a fragment thereof; (b) a Substrate-binding domain adapted wound treatment. US 9,593,160 B2 5 6 BRIEF DESCRIPTION OF THE DRAWINGS 20), followed by FcMBL-based ELISA analysis. “2" run” corresponds to the “input' blood sample incubated with 20 FIGS. 1A-1C shows a general scheme of engineering one ul/ml MYONETM FCMBL microbeads for microbe capture or more embodiments of engineered microbe-targeting or (with mixing on a shaker 10'), followed by FcMBL-based microbe-binding molecules and microbe-targeting Sub 5 ELISA analysis. strates described herein. FIG. 1A is a diagrammatic view of FIG. 10 is a schematic diagram of an exemplary ELISA a native (wild-type) mannose-binding lectin (MBL). FIG. assay comprising engineered microbe-targeting magnetic 1B shows one or more embodiments of the engineered microbeads according to one or more embodiments. The microbe-targeting molecules or engineered-binding mol ELISA assay can be used for any diagnostic applications, ecules, e.g., engineered MBL molecules. FIG. 1C shows one 10 e.g., for sepsis tests. or more embodiments of the microbe-targeting or microbe FIG. 11 is a graph showing results of detecting C. albicans binding molecules conjugated to a Substrate, e.g., a magnetic in blood. Serial dilutions of C. albicans were spiked into microbead or nanobead, to form a microbe-targeting Sub blood, captured by AKT-FcMBL magnetic microbeads (1 Strate. um) and detected by an ELISA method using HRP-labeled FIG. 2 shows a crystal structure of a portion of a wild-type 15 FCMBL. MBL, which is the “neck and carbohydrate recognition FIG. 12 is a graph showing bacterial detection sensitivity domain (CRD) head.” The crystal structure depicts three of one or more embodiments of the FcMBL-based ELISA MBL heads, and calcium binding sites (Chang et al. (1994) assay. Serial dilutions of E. coli were spiked into a buffer, J Mol Biol. 241:125-7). captured by AKT-FcMBL magnetic microbeads (about 128 FIG. 3 is a schematic diagram showing an exemplary nm in size) and detected by an ELISA method using HRP Fc-X vector construct for one or more embodiments of the labeled FcMBL. In some embodiments, the limit of detec engineered microbe-targeting or microbe-binding molecules tion (LOD) of the FcMBL-based ELISA colorimetric assay described herein. is about or below 160 E. coli bacteria. FIG. 4 shows a Western blot image indicating expression FIG. 13 is a schematic diagram showing one or more of the purified wild-type MBL (MBLWT) proteins and one 25 embodiments of a dipstick assay for microbial detection. or more embodiments of the engineered microbe-targeting The FcMBL can be attached to a membrane (for example or microbe-binding molecules described herein (FcMBL.81: Biodyne membrane). The membrane can be mixed with a SEQ ID NO. 6). test sample (e.g., blood sample), washed, incubated with a FIG. 5 shows the mannan-binding results of various desired detecting protein (e.g., AP-labeled FcMBL or spe embodiments of the engineered microbe-targeting or 30 cific antibody for certain microbes, e.g., bacteria or fungus), microbe-binding molecules described herein in the presence washed and added with a readout reagent for colorimetric or absence of calcium ions. A chelating agent (e.g., EDTA) development. The dipstickassay can be performed manually can be added to the sample to remove calcium ions. or modified for automation. FIGS. 6A and 6B are bar graphs showing the results of FIG. 14 is a schematic diagram showing one or more capturing microbes, e.g., C. albicans, with one or more 35 embodiments of an ELISA-based test for microbial detec embodiments of the microbe-targeting Substrates (e.g., AKT tion. A test sample (e.g., blood sample) can be added into a FcMBL.81 conjugated to magnetic microbeads having a size single tube (e.g., a blood collection container Such as EDTA of about 1 um at various microbe densities. FIG. 6A shows VACUTAINER(R) containing lyophilized FcMBL magnetic the percentage of microbes bound to microbe-targeting microbeads or FcMBL-coated magnetic microbeads. An substrates and controls at a low microbe density (e.g., 1500 40 exemplary protocol for microbial capture and detection is C. albicans cells). FIG. 6B shows the amount of unbound described in Example 10. The ELISA-based test can be microbes remained in the microbe samples after treatment performed manually or modified for automation. In some with different magnetic microbeads (including the engi embodiments, the single-tube based ELISA assay can be neered microbe-targeting magnetic microbeads) when the used to detect microbes or pathogens such as S. aureus and microbe is present at a much higher microbe density (e.g., 45 E. coli. greater than 10 cells). FIG. 15 is an image showing direct detection of bacteria FIG. 7 shows the size effect of one or more embodiments on a membrane by AP-labeled FcMBL. Serial dilutions of E. of the microbe-targeting Substrates (e.g., microbe-targeting coli and S. aureus (10' to 10) were spotted directly onto magnetic microbeads such as AKT-FcMBL.81 magnetic a Biodyne membrane, blocked for about 30 mins in 1% microbeads, wherein the size of the microbeads were varied 50 casein, washed twice in TBST containing Ca" (5 mM), from about 100 nm to about 1000 nm diameter) on the incubated with AP-labeled FcMBL (1:10,000 dilution) in efficiency of capturing microbes or pathogens, e.g., Can 3% BSA 1XTBST containing Ca" (for about 20 min), dida. washed twice in TBST containing Ca" (5 mM) and once in FIG. 8 shows the amount of unbound microbes remained TBS containing Ca" (5 mM), and reacted with BCIP/NBT in the microbe samples after treatment with different mag 55 for about 20 mins to develop a colorimetric readout. In this netic microbeads (including the engineered microbe-target example, maximum dilution allowed for detection of both ing magnetic microbeads) when the microbes (e.g., Can species was 10' after 30 min development (corresponding dida) are growing in a log phase VS. in a saturated phase. to detection of 130 E. coli and 343 S. aureus cells). FIG. 9 shows the depletion of microbe/microbial matter FIG. 16 is an image showing capture and detection of S. from a blood sample from a human donor as measured using 60 aureus by dot blot using a membrane coupled with FcMBL. FcMBL, ELISA. In the figure, “input corresponds to undi Dilutions of S. aureus (10° and 10) were captured by luted EDTA donor blood spiked with S. aureus or E. coli, FcMBL immobilized on a Biodyne membrane. For example, and supplemented with Ca" (final Ca"=5 mM) and 5uL of two indicated concentrations of FcMBL were spotted heparin (4 mg/ml), before addition of any FcMBL micro onto a Biodyne membrane, allowed to dry, blocked in 1% beads. "1 run” corresponds to the "input' blood sample 65 casein, and washed twice in TBST containing Ca" (5 mM). incubated with 20 ul/mL MYONETM FCMBL microbeads Each FcMBL concentration was assessed for capture (~10 for microbe capture (with mixing on a HULAMIXERTM min) of serial dilutions of S. aureus, washed, and detected US 9,593,160 B2 7 8 with 1:10,000 dilution of AP-labeled FcMBL in 3% BSA FIG. 27 is a bar graph comparing different microbial or 1XTBST containing Ca" (~20 min). Excess AP-labeled pathogenic species captured on FcMBL-coated Substrates FcMBL was removed by washes (e.g., washing three times (e.g., magnetic microbeads) in the presence or absence of a with TBST containing Ca" (5 mM) and once with TBS chelating agent (e.g., EDTA) and various Ca" concentra containing Ca" (5 mM)). Colorimetric detection was devel tions. oped with BCIP/NBT for -20 min. FIG. 28 is an image showing colorimetric outcomes of the FIG. 17 is a schematic of an exemplary microbial detec tube-based ELISA assay for S. aureus and E. coli binding to tion process or diagnosis process. FcMBL-coated Substrates (e.g., magnetic microbeads) in the FIGS. 18A and 18B are line graphs showing ELISA of E. presence or absence of a chelating agent (e.g., EDTA) and/or 10 a low pH buffer. coli on two different FcMBL, microbead formats. FIG. 18A FIG. 29 is an image showing dot blot determination of E. corresponds to FcMBL directly coupled to MYONETM Tosyl coli and S. aureus with or without EDTA in the capture activated beads and FIG. 18B corresponds to biotinylated and/or wash buffer. AKT-FcMBL coupled to Streptavidin MYONETMT1 micro FIGS. 30A-30B are images showing binding of one or beads (~1000 nm diameter). Three different dilutions of an 15 more embodiments of microbe-targeting Substrates to E. coli overnight culture were captured on FcMBL micro microbial matter, including live microbes and/or fragments beads, washed with one of four elution buffers and then run or matter derived from microbes. FIG. 30A shows that through one or more embodiments of the ELISA protocol microbial outgrowth is observed when one or more microbe described herein. A decrease in signal corresponds to fewer targeting Substrates (e.g., FcMBL-coated fluorescent micro E. coli bound to the microbeads prior to the ELISA detec beads) bind(s) to at least one live microbe, e.g., E. coli. FIG. tion. 30B is a set of fluorescent images showing that FcMBL FIG. 19 is an image showing plating out of equal titers of coated fluorescent microbeads bind to microbial matter (left S. aureus either mixed with FcMBL microbeads or control panel) including live microbes (indicated by the middle without FcMBL microbeads. panel) and fragments or matter derived from microbes. The FIGS. 20A and 20B are line graphs showing capture 25 right panel is an overlay of the first two fluorescent images efficiency of engineered microbe-targeting or microbe-bind in addition to a bright-field image. ing molecules (e.g., FcMBL) in clinical isolates of different FIGS. 31A-31B are images showing capture of microbes microbial species. FIG. 20A shows data for capture effi or fragments thereof on one or more embodiments of ciency of FcMBL in the clinical isolates of S. aureus and microbe-targeting Substrates from fluid samples, followed methicillin-resistant S. aureus (MRSA). FIG. 20B shows 30 by antibody characterization. FIG. 31A shows capture of E. data for capture efficiency of FcMBL in the clinical isolates coli or fragments thereof on FcMBL-coated microbeads of S. aureus, MRSA, N. meningitidis, and P. aeroginosa. (e.g., magnetic or fluorescent microbeads) from heparinized FIGS. 21A-21B are line graphs showing capture effi blood, followed by incubation with an antibody against E. ciency of engineered microbe-targeting or microbe-binding coli lipopolysaccharide lipid A (anti-LPS lipid A antibody. molecules (e.g., FcMBL) in clinical isolates obtained from 35 FIG. 31B shows capture of E. coli or fragments thereof on different types of fluids. FIGS. 21A and 21B shows data for FcMBL-coated microbeads (e.g., magnetic or fluorescent capture efficiency of FcMBL in the clinical isolates of S. microbeads) from blood containing EDTA anticoagulation aureus and E. coli, respectively, obtained from other body agent, followed by incubation with an antibody against E. fluids, e.g., urine, cerebrospinal fluid (CSF), and sputum. coli lipopolysaccharide lipid A (anti-LPS lipid A antibody). FIG. 22 is a schematic diagram showing mechanism by 40 Both FIGS. 31A-31B show that the anti-LPS lipid A anti which S. aureus avoids opsonophagocytosis. See additional body does not bind to FcMBL-coated microbeads in the details in Fraser T., Nature Reviews Microbiology 2005: absence of E. coli or fragments thereof. 3(12):948-58. FIGS. 32A-32B are images showing capture of microbes FIG. 23 is a bar graph showing detection signals of on one or more embodiments of microbe-targeting Sub various concentrations of S. aureus captured by AKT-Fc 45 strates from samples of a rat sepsis model, followed by MBL 1 uM magnetic microbeads and detected by FcMBL antibody characterization. FIG. 32A shows capture of HRP ELISA. Sensitivity of this embodiment of the assay microbes or fragments thereof on FcMBL-coated micro was about 149 CFU/mL. beads (e.g., magnetic or fluorescent microbeads) from rat FIG. 24 is a bar graph showing elution of S. aureus and blood (upper panel) or pleural (lower panel) fluids after E. coli bacteria bound onto FcMBL-coated substrates (e.g., 50 24-hr infection, followed by incubation with an anti-LPS magnetic microbeads) with different treatments, including lipid A antibody. FIG. 32B shows capture of microbes or chelation, pH and salt washes. fragments thereof on FcMBL-coated microbeads (e.g., mag FIGS. 25A and 25B are bar graphs showing elution of E. netic or fluorescent microbeads) from rat blood (upper coli and S. aureus off FcMBL-coated Substrates (e.g., mag panel) or pleural (lower panel) fluids after 72-hr infection, netic microbeads) using chelators. FIG. 25A shows the 55 followed by incubation with an anti-LPS lipid A antibody. results in OD450 and FIG. 25B shows the results as a FIG. 33 is a set of images showing the use of specific percent of bound bacteria remained on the FcMBL-coated antibodies to microbes to allow further discrimination or substrates after treatment. identification of samples that indicate positive signals with FIGS. 26A and 26B show results of tube-based ELISA for one or more embodiments of microbe-targeting Substrates. S. aureus and E. coli binding to FcMBL-coated substrates 60 De-identified clinical blood samples were screened by (e.g., magnetic microbeads) in the presence of a chelating FcMBL, ELISA described herein and the captured microbial agent (e.g., EDTA). FIG. 26A is an image showing colori matters (including intact cells and fragments thereof) on the metric outcomes of the tube-based ELISA for S. aureus and FcMBL-coated microbeads were further screened by using E. coli binding to FcMBL-coated Substrates (e.g., magnetic an anti-LPS lipid A antibody. The top panel indicates that no microbeads) in the presence or absence of a chelating agent 65 detection of anti-LPS lipid A antibody signal was observed (e.g., EDTA). FIG. 26B is a bar graph showing quantitative in clinical samples with Substantially negative or negligible measurement of the color developed in FIG. 26A. signal from FcMBL, ELISA, indicative of no microbial US 9,593,160 B2 10 infection detected in the clinical samples. The middle panel tation as to the contents of these documents is based on the indicates that the microbial matter producing positive signal information available to the applicants and does not consti (OD=-1.69) in FcMBL ELISA bound to anti-LPS lipid A tute any admission as to the correctness of the dates or antibody, which indicates that the microbial matter could be contents of these documents. derived from E. coli, and that the corresponding clinical Unless defined otherwise, all technical and scientific samples had a gram-negative infection (e.g., E. coli infec terms used herein have the same meaning as those com tion). In contrast, the bottom panel indicates that the micro monly understood to one of ordinary skill in the art to which bial matter producing positive signal (OD->3.9) did not bind this invention pertains. Although any known methods, to anti-LPS lipid A antibody, which indicates that the micro devices, and materials may be used in the practice or testing bial matter could be derived from microbes other than E. 10 of the invention, the methods, devices, and materials in this coli, e.g., when the clinical samples were infected with a regard are described herein. gram-positive microbe. Described herein are engineered microbe-targeting or FIGS. 34A-34D are data graphs showing that use of microbe-binding molecules, compositions comprising the FcMBL magnetic microbeads is a more sensitive and reli same, processes or assays, and kits for separating microbes able measure of blood-borne pathogens (including live and 15 from a test sample in vivo, in situ or in vitro, and/or detecting non-viable pathogens Such as dead pathogens and endotox the presence or absence of the microbes in the test sample. ins) than conventional blood cultures. FIG. 34A is a bar The engineered microbe-targeting or microbe-binding mol graph showing results of anaerobe cultures at Day 4 of blood ecules can bind or capture at least one microbe, e.g., an collected from five rats developed with intra-abdominal intact microbe, and/or “microbial matter.” The term “micro abscesses. FIG. 34B is a plot comparing the microbe detec bial matter as used herein refers to any matter or component tion results based on colorimetric ELISA using FcMBL that is derived, originated or secreted from a microbe. For magnetic microbeads and conventional blood cultures and example, microbial matter or a component derived or their correlations with morbidity of the rats. FIG. 34C is a secreted from a microbe that can bind to an engineered line graph showing correlation of pathogen load determined microbe-targeting or microbe-binding molecule can include, by the ELISA using FcMBL magnetic microbeads with 25 but are not limited to, a cell wall component, an outer morbidity ranking. FIG. 34D is a bar graph comparing the membrane, a plasma membrane, a ribosome, a microbial microbe detection results based on colorimetric ELISA capsule, a pili or flagella, any fragments of the aforemen using FcMBL magnetic microbeads and conventional blood tioned microbial components, any nucleic acid (e.g., DNA, cultures in a separate experiment. including 16S ribosomal DNA, and RNA) derived from a FIG. 35 is a bar graph showing percentages of microbe 30 microbe, and microbial endotoxin (e.g., lipopolysaccharide). depletion by one or more embodiments of the microbe In addition, microbial matter can encompass non-viable targeting magnetic microbeads. FcMBL-coated magnetic microbial matter that can cause an adverse effect (e.g., microbeads of different sizes (~1 um, ~128 nm, and ~50 nm) toxicity) to a host or an environment. were used to capture E. coli and S. aureus that were initially In accordance with various embodiments described spiked into a buffered solution. The microbe-bound FcMBL 35 herein, the engineered microbe-targeting molecules or coated magnetic microbeads were then removed from the microbe-binding molecules comprise a microbe Surface buffered solution. After removal of the magnetic micro binding domain (e.g., a carbohydrate recognition domain), beads, the buffered solution was used for inoculation on LB directly or indirectly, conjugated to a linker (e.g., a Fc plates to determine the level of microbe depletion by fragment), which can further comprise a substrate-binding FcMBL-coated magnetic microbeads of different sizes. 40 domain for immobilization. Thus, the engineered microbe targeting molecules or microbe-binding molecules described DETAILED DESCRIPTION OF THE herein can be used as Soluble proteins, e.g., in therapeutic INVENTION compositions, or be immobilized to a Substrate for various applications ranging from diagnosis and/or treatment of a It should be understood that this invention is not limited 45 microbial infection or disease, to microbe-clearing compo to the particular methodology, protocols, and reagents, etc., sitions or devices, to drug delivery. described herein and as Such may vary. The terminology In one aspect, provided herein is an engineered microbe used herein is for the purpose of describing particular targeting molecule (or an engineered microbe-binding mol embodiments only, and is not intended to limit the scope of ecule) comprising at least one microbe Surface-binding the present invention, which is defined solely by the claims. 50 domain, a Substrate-binding domain adapted for orienting As used herein and in the claims, the singular forms the carbohydrate recognition domain away from the Sub include the plural reference and vice versa unless the context strate, and at least one linker between the microbe surface clearly indicates otherwise. Other than in the operating binding domain and the Substrate-binding domain. In some examples, or where otherwise indicated, all numbers embodiments, the microbe Surface-binding domain can expressing quantities of ingredients or reaction conditions 55 comprise a carbohydrate recognition domain or a fragment used herein should be understood as modified in all thereof. In some embodiments, the microbe Surface-binding instances by the term “about.” domain can further comprise at least a portion of mannose All patents and other publications identified are expressly binding lectin (MBL). Accordingly, another aspect provided incorporated herein by reference for the purpose of describ herein is an engineered MBL molecule comprising at least ing and disclosing, for example, the methodologies 60 a fragment of a carbohydrate recognition domain derived described in Such publications that might be used in con from MBL: a substrate-binding domain adapted for orient nection with the present invention. These publications are ing the carbohydrate domain away from the Substrate; and at provided solely for their disclosure prior to the filing date of least one linker between the fragment of the MBL carbo the present application. Nothing in this regard should be hydrate recognition domain and the Substrate-binding construed as an admission that the inventors are not entitled 65 domain. The terms “microbe-binding molecule(s) and to antedate such disclosure by virtue of prior invention or for “microbe-targeting molecule(s) are used interchangeably any other reason. All Statements as to the date or represen herein. US 9,593,160 B2 11 12 In some embodiments of any aspects described herein, the For example, an interspecies hybrid microbe-targeting mol Substrate-binding domain adapted for orienting the carbo ecule can contain a linker, e.g., a peptide linker, from a hydrate recognition domain away from the Substrate is not murine species, and a human sequence from a carbohydrate always necessary and thus can be excluded under certain recognition domain protein, provided that they do not pro circumstances, e.g., using the engineered microbe-targeting vide unacceptable levels of deleterious effects. The engi molecules in a soluble format, e.g., for therapeutic purposes. neered microbe-targeting molecules described herein can Further, it should be noted that the engineered microbe also include those that are made entirely from murine binding molecules excluding the Substrate-binding domain derived sequences or fully human. adapted for orienting the carbohydrate recognition domain Microbe Surface-Binding Domain and Carbohydrate Rec away from the Substrate does not necessarily mean that the 10 ognition Domain engineered microbe-binding molecules cannot bind to a As disclosed herein, an engineered microbe-targeting Substrate Surface. In some embodiments, the engineered molecule can comprise at least one microbe Surface-binding microbe-binding molecules excluding the Substrate-binding domain, including at least two, at least three, at least four, at domain adapted for orienting the carbohydrate recognition least five, at least six, at least seven, at least eight, at least domain away from the substrate can still bind to a substrate 15 nine, at least ten or more microbe Surface-binding domains. surface, but the orientation of the carbohydrate recognition The term “microbe surface-binding domain” as used herein domain relative to the Substrate Surface can be random. refers to any molecule or a fragment thereof that can In some embodiments of any aspects described herein, the specifically bind to the Surface of a microbe or pathogen, engineered microbe-targeting molecule can further comprise e.g., any component present on a surface of a microbe or a detectable label, e.g., to facilitate detection of the presence pathogen, and/or any microbial matter, e.g., any matter or or absence of a microbe and/or microbial matter. Detectable component/fragment that is derived, originated or secreted labels suitable for conjugation to some embodiments of the from a microbe. Molecules that can be used in the microbe engineered microbe-targeting molecule can include any Surface-binding domain can include, for example, but are composition detectable by spectroscopic, photochemical, not limited to, peptides, polypeptides, proteins, peptidomi biochemical, immunochemical, electrical, magnetic, optical 25 metics, antibodies, antibody fragments (e.g., antigen binding or chemical means, as well as any examples of detectable fragments of antibodies), carbohydrate-binding protein, e.g., labels described herein and any equivalent thereof. In some a lectin, glycoproteins, glycoprotein-binding molecules, embodiments, the detectable labels also encompass any amino acids, carbohydrates (including mono-, di-, tri- and imaging agent (e.g., but not limited to, a bubble, a liposome, poly-saccharides), lipids, steroids, hormones, lipid-binding a sphere, a contrast agent, or any detectable label described 30 molecules, cofactors, nucleosides, nucleotides, nucleic acids herein) that can facilitate imaging or visualization of a tissue (e.g., DNA or RNA, analogues and derivatives of nucleic or an organ in a subject, e.g., for diagnosis of an infection. acids, or aptamers), peptidoglycan, lipopolysaccharide, In some embodiments, the detectable label conjugated to Small molecules, and any combinations thereof. In some the engineered microbe-targeting molecule can include an embodiments, the microbe Surface-binding domain can enzyme of horseradish peroxidase (HRP), alkaline phos 35 comprise a carbohydrate recognition domain or a fragment phatase (AP), or any combinations thereof. Conjugation of thereof. In some embodiments, a microbe Surface-binding the detectable label (e.g., HRP or AP) to any proteins and domain can comprise a peptidomimetic that mimics any antibodies are known in the art. In one embodiment, molecule or a fragment thereof that can specifically bind to FcMBL-HRP or FcMBL-AP construct is generated using the Surface of a microbe or pathogen, and/or any microbial any art-recognized methods for direct coupling HRP or AP 40 matter. For example, a microbe Surface-binding domain can to FCMBL. comprise a peptidomimetic that mimics any carbohydrate In some embodiments, the detectable label conjugated to recognition domain or a fragment thereof, e.g., carbohydrate the engineered microbe-targeting molecule can include a recognition domain of MBL or a fragment thereof, or any microbial enzyme Substrate conjugated to a detectable agent. carbohydrate recognition domain that is known in the art or For example, the detectable agent can be any moiety that, 45 a fragment thereof. In some embodiments, the microbe when cleaved from a microbial enzyme substrate by the Surface binding domain comprises the full amino acid enzyme possessed or secreted by the microbe, forms a sequence of a carbohydrate-binding protein. detectable moiety (e.g., a light-emitting signal), but that is In some embodiments, the microbe Surface-binding not detectable in its conjugated State. The microbial enzyme domain can have an amino acid sequence of about 10 to Substrate is a Substrate specific for one or more types of 50 about 300 amino acid residues, or about 50 to about 150 microbes to be detected, and it can be selected depending amino acid residues. In some embodiments, the microbe upon what enzymes the microbe possesses or secretes. See, Surface-binding domain can have an amino acid sequence of e.g., International Patent Application: WO 2011/103144 for at least about 5, at least about 10, at least about 15, at least the use of such detectable label in detection of microbes, the about 20, at least about 30, at least about 40, at least about content of which is incorporated herein by reference. 55 50, at least about 60, at least about 70, at least about 80, at General methods of preparing any embodiments of the least about 90, at least about 100 amino acid residues or engineered microbe-targeting molecules are known in the art more. For any known sequences of microbe Surface-binding (Ashkenazi, A. and S. M. Chamow (1997), “Immunoad molecules, one of skill in the art can determine the optimum hesins as research tools and therapeutic agents. Curr. Opin. length of amino acid sequence for the microbe Surface Immunol. 9(2): 195-200, Chamow, S. M. and A. Ashkenazi 60 binding domain. (1996). “Immunoadhesins: principles and applications.” In some embodiments, the microbe Surface-binding Trends Biotechnol. 14(2):52-60). In one example, an engi domain can comprise an opsonin or a fragment thereof. The neered microbe-targeting molecule can be made by cloning term “opsonin’ as used herein refers to naturally-occurring into an expression vector Such as Fc-X vector as discussed and synthetic molecules which are capable of binding to or in Lo et al. (1998) 11:495 and Example 1. 65 attaching to the Surface of a microbe or a pathogen, of acting The engineered microbe-targeting molecules can contain as binding enhancers for a process of phagocytosis. sequences from the same species or from different species. Examples of opsonins which can be used in the engineered US 9,593,160 B2 13 14 molecules described herein include, but are not limited to, mannose-containing glycoproteins on their envelopes and/or vitronectin, fibronectin, complement components such as function as a receptor for several viruses such as HIV and Cld (including any of its component polypeptide chains A, Hepatitis C. B and C), complement fragments such as C3d, C3b and C4b, Collectins are soluble pattern recognition receptors mannose-binding protein, conglutinin, Surfactant proteins. A (PRRS) belonging to the Superfamily of collagen containing and D, C-reactive protein (CRP), alpha2-macroglobulin, and C-type lectins. Exemplary collectins include, without limi immunoglobulins, for example, the Fc portion of an immu tations, mannose-binding lectin (MBL) (also known as noglobulin. mannan-binding lectin, mannan-binding protein, or man In some embodiments, the microbe Surface-binding nose-binding protein), Surfactant protein A (SP-A), Surfac 10 tant protein D (SP-D), collectin liver 1 (CL-L1), collectin domain can comprise a carbohydrate recognition domain. In placenta 1 (CL-P1), conglutinin, collectin of 43 kDa (CL Some embodiments, the microbe Surface-binding domain 43), collectin of 46 kDa (CL-46), and a fragment thereof. can further comprise at least a portion of a carbohydrate Mannose-binding lectin (MBL), also known as mannose binding protein or a portion thereof. In some embodiments, binding protein (MBP), or mannan-binding lectin or man the portion of the carbohydrate-binding proteins can activate 15 nan-binding protein, is a calcium-dependent serum protein the complement system. In alternative embodiments, the that can play a role in the innate immune response by portion of the carbohydrate-binding protein cannot activate binding to carbohydrates on the Surface of a wide range of the complement system. In some embodiments, the portion microbes or pathogens (viruses, bacteria, fungi, protozoa) of the carbohydrate-binding protein can be selected or where it can activate the complement system. MBL can also configured such that it cannot activate the complement serve as a direct opsonin and mediate binding and uptake of system, e.g., via modification. Examples of carbohydrate pathogens by tagging the Surface of a pathogen to facilitate binding proteins include, but are not limited to, lectin, recognition and ingestion by phagocytes. collectin, ficolin, mannose-binding lectin (MBL), maltose MBL is a member of the collectin family of proteins. A binding protein, arabinose-binding protein, and glucose native MBL is a multimeric structure (e.g., about 650 kDa) binding protein. Additional carbohydrate-binding proteins 25 composed of subunits, each of which contains three identical that can be included in the microbe Surface-binding domain polypeptide chains (FIG. 1A). Each MBL polypeptide chain described herein can include, but is not limited to, lectins or (containing 248 amino acid residues in length with a signal agglutinins that are derived from a plant, e.g., Galanthus sequence: SEQ ID NO. 1) comprises a N-terminal cysteine inivalis agglutinin (GNA) from the Galanthus (Snowdrop) rich region, a collagen-like region, a neck region, and a plant, and peanut lectin. In some embodiments, pentraxin 30 carbohydrate recognition domain (CRD). The sequence of each region has been identified and is well known in the art. family members, e.g., C-reactive protein, can also be used as SEQID NO. 2 shows a full-length amino acid sequence of a carbohydrate-binding protein. Pentraxin family members MBL without a signal sequence. can generally bind capsulated microbes. The carbohydrate The Surface or carbohydrate recognition function of a binding proteins can be wild-type, recombinant or a fusion 35 native MBL is mediated by clusters of three C-type carbo protein. The respective carbohydrate recognition domains hydrate-recognition domains (CRDs) held together by for Such carbohydrate-binding proteins are known in the art, coiled-coils of a-helices. The N-terminal portion collagen and can be modified for various embodiments of the engi like domain is composed of Gly-X-Y triplets. The short neered microbe-targeting molecules described herein. In N-terminal domain contains several cysteine residues that Some embodiments, peptidomimetics or any structural mim 40 form interchain disulfide bonds. Serum MBLs assemble into ics mimicking a microbe Surface-binding domain (e.g., a larger forms containing 2-4 trimeric subunits in rodents and carbohydrate recognition domain or a fragment thereof) and as many as six subunits in humans. All three oligomeric capable of binding to a microbe Surface can also be used as forms of rat serum MBP, designated MBPA, can fix comple a microbe Surface-binding domain described herein. ment, although the larger oligomers have higher specific The term “lectin’ as used herein refers to any molecules 45 activity. Many species express a second form of MBP. In including proteins, natural or genetically modified (e.g., rats, the second form, MBP-C, is found in the liver. MBP-C recombinant), that interact specifically with Saccharides does not form higher oligomers beyond the simple Subunit (e.g., carbohydrates). The term “lectin’ as used herein can that contains three polypeptides. also refer to lectins derived from any species, including, but When a native MBL interacts with carbohydrates on the not limited to, plants, animals, insects and microorganisms, 50 Surface of microbes or pathogens, e.g., calcium-dependent having a desired carbohydrate binding specificity. Examples binding to the carbohydrates mannose, N-acetylglu of plant lectins include, but are not limited to, the Legumi cosamine, and/or fucose, it can form the pathogen recogni nosae lectin family, such as ConA, soybean agglutinin, tion component of the lectin pathway of complement acti peanut lectin, lentil lectin, and Galanthus nivalis agglutinin vation. The MBL binds to surface arrays containing repeated (GNA) from the Galanthus (snowdrop) plant. Other 55 mannose or N-acetylglucosamine residues. It circulates as a examples of plant lectins are the Gramineae and Solanaceae complex with one or more MBP-associated serine proteases families of lectins. Examples of animal lectins include, but (MASPs) that autoactivate when the complex binds to an are not limited to, any known lectin of the major groups appropriate surface. The MBL and associated MASP pro S-type lectins, C-type lectins, P-type lectins, and I-type teins can activate C2/C4 convertase leading to the deposition lectins, and galectins. In some embodiments, the carbohy 60 of C4 on the pathogen Surface and opSonization for phago drate recognition domain can be derived from a C-type cytosis. The native MBL can also activate coagulation lectin, or a fragment thereof. C-type lectin can include any function through MASP proteins. carbohydrate-binding protein that requires calcium for bind While native MBL can detect microbes or pathogens and ing. In some embodiments, the C-type lectin can include, but act as opsonins for tagging the microbes for phagocytosis, are not limited to, collectin, DC-SIGN, and fragments 65 native MBLs may not be desirable for use in treatment of thereof. Without wishing to be bound by theory, DC-SIGN microbe-induced inflammatory diseases or infections, e.g., can generally bind various microbes by recognizing high sepsis, because native MBLS can activate complement sys US 9,593,160 B2 15 16 tem and induce an inflammatory response. Provided herein In some embodiments, the nucleic acid encodes a carbo is an engineered MBL molecule that binds to microbes or hydrate recognition domain having greater than 50% homol pathogens, comprising at least one carbohydrate recognition ogy, including greater than 60%, greater than 70%, greater domain or a fragment thereof, e.g., derived from MBL. In than 80%, greater than 90% homology or higher, to a some embodiments, the engineered MBL molecule can fragment of at least 50, at least 60, at least 70, at least 80, at comprises at least two, at least three or at least four carbo least 90, at least 100, at least 150 contiguous amino acids or hydrate recognition domains or a fragment thereof. In some more, of any known carbohydrate-binding molecules (e.g., embodiments, the engineered MBL molecules do not acti mannose-binding lectins). vate complement system or coagulation side effects that are The term “carbohydrate recognition domain as used present in a native MBL. Such embodiments can be used as 10 herein refers to a region, at least a portion of which, can bind dominant-negative inhibitors of downstream responses in to carbohydrates on a Surface of microbes or pathogens. For Vivo or as microbe-binding proteins that do not induce example, as shown in FIG. 1B, the carbohydrate recognition coagulation or complement fixation in vitro. For example, domain, in Some embodiments, can encompass MBL CRD the engineered MBL molecules that do not have complement 102. However, in some embodiments, the carbohydrate fixation and/or coagulation domains can act as a dominant 15 recognition domain can be also construed to encompass a negative protein in terms of activating cytokine and/or neck region 104 in addition to MBL CRD 102. In some inflammatory cascades, and thus reduce system inflamma embodiments, the carbohydrate recognition domain can tory syndrome and/or sepsis symptoms. comprise at least about 50% of its domain, including at least FIG. 1B shows a diagrammatic view of a dimeric engi about 60%, at least about 70%, at least about 80%, at least neered MBL molecule 100 according to one or more about 90% or higher, capable of binding to carbohydrates on embodiments of the engineered MBL molecules described a microbe surface. In some embodiments, 100% of the herein. The dimeric molecule 100 comprises at least two carbohydrate recognition domain can be used to bind to carbohydrate recognition domains 102 (e.g., MBL CRD) microbes or pathogens. In other embodiments, the carbohy connected, directly or indirectly, to a linker, e.g., a Fc region drate recognition domain can comprise additional regions 106. The N-terminal of the Fc region 106 can further 25 that are not capable of carbohydrate binding, but can have comprise an oligopeptide 108, e.g., comprising an amino other characteristics or perform other functions, e.g., to acid sequence AKT. In some embodiments, the carbohydrate provide flexibility to the carbohydrate recognition domain recognition domains 102 can further comprise neck regions when interacting with microbes or pathogens. 104 such as MBL neck to provide flexibility of the CRD Accordingly, in some embodiments, the carbohydrate interacting with microbes. 30 recognition domain can further comprise a neck region of The full-length amino acid sequence of carbohydrate the MBL with an amino acid sequence pdgdsslaaserka recognition domain (CRD) of MBL is shown in SEQID NO. ldtema rikkwltfslgkg (SEQ ID NO. 15) or a fragment 4. The carbohydrate recognition domain of an engineered thereof. Without wishing to be bound by theory, the neck MBL described herein can have an amino acid sequence of region can provide flexibility and proper orientation of the about 10 to about 300 amino acid residues, or about 50 to 35 CRD to bind to a microbe surface. In some embodiments, about 160 amino acid residues. In some embodiments, the the carbohydrate recognition domain can comprises a full microbe Surface-binding domain can have an amino acid length CRD of MBL (SEQID NO. 4; termed as “CRD head” sequence of at least about 5, at least about 10, at least about 102) and the neck region thereof 104, as shown in FIG. 1B. 15, at least about 20, at least about 30, at least about 40, at The amino acid sequence encoding a full-length CRD of least about 50, at least about 60, at least about 70, at least 40 MBL and the neck region thereof is shown in SEQ ID NO. about 80, at least about 90, at least about 100, at least about 5. The crystal structure of a native MBL “neck and CRD 150 amino acid residues or more. Accordingly, in some head' has been previously shown in Chang et al. (1994) J embodiments, the carbohydrate recognition domain of the Mol Biol. 241:125-7 (FIG. 2). A skill artisan can readily engineered MBL molecule can comprise SEQID NO. 4. In modify the identified CRD and fragments thereof to modu Some embodiments, the carbohydrate recognition domain of 45 late its orientation and binding performance to carbohy the engineered MBL molecule can comprise a fragment of drates on a microbe Surface, e.g., by theoretical modeling SEQ ID NO. 4. Exemplary amino acid sequences of such and/or in vitro carbohydrate-binding experiments. In addi fragments include, but are not limited to, ND (SEQID NO. tion, based on the crystal structure of the native MBL “neck 10), EZN (SEQID NO. 11: where Z is any amino acid, e.g., and CRD head', peptidomimetics that can effectively mimic P), NEGEPNNAGS (SEQID NO. 12) or a fragment thereof 50 at least a fragment of the CRD head and optionally the neck comprising EPN, GSDEDCVLL (SEQ ID NO. 13) or a region can be also used as a carbohydrate recognition fragment thereof comprising E, and LLLKNGOWND domain of the engineered microbe-targeting molecule or VPCST (SEQID NO. 14) or a fragment thereof comprising MBL molecule described herein. One of skill in the art can ND. Modifications to such CRD fragments, e.g., by conser readily determine such peptidomimetic structure without vative substitution, are also within the scope described 55 undue experimentations, using any methods known in the art herein. In some embodiments, the MBL or a fragment and the known crystal structure. thereof used in the microbe surface-binding domain of the In some embodiments, the carbohydrate recognition engineered microbe-targeting molecules described herein domain of the microbe-targeting molecule can further com can be a wild-type molecule or a recombinant molecule. prise a portion of a carbohydrate-binding protein. However, The exemplary sequences provided herein for the carbo 60 in Some circumstances, complement or coagulation activa hydrate recognition domain of the engineered microbe tion induced by a carbohydrate-binding protein or a frag targeting molecules are not construed to be limiting. For ment thereof can be undesirable depending on various example, while the exemplary sequences provided herein applications, e.g., in vivo administration for treatment of are derived from a human species, amino acid sequences of sepsis. In such embodiments, the portion of the carbohy the same carbohydrate recognition domain in other species 65 drate-binding protein can exclude at least one of comple Such as mice, rats, porcine, bovine, feline, and canine are ment and coagulation activation regions. By way of known in the art and within the scope described herein. example, when the carbohydrate-binding protein is man US 9,593,160 B2 17 18 nose-binding lectin or a fragment thereof, the mannose Immun. 688 (2000). The capsular polysaccharide of Neis binding lectin or a fragment thereof can exclude at least one seria meningitides serogroup B. H. influenzae type b and of the complement and coagulation activation regions Cryptococcus neoformans are thought to decrease MBL located on the collagen-like region. In such embodiments, binding, as does bacterial endotoxin. Id.; Van Emmerik et the mannose-binding lectin or a fragment thereof can al., 97 Clin. Exp. Immunol. 411 (1994); Schelenz et al., 63 exclude at least about one amino acid residue, including at Infect. Immun. 3360 (1995). least about two amino acid residues, at least about three Others have reported that MBL facilitates opsonophago amino acid residues, at least about four amino acid residues, cytosis of yeasts but not of bacteria, despite MBL binding: at least about five amino acid residues, at least about six MBL (Lectin) pathway of complement was critical for the amino acid residues, at least about seven amino acid resi 10 opsonophagocytosis of yeast, but the classical complement dues, at least about eight amino acid residues, at least about pathway was critical for opsonophagocytosis of bacteria. nine amino acid residues, at least about ten amino acid Brouwer et al., 180 J. Immunol. 4124 (2008). It was not residues or more, around amino acid residue K55 or L56 of reported that MBL bound to the bacterial species tested, SEQ ID NO. 2. Exemplary amino sequences comprising however, only that MBL binding did not promote significant K55 or L56 of SEQID NO. 2 that can be excluded from the 15 complement activation and opsonophagocytosis. engineered MBL molecule include, but are not limited to, Derivatives of MBL with a particular specificity can be EPGQGLRGLQGPPGKLGPPGNPGPSGS (SEQ ID NO. isolated, e.g., by the following approach, which is a standard 16), GKLG (SEQ ID NO. 17), GPPGKLGPPGN (SEQ ID phage display strategy: First, express a set of MBL variants NO. 18), RGLQGPPGKL (SEQ ID NO. 19), GKLGPPGN from a phagemid vector; then bind this library to a target of PGPSGS (SEQ ID NO. 20), GLRGLQGPPGKLGPPGN interest (e.g., E. coli) and perform one or two rounds of PGP (SEQ ID NO. 21), or any fragments thereof. selection; and then perform a round of negative selection Further regarding the carbohydrate recognition domain against a related target (e.g., Candida), taking those (CRD) or a fragment thereof its binding characteristics can phagemids that fail to bind. These cycles of positive and be manipulated by directed evolution for altered binding negative selection are then repeated until a population of specificity. By way of example only, MBL can be modified 25 phages that generally bind to the target and do not bind to the so that it binds to a more limited set of sugars or other non-target is generated. This method can be applied to any molecular features, with the result that the modified MBL pair of microbial strains against which differential binding is will bind to a more limited set of microbes to provide a desired. Such as bacteria that are resistant and sensitive to a capability for pathogen class identification (e.g., one of given antibiotic. This positive/negative enrichment strategy virus, bacteria, fungi, or protozoan), Subclass typing (e.g., 30 can also be used with an antibody-phage display library, gram negative or gram positive bacteria) or specific species which is an even more standard way to isolate such specific determination. Numerous strategies of directed evolution are binders. available in the art. The directed evolution and selection approach described For example, a straightforward directed evolution strategy above also can potentially be used to generate human visually examines an atomic structure of MBL complexed 35 antibody fragments or peptides that provide the class, Sub with a Sugar, and then mutates appropriate amino acids that class and species specificity described above. make contact in a Sugar-specific manner, so that distinctive In some embodiments, at least two microbe Surface contacts are lost or particular types of steric hindrance are binding domains (e.g., carbohydrate recognition domains), created. The three dimensional structure of rat MBL has including at least three, at least four, at least five, at least six, been solved in a complex with a high-mannose oligosac 40 at least seven, at least eight, at least nine, at least ten or more charide and with N acetylglucosamine, a methylated fucose, microbe Surface-binding domains, can be linked together to and so on. His 189Val and Ile207Val are examples of sub form a multimeric microbe Surface-binding domain or car stitutions that modifications alter specificity. bohydrate recognition domain. In such embodiments, the In another strategy of directed evolution, the protein is distances between microbe Surface-binding domains (e.g., Subjected to random mutagenesis and the resulting proteins 45 carbohydrate recognition domains) can be engineered to are screened for desired qualities. This is a particularly match with the distance between the binding sites on the useful technology for affinity maturation of phage display target microbe Surface. antibodies, where the antibody complementary determining A multimeric microbe Surface-binding domain can have regions (CDRS) are mutated by Saturation mutagenesis and each of the individual microbe surface-binding domains the successful variants of the six CDRs are shuffled together to 50 same. Alternatively, a multimeric microbe Surface-binding form the highest affinity antibodies. domain can have at least one, at least two, or at least three The directed evolution paradigm can be applied to MBL microbe surface-binding domains different from the rest. In in order to select MBL variants with specific binding to, e.g., Such embodiments, microbe Surface-binding domains that but not limited to, yeast, gram-positive bacteria, gram share a common binding specificity for carbohydrates on a negative, coagulase negative, and aerobic bacteria. For this 55 microbe Surface can be used. By way of example only, the to work, however, the pattern and nature of the target Sugars fibrinogen-like domain of several lectins has a similar func or related Surface features on these target microorganisms tion to the CRD of C-type lectins including MBL, and can differ between the classes or species. function as pattern-recognition receptors to discriminate MBL is known to bind strongly to mannose and N-acetyl pathogens from self. One of Such lectins comprising the glucosamine Sugars on fungi, gram-positive, and gram 60 fibrinogen-like domain is serum ficolins. negative bacteria. For example, MBL binds strongly to Serum ficolins have a common binding specificity for Candida spp., Aspergillus filmigatus, Staphylococcus GlcNAc (N-acetylglucosamine), elastin or GalNAc aureus, and B hemolytic group A Streptococci. MBL has (N-acetyl-galactosamine). The fibrinogen-like domain is intermediate affinity to Escherichia coli, Klebsiella spp., and responsible for the carbohydrate binding. In human serum, Haemophilus influenzae type b. MBL binds weakly to B 65 two types of ficolin, known as L-ficolin (also called P35, hemolytic group B Streptococci, Streptococcus pneumoniae, ficolin L, ficolin 2 or hucolin) and H-ficolin (also called and Staphylococcus epidermidis. Neth et al., 68 Infect. & Hakata antigen, ficolin 3 or thermolabile b2-macroglyco US 9,593,160 B2 19 20 protein), have been identified, and both of them have lectin - Continued activity. L-ficolin recognises GlcNAc and H-ficolin recog nises GalNAc. Another ficolin known aS M-ficolin (also ciohydrate recognition domain (CRD) of MBL (SEQ called P3 5-related protein, ficolin 1 or ficolin A) is not ID NO. 4) : considered to be a serum protein and is found in leucocytes 5 VGNKFFLTNG EIMTFEKVKA LCVKFQASVA TPRNAAENGA and in the lungs. L-ficolin and H-ficolin activate the lectin complement pathway in association with MASPs. M-Fico IONLIKEEAF LGITDEKTEG OFVDLTGNRL TYTNWNEGEP lin, L-ficolin and H-ficolin has calcium-independent lectin NNAGSDEDCV LLLKNGOWND VPCSTSHLAV CEFPI activity. Accordingly, in Some embodiments, an engineered (v) 10 (v microbe-targeting, e.g., an engineered MBL molecule, Ca Neck + Carbohydrate recognition domain of MBL (SEQ comprise MBL and L-ficolin carbohydrate recognition ID NO. 5) : domains, MBL and H-ficolin carbohydrate recognition PDGDSSLAAS ERKALOTEMA RIKKWLTFSL GKOVGNKFFL domains, or a combination thereof. TNGEIMTFEK VKALCVKFOA SWATPRNAAE NGAIONLIKE Any art-recognized recombinant carbohydrate-binding 15 proteins or carbohydrate recognition domains can also be EAFLGITDEK TEGOFWDLTG NRLTYTNWNE GEPNNAGSDE used in the engineered microbe-targeting molecules. For DCVLLLKNGO WNDVPCSTSH LAVCEFPI example, recombinant mannose-binding lectins, e.g., but not limited to, the ones disclosed in the U.S. Pat. Nos. 5,270, FoMBL.(vi) 81 (SEQ ID NO. 6) : 199; 6,846,649; and U.S. Patent Application No. US 2004/ 20 EPKSSDKTHT CPPCPAPELL GGPsv FPP 0229212, the contents of which are incorporated herein by reference, can be used in constructing the engineered MBL KPKDTLMSR TPEWTCVWWD WSHEDPEWKFNWYWDGWEVH molecules described herein. NAKTKPREEO YNSTYRVVSV LTVLHODWLN GKEYKCKVSN In one embodiment, the microbe-binding molecule com- as prises an MBL, a carbohydrate recognition domain of an KALPAPIEKT ISKAKGOPRE POVYTLPPSR DELTKNOWSL MBL, or a genetically engineered version of MBL (FcMBL) TCLVKGFYPS DIAVEWESNG OPENNYKTTPPVLDSDGSFF as described in International Application No. WO 2011/ 090954, filed Jan. 19, 2011, the content of both of which is LYSKLTVDKS RWOOGNWFSC SVMHEALHNH YTOKSLSLSP incorporated herein by reference. Amino acid sequences for 30 GAPDGDSSLAASERKALQTE MARIKKWLTF SLGKQVGNKF MBL and engineered MBL include, but are not limited to: FLTNGEIMTF EKVKALCVKF OASWATPRNA. AENGAIONLI

KEEAFLGITD EKTEGOFWDL TGNRLTYTNW NEGEPNNAGS (i) MBL full length (SEQ ID NO. 1) : 35 DEDCVLLLKN GOWNDVPCST SHLAVCEFPI MSLFPSLPLL LISMWAASYS (vii) ETVTCEDAOK TCPAVIACSS PGINGFPGKD GRDGTKGEKG AKT-FoMBL (SEO ID NO. 7) : AKTEPKSSDKTHT CPPCPAPELL EPGQGLRGLQ GPPGKLGPPG NPGPSGSPGP KGOKGDPGKS GGPSWFLFPP KPKDTLMISR TPEWTCWWWD WSHEDPEWKF PDGDSSLAAS ERKALOTEMA RIKKWLTFSL GKOVGNKFFL 40 NWYWDGVEVH NAKTKPREEO YNSTYRVVSV LTVLHODWLN TNGEIMTFEK VKALCVKFOA SWATPRNAAE NGAIONLIKE GKEYKCKVSN KALPAPIEKT ISKAKGOPRE POVYTLPPSR EAFLGITDEK TEGOFWDLTG NRLTYTNWNE GEPNNAGSDE DELTKNOWSL TCLVKGFYPS DIAVEWESNG OPENNYKTTP DCVLLLKNGO WNDVPCSTSH LAVCEFPI 45 PVLDSDGSFF LYSKLTVDKS RWOOGNWFSC SVMHEALHNH (ii) MBL without the signal sequence (SEQ ID NO. 2) : YTOKSLSLSP GAPDGDSSLA ASERKALOTE MARIKKWLTF ETVTCEDAOK SLGKQVGNKF FLTNGEIMTF EKVKALCVKF OASWATPRNA TCPAVIACSS PGINGFPGKD GRDGTKGEKG EPGOGLRGLO 50 AENGAIONLI KEEAFLGITD EKTEGOFVDL TGNRLTYTNW GPPGKLGPPG NPGPSGSPGP KGOKGDPGKS PDGDSSLAAS NEGEPNNAGS DEDCVLLLKN GOWNDVPCST SHLAVCEFPI ERKALOTEMA RIKKWLTFSL GKOVGNKFFL TNGEIMTFEK (viii) WKALCVKFOA SWATPRNAAE NGAIONLIKE EAFLGITDEK 55 FoMBL. 111 (SEQ ID NO. 8) : EPKSSDKTHT CPPCPAPELL GGPSWFLFPP TEGOFVDLTG NRLTYTNWNE GEPNNAGSDE DCVLLLKNGO KPKDTLMSR TPEWTCVWWD WSHEDPEWKF NWYWDGWEVH WNDWPCSTSH LAWCEFPI NAKTKPREEO YNSTYRVVSV LTVLHODWLN GKEYKCKVSN (iii) Truncated MBL (SEQ ID NO. 3) : KALPAPIEKT ISKAKGQPRE POVYTLPPSR DELTKNOVSL AASERKALOT EMARIKKWLT TCLVKGFYPS DIAVEWESNG OPENNYKTTP PVLDSDGSFF FSLGKOVGNK FFLTNGEIMT FEKVKALCVK FOASWATPRN LYSKLTVDKS RWOOGNWFSC SVMHEALHNH YTOKSLSLSP AAENGAIONL IKEEAFLGIT DEKTEGOFWD LTGNRLTYTN 65 GATSKQVGNKF FLTNGEIMTF EKVKALCVKF QASVATPRNA WNEGEPNNAG SDEDCVLL.L.K NGOWNDVPCS TSHLAVCEFP I US 9,593,160 B2 21 22 - Continued (Thr), methionine (Met) or alanine (Ala), or at least one of AENGAIONLI KEEAFLGITD EKTEGOFVDL TGNRLTYTNW codon sequences encoding the aforementioned amino acids (i.e., Gly, Ser, Asn. Thr, Met or Ala). Such amino acids and NEGEPNNAGS DEDCVLLLKN GOWNDVPCST SHLAVCEFPI corresponding nucleic acid sequences are generally used to In some embodiments, a microbe-binding molecule com- 5 provide flexibility of a linker. However, in some embodi prises an amino acid sequence selected from SEQ ID NO. ments, other uncharged polar amino acids (e.g., Gln, CyS or 1-SEQ ID NO. 8. Tyr), nonpolar amino acids (e.g., Val, Leu, Ile, Pro, Phe, and Without wishing to be bound by a theory, microbe Trp), or nucleic acid sequences encoding the amino acids binding molecules comprising lectins or modified versions thereof can also be included in a linker sequence. In alter thereof can act as broad-spectrum pathogen binding mol 10 native embodiments, polar amino acids or nucleic acid ecules. Accordingly, microbes and/or microbial matter pres sequence thereof can be added to modulate the flexibility of ent in a test sample can be captured using lectin-based a linker. One of skill in the art can control flexibility of a microbe-binding molecules without identifying the microbe. linker by varying the types and numbers of residues in the Linkers linker. See, e.g., Perham, 30 Biochem. 8501 (1991); Wrig 15 gers et al., 80 Biopolymers 736 (2005). As used herein, the term “linker generally refers to a In alternative embodiments, a linker can be a chemical molecular entity that can directly or indirectly connect at linker of any length. In some embodiments, chemical linkers two parts of a composition, e.g., at least one microbe can comprise a direct bond or an atom such as oxygen or Surface-binding domain and at least one Substrate-binding sulfur, a unit such as NH, C(O), C(O)NH, SO, SO, SONH, domain. In some embodiments, the linker can directly or 20 or a chain of atoms, such as Substituted or unsubstituted indirectly connect to one or more microbe Surface-binding C-C alkyl, Substituted or unsubstituted C-C alkenyl, domains. Without limitations, in some embodiments, the substituted or unsubstituted C-C alkynyl, substituted or linker can also provide binding sites to one or more microbes unsubstituted C-C aryl, Substituted or unsubstituted and/or microbial matter. In such embodiments, the microbe Cs-C heteroaryl, Substituted or unsubstituted Cs-C het binding sites on the linker can bind to the same types and/or 25 erocyclyl. Substituted or unsubstituted C-C cycloalkyl, species of microbes as the microbes bind to a microbe where one or more methylenes can be interrupted or termi Surface-binding domain. Alternatively or additionally, the nated by O, S, S(O), SO, NH, or C(O). In some embodi microbe-binding sites on the linker can capture different ments, the chemical linker can be a polymer chain (branched types and/or species of microbes than the ones that bind to or linear). a microbe Surface-binding domain described herein. 30 In some embodiments where the linker is a peptide. Such Linkers can be configured according to a specific need, peptidyl linker can comprise at least a portion of an immu e.g., based on at least one of the following characteristics. noglobulin, e.g., IgA, Ig|D, IgE, IgG and IgM including their By way of example only, in some embodiments, linkers can Subclasses (e.g., IgG1), or a modified molecule or recom be configured to have a sufficient length and flexibility such binant thereof. In some embodiments, the peptide linker can that it can allow for a microbe Surface-binding domain to 35 comprise a portion of fragment crystallization (Fc) region of orient accordingly with respect to at least one carbohydrate an immunoglobulin or a modified thereof. In Such embodi on a microbe Surface. In some embodiments, linkers can be ments, the portion of the Fc region that can be used as a configured to allow multimerization of at least two engi linker can comprise at least one region selected from the neered microbe-targeting molecules (e.g., to from a di-, tri-, group consisting of a hinge region, a CH2 region, a CH3 tetra-, penta-, or higher multimeric complex) while retaining 40 region, and any combinations thereof. By way of example, biological activity (e.g., microbe-binding activity). In some in some embodiments, a CH2 region can be excluded from embodiments, linkers can be configured to facilitate expres the portion of the Fc region as a linker. In one embodiment, sion and purification of the engineered microbe-targeting Fc linker comprises a hinge region, a CH2 domain and a molecule described herein. In some embodiments, linkers CH3 domain, e.g., Fc IgG 106 as shown in FIG. 1B and FIG. can be configured to provide at least one recognition site for 45 3. Such Fc linker can be used to facilitate expression and proteases or nucleases. In addition, linkers are preferably purification of the engineered microbe-targeting molecules non-reactive with the functional components of the engi described herein. The N terminal Fc has been shown to neered molecule described herein (e.g., minimal hydropho improve expression levels, protein folding and secretion of bic or charged character to react with the functional protein the fusion partner. In addition, the Fc has a staphylococcal domains such as a microbe Surface-binding domain or a 50 protein A binding site, which can be used for one-step Substrate-binding domain). purification protein A affinity chromatography. See Lo KM In some embodiments, a linker can be configured to have et al. (1998) Protein Eng. 11:495-500. Further, the protein any length in a form of a peptide, peptidomimetic, an A binding site can be used to facilitate binding of protein aptamer, a protein, a nucleic acid (e.g., DNA or RNA), or A-expressing or protein G-expressing microbes in the any combinations thereof. In some embodiments, the pep- 55 absence of calcium ions. Such binding capability can be tidyl or nucleic acid linker can vary from about 1 to about used to develop methods for distinguishing protein A-ex 1000 amino acids long, from about 10 to about 500 amino pressing microbes (e.g., S. aureus) from non-protein A-ex acids long, from about 30 to about 300 amino acids long, or pressing or non-protein G-expressing microbes (e.g., E. coli) from about 50 to about 150 amino acids long. Longer or present in a test sample, and various embodiments of Such shorter linker sequences can be also used for the engineered 60 methods will be described in detail later. Further, such Fc microbe-targeting molecules described herein. In one linker have a molecule weight above a renal threshold of embodiment, the peptidyl linker has an amino acid sequence about 45 kDa, thus reducing the possibility of engineered of about 200 to 300 amino acids in length. microbe-targeting molecules being removed by glomerular In some embodiments, a peptide or nucleic acid linker can filtration. Additionally, the Fc linker can allow dimerization be configured to have a sequence comprising at least one of 65 of two engineered microbe-targeting molecules to form a the amino acids selected from the group consisting of dimer, e.g., the dimeric engineered MBL molecule 100 as glycine (Gly), serine (Ser), asparagine (ASn), threonine shown in FIG. 1B. US 9,593,160 B2 23 24 In some embodiments where the linker comprises a Fc effectively with microbes. In some embodiments, the dis region or a fragment thereof, the Fc region or a fragment tance between the microbe Surface-binding domain and the thereof can comprise at least one mutation, e.g., to modify substrate can range from about 50 angstroms to about 5000 the performance of the engineered microbe-targeting mol angstroms, from about 100 angstroms to about 2500 ang ecules. For example, in some embodiments, a half-life of the stroms, or from about 200 angstroms to about 1000 ang engineered microbe-targeting molecules described herein StrOmS. can be increased, e.g., by mutating an amino acid lysine (K) The linkers can be of any shape. In some embodiments, at the residue 232 of SEQ ID NO. 9 to alanine (A). Other the linkers can be linear. In some embodiments, the linkers mutations, e.g., located at the interface between the CH2 and can be folded. In some embodiments, the linkers can be CH3 domains shown in Hinton et al (2004) J Biol Chem. 10 branched. For branched linkers, each branch of a microbe 279:6213-6216 and Vaccaro C. et al. (2005) Nat Biotechnol. Surface-binding domain can comprise at least one microbe 23: 1283-1288, can be also used to increase the half-life of Surface-binding domain. In other embodiments, the linker the IgG1 and thus the engineered microbe-targeting mol adopts the shape of the physical Substrate. ecules. In some embodiments provided herein, the linker can In some embodiments, the linker can be albumin, trans 15 further comprise a detectable label. In some embodiments, ferrin or a fragment thereof. Such linkers can be used to the detectable label can be a chromogenic or fluorogenic extend the plasma half-life of the engineered microbe microbe enzyme substrate so that when a microbe binds to targeting molecules and thus are good for in vivo adminis the engineered microbe-targeting molecule, the enzyme that tration. See Schmidt S R (2009) Curr Opin Drug Discov the microbe releases can interact with the detectable label to Devel. 12: 284. induce a color change. Examples of Such microbe enzyme When the engineered microbe-targeting molecules are Substrate can include, but are not limited to, indoxyl used as therapeutics in vivo, the linker can be further butyrate, indoxyl glucoside, esculin, magneta glucoside, modified to modulate the effector function such as antibody red-f-glucuronide, 2-methoxy-4-(2-nitrovinyl) phenyl B-D- dependent cellular cytotoxicity (ADCC) and complement glu-copyranoside, 2-methoxy-4-(2-nitrovinyl) phenyl B-D- dependent cytotoxicity (CDC). By way of example only, the 25 cetamindo-2-deoxyglucopyranoside, and any other art-rec Fc region for use as a linker can mediate ADCC and CDC. ognized microbe enzyme Substrates. Such embodiments can In ADCC, the Fc region can generally bind to Fc receptors act as an indicator for the presence of a microbe or pathogen. on the Surface of immune effector cells such as natural Conjugation of Engineered Microbe-Targeting Molecules to killers and macrophages, leading to the phagocytosis or lysis a Substrate of a targeted cell. In CDC, the Fc region can generally 30 The engineered microbe-targeting molecules can be trigger the complement cascade at the cell Surface to kill the immobilized on any Substrate for various applications and/or targeted cell. Accordingly, modulating effector functions can purposes. For example, when the affinity of a single microbe be achieved by engineering the Fc region to either increase Surface-binding domain for a target molecule (e.g., a carbo or decrease their binding to the Fc receptors on the surface hydrate recognition domain for a Sugar/carbohydrate mol of the immune effector cells or the complement factors. For 35 ecule) is relatively low, and Such binding is generally driven example, numerous mutations within a Fc region for modu by avidity and multivalency, multivalency of Such engi lating ADCC and CDC are well known to a skilled artisan, neered microbe-targeting molecules can be effectively e.g., see Armour K L. et al. (1999) Eur J Immmunol 29: increased by attachment of a plurality of the engineered 2613-2624; Shields R L. et al. (2001) J Biol Chem. 276: microbe-targeting molecules (e.g., each with one or two or 6591-6604: Idusogie E. E. et al. (2001) J Immunol. 166: 40 more carbohydrate recognition domains) to a solid Substrate 2571-2575: Idusogie E. E. et al. (2000) J Immunol. 155: (e.g., a nanometer- or micrometer-sized bead) at a high 1165-1174; and Steurer W. et al. (1995) J Immunol. 155: density, which can be varied to provide optimal functional 1165-1674. In one embodiment, the amino acid asparagine ity. Alternatively, the engineered microbe-targeting mol (N) at the residue 82 of the SEQ ID NO. 6 can be mutated ecules can be immobilized on a solid substrate for easy to aspartic acid (D), e.g., to remove the glycosylation of Fc 45 handling during usage, e.g., for isolation, observation or and thus, in turn, reduce ADCC and CDC functions. microscopic imaging. In various embodiments, the N-terminus or the C-termi The attachment of the engineered microbe-binding mol nus of the linker, e.g., the portion of the Fc region, can be ecule (e.g., FcMBL) to a Substrate Surface (e.g., membrane modified. By way of example only, the N-terminus or the Surface, glass Surface, tubing Surface) can be performed with C-terminus of the linker can be extended by at least one 50 multiple approaches, for example, by direct cross-linking the additional linker described herein, e.g., to provide further engineered microbe-binding molecule (e.g., FcMBL) to the flexibility, or to attach additional molecules. In some Substrate Surface; cross-linking the engineered microbe embodiments, the N-terminus of the linker can be linked binding molecule (e.g., FcMBL) to the substrate surface via directly or indirectly (via an additional linker) with a sub a nucleic acid matrix (e.g., DNA matrix or DNA/oligonucle strate-binding domain adapted for orienting the carbohy 55 otide origami structures) for orientation and concentration to drate recognition domain away from the Substrate. increase detection sensitivity; cross-linking FcMBL to the In some embodiments, the linker can be embodied as part substrate surface via a dendrimer-like structure (e.g., PEG/ of the microbe Surface-binding domain, or part of the Chitin-structure) to increase detection sensitivity; attracting carbohydrate-binding protein (e.g., MBL and/or the neck FcMBL-coated magnetic microbeads to the substrate sur region thereof). 60 face with a focused magnetic field gradient applied to the In some embodiments, the linker can be a physical Substrate surface, attaching an engineered microbe-binding Substrate, e.g., microparticles or magnetic microbes, to molecule (e.g., FcMBL) to a substrate via biotin-avidin or which a plurality of microbe Surface-binding domains (in biotin-avidin-like interaction, or any other art-recognized cluding carbohydrate recognition domain) can bind, pro methods. vided that there is at least a certain distance between the 65 For engineered microbe-targeting molecules or mannose microbe Surface-binding domain and the Substrate surface binding lectin molecules to be immobilized on or conjugated sufficient for the microbe surface-binding domain to interact to a Substrate, the engineered molecules described herein can US 9,593,160 B2 25 26 further comprise at least one (e.g., one, two, three, four, five, ordinary skill in the art. The oligonucleotides including six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, aptamers can be of any length, e.g., from about 1 nucleotide fifteen, sixteen, seventeen, eighteen, nineteen, twenty or to about 100 nucleotides, from about 5 nucleotides to about more) Substrate-binding domain, e.g., adapted for orienting 50 nucleotides, or from about 10 nucleotides to about 25 the carbohydrate recognition domain away from the Sub nucleotides. Generally, a longer oligonucleotide for hybrid strate. Without limitations, exemplary types of substrates ization to a nucleic acid scaffold can generate a stronger can be a nucleic acid scaffold, a biological molecule (e.g., a binding strength between the engineered microbe Surface living cell), or a solid Surface. In some embodiments, the binding domain and Substrate. Solid Surface can be functionalized with a coupling mol Alternatively or additionally, the surface of a substrate can ecule, e.g., an amino group, to facilitate the conjugation of 10 be functionalized to include coupling molecules described engineered microbe Surface-binding domains to the Solid herein. As used herein, the term “coupling molecule” refers Surface. to any molecule or any functional group that is capable of As used herein, the term “substrate-binding domain selectively binding with an engineered microbe Surface refers to any molecule that facilitates the conjugation of the binding domain described herein. Representative examples engineered molecules described herein to a Substrate or a 15 of coupling molecules include, but are not limited to, functionalized Substrate. In some embodiments, the Sub antibodies, antigens, lectins, proteins, peptides, nucleic acids strate-binding domain can comprise at least one amino (DNA, RNA, PNA and nucleic acids that are mixtures group that can non-covalently or covalently coupled with thereof or that include nucleotide derivatives or analogs); functional groups on the surface of the substrate. For receptor molecules, such as the insulin receptor, ligands for example, the primary amines of the amino acid residues receptors (e.g., insulin for the insulin receptor); and biologi (e.g., lysine or cysteine residues) at the N-terminus or in cal, chemical or other molecules that have affinity for close proximity to the N-terminus of the engineered microbe another molecule, such as biotin and avidin. The coupling Surface-binding domains (e.g., engineered mannose-binding molecules need not comprise an entire naturally occurring lectins) can be used to couple with functional groups on the molecule but may consist of only a portion, fragment or Substrate Surface. 25 Subunit of a naturally or non-naturally occurring molecule, In some embodiments, the Substrate-binding domain can as for example the Fab fragment of an antibody. The comprise at least one, at least two, at least three or more coupling molecule can further comprise a detectable label. oligopeptides. The length of the oligonucleotide can vary The coupling molecule can also encompass various func from about 2 amino acid residues to about 10 amino acid tional groups that can couple the Substrate to the engineered residues, or about 2 amino acid residues to about 5 amino 30 microbe Surface-binding domains. Examples of Such func acid residues. Determination of an appropriate amino acid tional groups include, but are not limited to, an amino group, sequence of the oligonucleotide for binding with different a carboxylic acid group, an epoxy group, and a tosyl group. substrates is well within one of skill in the art. For example, In some embodiments, the engineered microbe-targeting as shown in FIG. 1B, according to one or more embodi molecule can be conjugated to a substrate surface through a ments, the Substrate-binding domain 108 can comprise an 35 covalent or non-covalent interaction. The engineered oligopeptide comprising an amino acid sequence of AKT, microbe-targeting molecule and/or coupling molecule can which provides a single biotinylation site for Subsequent be conjugated to the Surface of a solid Substrate covalently binding to streptavidin-coated Substrate, e.g., a magnetic or non-covalently using any of the methods known to those microbead 110. Such single biotinylation site can also of skill in the art. For example, covalent immobilization can enable the carbohydrate recognition domain of an engi 40 be accomplished through, for example, silane coupling. See, neered microbe Surface-binding domain to orient away from e.g., Weetall, 15 Adv. Mol. Cell Bio. 161 (2008); Weetall, 44 the Substrate, and thus become more accessible to microbes Meths. Enzymol. 134 (1976). The covalent interaction or pathogens. See, e.g., Witus et al. (2010).JACS 132: 16812. between the engineered microbe-targeting molecule and/or In some embodiments, the Substrate-binding domain can coupling molecule and the Surface can also be mediated by comprise at least one oligonucleotide. The sequence and 45 other art-recognized chemical reactions, such as NHS reac length of the oligonucleotides can be configured according tion or a conjugation agent. The non-covalent interaction to the types of the Substrate, binding density, and/or desired between the engineered microbe-targeting molecule and/or binding strength. For example, if the Substrate is a nucleic coupling molecule and the Surface can be formed based on acid scaffold, e.g., a DNA scaffold, the oligonucleotide ionic interactions, van der Waals interactions, dipole-dipole sequence of the Substrate-binding domain can be designed 50 interactions, hydrogen bonds, electrostatic interactions, and/ Such that it is complementary to a Sub-sequence of the or shape recognition interactions. nucleic acid scaffold to where the substrate-binding domain Without limitations, conjugation can include either a can hybridize. stable or a labile (e.g. cleavable) bond or conjugation agent. In some embodiments, the oligonucleotides can include Exemplary conjugations include, but are not limited to, aptamers. As used herein, the term "aptamer” means a 55 covalent bond, amide bond, additions to carbon-carbon single-stranded, partially single-stranded, partially double multiple bonds, azide alkyne Huisgen cycloaddition, Diels Stranded or double-stranded nucleotide sequence capable of Alder reaction, disulfide linkage, ester bond, Michael addi specifically recognizing a selected non-oligonucleotide mol tions, silane bond, urethane, nucleophilic ring opening reac ecule or group of molecules by a mechanism other than tions: epoxides, non-aldol carbonyl chemistry, cycloaddition Watson-Crick base pairing or triplex formation. Aptamers 60 reactions: 1,3-dipolar cycloaddition, temperature sensitive, can include, without limitation, defined sequence segments radiation (IR, near-IR, UV) sensitive bond or conjugation and sequences comprising nucleotides, ribonucleotides, agent, pH-sensitive bond or conjugation agent, non-covalent deoxyribonucleotides, nucleotide analogs, modified nucleo bonds (e.g., ionic charge complex formation, hydrogen tides and nucleotides comprising backbone modifications, bonding, pi-pi interactions, cyclodextrin/adamantly host branchpoints and nonnucleotide residues, groups or bridges. 65 guest interaction) and the like. Methods for selecting aptamers for binding to a molecule are As used herein, the term "conjugation agent’ means an widely known in the art and easily accessible to one of organic moiety that connects two parts of a compound. US 9,593,160 B2 27 28 Linkers typically comprise a direct bond or an atom Such as carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme oxygen or sulfur, a unit such as NR", C(O), C(O)NH, SO, inhibitor, and complementary oligonucleotide pairs capable SO, SONH or a chain of atoms, such as substituted or of forming nucleic acid duplexes). The coupling molecule unsubstituted alkyl, substituted or unsubstituted alkenyl, pair can also include a first molecule that is negatively Substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, charged and a second molecule that is positively charged. arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroaryl One example of using coupling pair conjugation is the alkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocy biotin-avidin or biotin-streptavidin conjugation. In this clylalkynyl, aryl, heteroaryl, heterocyclyl cycloalkyl, approach, one of the members of the coupling pair (e.g., a cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylal portion of the engineered microbe-targeting molecule Such kynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylal 10 as Substrate-binding domain, or a Substrate) is biotinylated kynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylal and the other (e.g., a Substrate or the engineered microbe kynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, targeting molecule) is conjugated with avidin or streptavi alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylhet din. Many commercial kits are also available for biotinylat eroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroary ing molecules, such as proteins. For example, an aminooxy lalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, 15 biotin (AOB) can be used to covalently attach biotin to a alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkyl molecule with an aldehyde or ketone group. In one embodi hererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylhet ment, AOB is attached to the Substrate-binding domain (e.g., erocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylhet comprising AKT oligopeptide) of the engineered microbe erocyclylalkyl, alkynylheterocyclylalkenyl, targeting molecule. alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alky One non-limiting example of using conjugation with a nylaryl, alkylheteroaryl, alkenylheteroaryl, alkynyl coupling molecule pair is the biotin-sandwich method. See, hereroaryl, where one or more methylenes can be interrupted e.g., Davis et al., 103 PNAS8155 (2006). The two molecules or terminated by O, S, S(O), SO, NH, C(O)N(R'), C(O), to be conjugated together are biotinylated and then conju cleavable linking group, Substituted or unsubstituted aryl, gated together using tetravalent Streptavidin. In addition, a substituted or unsubstituted heteroaryl, substituted or unsub 25 peptide can be coupled to the 15-amino acid sequence of an stituted heterocyclic; where R' is hydrogen, acyl, aliphatic acceptor peptide for biotinylation (referred to as AP; Chen et or substituted aliphatic. al., 2 Nat. Methods 99 (2005)). The acceptor peptide Without limitations, any conjugation chemistry known in sequence allows site-specific biotinylation by the E. coli the art for conjugating two molecules or different parts of a enzyme biotin ligase (BirA. Id.). An engineered microbe composition together can be used for linking at least one 30 Surface-binding domain can be similarly biotinylated for engineered microbe-targeting molecule to a substrate. conjugation with a solid Substrate. Many commercial kits Exemplary coupling molecules and/or functional groups for are also available for biotinylating proteins. Another conjugating at least one engineered microbe-targeting mol example for conjugation to a solid Surface would be to use ecule to a substrate include, but are not limited to, a PLP-mediated bioconjugation. See, e.g., Witus et al., 132 polyethylene glycol (PEG, NH-PEG-COOH which can 35 JACS 16812 (2010). As described earlier, an AKT sequence have a PEG spacer arm of various lengths X, where on the N terminal of the engineered microbe-targeting 1lactic acid)S, poly(glycolic acid)S. microbe-targeting magnetic microbeads can be used to sepa poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, 30 rate microbes or pathogens from a test sample, e.g., but not polycaprolactone, polyesteramides, polycarbonate, polycya limited to, any fluid, including a biological fluid Such as noacrylate, polyurethanes, polyacrylate, blends and copoly blood. In some embodiments, the microbe-targeting mag mers thereof. netic microbeads can be used to remove living microbes or Other additional biodegradable polymers include biode pathogens. Using magnetic microbeads as a Substrate can be gradable polyetherester copolymers. Generally speaking, the 35 advantageous because the microbe-bound magnetic micro polyetherester copolymers are amphiphilic block copoly beads can be easily separated from a sample fluid using a mers that include hydrophilic (for example, a polyalkylene magnetic field gradient, be examined for the presence of the glycol. Such as polyethylene glycol) and hydrophobic blocks microbe, and/or be used to transfer the collected microbes to (for example, polyethylene terephthalate). An exemplary conventional pathogen culture and sensitivity testing assays. block copolymer is, but is not limited to, poly(ethylene 40 Thus, in some embodiments, the microbe-targeting mag glycol)-based and poly(butylene terephthalate)-based blocks netic microbeads can be used to remove microbe contami (PEG/PBT polymer). PEG/PBT polymers are commercially nants from any source or in any fluid, e.g., a biological fluid available from OctoPlus Inc, under the trade designation (e.g., blood sample), environmental fluid or Surface (e.g., Poly ActiveTM. Non-limiting examples of biodegradable wastewater, building or machine Surface), or an edible copolymers or multiblock copolymers include the ones 45 Substance or fluid (e.g., food, water). In some embodiments described in U.S. Pat. Nos. 5,980,948 and 5,252,701, the where the fluid is blood, after removal of the microbef contents of which are incorporated herein by reference. pathogen from the blood collected from a subject with the Other biodegradable polymer materials include biode microbe-targeting magnetic microbeads, the blood can be gradable terephthalate copolymers that include a phospho circulated back to the same subject as a therapeutic inter rus-containing linkage. Polymers having phosphoester link 50 vention. In some embodiments, the microbe-targeting mag ages, called poly(phosphates), poly(phosphonates) and poly netic microbeads can be used in diagnostics as a means of (phosphites), are known in the art. See, for example, collecting potential pathogens for identification; not only in Penczek et al., Handbook of Polymer Synthesis, Chapter 17: the diagnosis of disease, but in the identification of water- or “Phosphorus-Containing Polymers. 1077-1132 (Hans R. food-borne pathogens, particulates or other contaminants. Kricheldorf ed., 1992), as well as U.S. Pat. Nos. 6,153,212: 55 Alternatively, the solid substrate can comprise a hollow 6,485,737; 6,322,797; 6,600,010; 6,419,709; 6,419,709; fiber reactor or any other blood filtration membrane or flow 6,485,737; 6,153,212; 6,322,797 and 6,600,010, the contents device (e.g., a simple dialysis tube, spiral mixer or static of which are incorporated herein by reference. mixer) or other resins, fibers, or sheets to selective bind and Biodegradable polyhydric alcohol esters can also be used sequester the biological pathogens. as a material of a Substrate (e.g., a microparticle) (See U.S. 60 The magnetic microbeads can be of any shape, including Pat. No. 6,592,895, which is incorporated herein by refer but not limited to spherical, rod, elliptical, cylindrical, and ence). In some embodiments, the biodegradable polymer can disc. In some embodiments, magnetic beads having a Sub be a three-dimensional crosslinked polymer network con stantially spherical shape and defined Surface chemistry can taining hydrophobic and hydrophilic components which be used to minimize chemical agglutination and non-specific forms a hydrogel with a crosslinked polymer structure. Such 65 binding. As used interchangeably herein, the terms “mag as the one described in U.S. Pat. No. 6,583,219. In yet netic microbeads' and “magnetic beads' can refer to a nano further embodiments, the biodegradable polymer can com or micro-scale particle that is attracted or repelled by a US 9,593,160 B2 35 36 magnetic field gradient or has a non-zero magnetic suscep lial system, etc. In some embodiments, the Surface to which tibility. The magnetic microbeads can be ferromagnetic, the engineered microbe-targeting molecules bind can also be paramagnetic or Super-paramagnetic. In some embodiments, the extracellular matrix of one or more of these tissues or magnetic microbeads can be Super-paramagnetic. In some Organs. embodiments, magnetic microbeads can have a polymer 5 Microbe-Binding Microtiter Plates: shell for protecting the microbe-targeting molecule from In some embodiments, the bottom surface of microtiter exposure to iron provided that the polymer shell has no wells can be coated with the engineered microbe-targeting adverse effect on the magnetic property. For example, bio molecules described herein, e.g., for detecting and/or deter compatible polymer-coated magnetic microbeads can be mining the amount of microbes in a sample. After microbes used to remove microbes/pathogens from a test sample, e.g., 10 or pathogens in the sample binding to the engineered a biological fluid. Such as blood. microbe-targeting molecules bound to the microwell Sur The magnetic microbeads can range in size from 1 nm to face, the rest of the sample can be removed. Detectable 1 mm. For example, magnetic microbeads can be about 2.5 molecules that can also bind to microbes or pathogens (e.g., nm to about 500 um, or about 5 nm to about 250 um in size. an engineered microbe-targeting molecule conjugated to a In some embodiments, magnetic microbeads can be about 5 15 detectable molecule as described herein) can then be added nm to about 100 um in size. In some embodiments, magnetic to the microwells with microbes/pathogens for detection of microbeads can be about 0.01 um to about 10 um in size. In microbes/pathogens. Various signal detection methods for Some embodiments, magnetic microbeads can be about 0.05 determining the amount of proteins, e.g., using enzyme um to about 5 um in size. In some embodiments, magnetic linked immunosorbent assay (ELISA), with different detect microbeads can be about 0.08 um to about 1 um in size. In 20 able molecules have been well established in the art, and one embodiment, magnetic microbeads can be about 10 nm those signal detection methods can also be employed herein to about 10 um in size. In some embodiments, the magnetic to facilitate detection of the signal induced by microbes/ microbeads can be magnetic nanobeads, e.g., with a size pathogens binding on the engineered microbe-targeting mol ranging from about 1 nm to about 1000 nm, from about 10 ecules. nm to about 500 nm, from about 25 nm to about 300 nm, 25 Microbe-Binding Dipsticks/Test Strips: from about 40 nm to about 250 nm, or from about 50 nm to In some embodiments, the engineered microbe-targeting about 200 nm. In one embodiment, the magnetic microbeads molecules can be adapted for use in a dipstick and/or a test can be magnetic nanobeads with a size of about 50 nm to strip for detection of microbes or pathogens. For example, a about 200 nm. Magnetic microbeads can be manipulated dipstick and/or a test strip can include at least one test area using magnetic field or magnetic field gradient. Such par- 30 containing one or more engineered microbe-targeting mol ticles commonly consist of magnetic elements such as iron, ecules described herein. In some embodiments, the engi nickel and cobalt and their oxide compounds. Magnetic neered microbe-targeting molecules can be conjugated or microbeads are well-known and methods for their prepara attached to a test area surface of the dipstick and/or a test tion have been described in the art. See, e.g., U.S. Pat. No. strip. Methods for conjugating a protein to a substrate 6,878,445; U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,578,325; 35 Surface are known in the art, including, but not limited to U.S. Pat. No. 6,676,729; U.S. Pat. No. 6,045,925; and U.S. direct cross-linking, indirect cross-linking via a coupling Pat. No. 7,462.446; and U.S. Patent Publications No. 2005/ agent (e.g., a functional group, a peptide, a nucleic acid 0025971; No. 2005/0200438; No. 2005/0201941; No. 2005/ matrix Such as DNA matrix), absorption, or any other 0271745; No. 2006/0228551: No. 2006/0233712: No. 2007/ art-recognized methods known in the art. 01666.232; and No. 2007/0264.199, the contents of which are 40 In one embodiment, about 1 Jug to about 100 g microbe incorporated herein by reference. binding molecules can be coated on or attached to a dipstick Magnetic microbeads are also widely and commercially or membrane Surface. In another embodiment, about 3 ug to available, with or without functional groups capable of about 60 g microbe-binding molecules can be coated on or binding to coupling molecules. Magnetic microbeads func attached to a dipstick or membrane Surface. In some embodi tionalized with various functional groups, e.g., amino 45 ments, about 0.1 mg/mL to about 50 mg/mL, about 0.5 groups, carboxylic acid groups, epoxy groups, tosyl groups, mg/mL to about 40 mg/mL, about 1 mg/mL to about 30 or silica-like groups, are also widely and commercially mg/mL, about 5 mg/mL to about 20 mg/mL microbe-binding available. Suitable magnetic microbeads are commercially molecules can be coated on or attached to a dipstick or available such as from AdemTech, Miltenyi, PerSeptive membrane Surface. In one embodiment, about 11.5 mg/mL Diagnostics, Inc. (Cambridge, Mass.); Invitrogen Corp. 50 microbe-binding molecules can be coated on or attached to (Carlsbad, Calif); Cortex Biochem Inc. (San Leandro, a dipstick or membrane Surface. Calif.); and Bangs Laboratories (Fishers, Ind.). In particular In some embodiments, the engineered microbe-targeting embodiments, magnetic microbeads that can be used herein molecule(s) conjugated to the dipstick and/or a test strip can can be any DYNABEADSR) magnetic microbeads (Invitro further comprise a detectable label as described herein. In gen Inc.), depending on the Substrate Surface chemistry. 55 one embodiment, the detectable label can include a micro Microbe-Targeting Cells: bial enzyme Substrate conjugated to a detectable moiety. In some embodiments, the Substrate to which the engi Such detectable moiety is undetectable when conjugated to neered microbe-targeting molecule binds can be a living the microbial enzyme substrate, but becomes a detectable cell, or a biological tissue or organ. For example, the living entity (e.g., a light-emitting signal) in the presence of an cells can be associated with an immune response, and Such 60 enzyme possessed or secreted by the microbe. See, e.g., WO cells include, but are not limited to, a phagocyte (macro 2011/103144, for the use of such detectable label in detec phage, neutrophil, and dendritic cell), mast cell, eosinophil. tion of microbes, the content of which is incorporated herein basophil, and/or natural killer cell. Alternatively, the living by reference. cell can be the cell of biological tissues or organs of the In some embodiments, the dipstick and/or a test strip can immune system, such as spleen, lymph nodes, lymphatic 65 further comprise at least one reference area or control area vessels, tonsils, thymus, bone marrow, Peyer's patches, for comparison with a readout signal determined from the connective tissues, mucous membranes, the reticuloendothe test area. The reference area generally excludes the engi US 9,593,160 B2 37 38 neered microbe-targeting molecules, e.g., to account for any not non-living microbial matter, Such as endotoxins, that can background signal. In some embodiments, the reference area have devastating effects on patient Survival. can include one or more known amounts of the detectable Specific antigen detection is widely used for a variety of label that the engineered microbe-targeting molecules in the infectious diseases, most commonly for legionellosis (Le test area encompass. In such embodiments, the reference 5 gionella pneumophila Serotype 1 in urine), malaria (Plas area can be used for calibration such that the amount of modium falciparum in blood) and with less success with microbes in a test sample can be estimated or quantified. Streptococcus pneumonia infection (in urine). However, The dipstick and/or a test strip can be in any shape and/or directantigen detection can only be used to rule in or rule out in any format, e.g., a planar shape such as a rectangular strip a specific etiology and cannot identify most bacteria. 10 As described herein, engineered microbe-binding mol or a circular disk, or a curved surface such as a stick. ecules or substrates (e.g., FcMBL molecules or FcMBL Alternatively, a continuous roll can be utilized, rather than bound magnetic microbeads) can bind to the Surface of a discrete test strips, on which the test area(s) and optionally wide array of microbes including pathogens, e.g., but not reference area(s) are present in the form of continuous lines limited to, bacterial, fungal, parasitic or viral. For example, or a series of spots. 15 in some embodiments, blood or urine or any other biological The dipstick and/or a test strip can be made of any fluid can be subjected to microbial capture by the engineered material, including, without limitations, paper, nitrocellu microbe-binding molecules or substrates (e.g., FcMBL mol lose, glass, plastic, polymer, membrane material, nylon, and ecules or FcMBL-bound magnetic microbeads) and any combinations thereof. In one embodiment, the dipstick adequate controls (e.g., non-specific binding control by and/or a test strip can include paper. In one embodiment, the non-relevant protein coated magnetic microbeads). Accord dipstick and/or a test strip can include nylon. ingly, engineered microbe-binding molecules or Substrates The microbe-binding dipsticks and/or test strips described (e.g., FcMBL or FcMBL-coated magnetic microbeads) can herein can be used as point-of-care diagnostic tools for be used to bind microbes such as bacteria for diagnostic or microbe or pathogen detection. By way of example only, a therapeutic applications. microbe-binding dipstick or test strip (e.g., made of mem 25 Not only can the engineered microbe-binding molecules brane material Such as nylon) can be brought into contact or substrates bind to at least a portion of a cell surface of a with a test sample (e.g., a blood sample) from a patient or a microbe, the engineered microbe-binding molecules or Sub Subject, and incubated for a period of time, e.g., at least strates can also capture microbial matter (e.g., microbe about 15 seconds, at least about 30 seconds, at least about 1 originating cell fragments or matter derived from microbes min, at least about 2 mins, at least about 5 mins, at least 30 circulating in biological fluids including endotoxins, e.g., about 10 mins, at least about 15 mins, at least about 30 mins, during the course of an infection, even in the absence of at least about 1 hour or more. In some embodiments, the bacteremia, or found on an environmental surface, food or incubated dipstick or test strip can then be incubated in a water, a pharmaceutical product or a medical device). The blocking agent (e.g., BSA, normal serum, casesin, non-fat presence of Such microbial cell fragments or microbe dry milk, and/or any commercially-available blocking 35 derived matter can be used, alone or in combination with agents to minimize non-specific binding). Depending on detection of an intact microbe, for diagnostic applications, different embodiments of the engineered microbe-targeting e.g., the presence of pathogen-originating cell fragments or molecules, in some embodiments, the microbe-binding dip matter derived from pathogens can be diagnostic of an Stick or test strip after contact with a test sample (e.g., a infectious disease in a Subject, or a microbial contamination blood sample) can be further contacted with at least one 40 on an environmental Surface, food or water, a pharmaceu additional agent to facilitate detection of pathogen, and/or to tical product, or a medical device. Moreover, the biochemi increase specificity of the pathogen detection. For example, cal/proteomic (MALDI-TOF, multiple mass spectrometry some embodiments of the dipstick or test strip after contact (e.g., MSn) or specific antibody or aptamer based) analysis with a test sample (e.g., a blood sample) can be further of the bound products (e.g., microbial matter or microbes contacted with a detectable label that is conjugated to a 45 bound onto an engineered microbe-binding molecule or molecule that binds to a microbe and/or microbial matter. Substrate) can allow recognition of elements pathognomonic Examples of Such molecules can include, but are not limited for microbes. to, one or more embodiments of the engineered microbe Accordingly, provided herein also include methods for targeting molecule described herein, an antibody specific for detection of the presence or absence of a microbe and/or the microbes or pathogens to be detected, a protein, a 50 microbial matter in an organ, a tissue, and/or a cell in a peptide, a carbohydrate or a nucleic acid that is recognized subject (including blood, normally sterile fluids or virtual by the microbes or pathogens to be detected, and any cavities). For example, the presence or absence of a microbe combinations thereof. and/or microbial matter can be detected by capture of a In some embodiments, the readout of the microbe-binding microbe and/or non-viable microbial matter or particles dipsticks and/or test strips can be performed in a system or 55 circulating in the Subject’s body fluid, e.g., blood, or found device, e.g., a portable device. The system or device can in other fluids Such as urine, or in any other organ sampled display a signal indicating the presence or the absence of a by any appropriate means (e.g., but not limited to, biopsy, microbial infection in a test sample, and/or the extent of the puncture, aspiration, and lavage). microbial infection. The inventors have discovered that, in some embodiment, Generally, the diagnosis of infection relies on indirect or 60 FcMBL captured not only whole bacteria for concentration direct evidence. The indirect evidence relies on the detection and direct analysis but also non-viable microbial matter. of an adapted and specific host response directed against the Such binding can be quantified by a microbe binding assay pathogen. The direct evidence relies on the culture of the based on the capture of this microbial matter on the engi microorganism from the infected site, amplification and neered microbe-binding molecules or Substrates (e.g., detection of pathogen-specific nucleic acids or the detection 65 FcMBL-coated microbeads). The detection of this material of a specific antigen in blood or urine; however, existing can be performed using enzyme-linked engineered microbe technologies only allow detection of living pathogens and binding molecules described herein (e.g., FcMBL) or fluo US 9,593,160 B2 39 40 rescent-linked engineered microbe-binding molecules microbes can reflect deep tissue infection as they generally described herein (e.g., FcMBL). The engineered microbe find its way into the bloodstream and most likely the urine. binding molecules (e.g., FcMBL) can be multimerized on The capture and characterization of this microbial matter or the Surface of a desired Substrate (e.g., a magnetic bead) to fragments of microbes can be used as evidence markers form a microbe-binding substrate for enhanced avidity. specific for a given microbial species, thus allowing the Examples 16-17 show that engineered microbe-binding diagnosis and/or identification of a microbe causing infec molecules described herein (e.g., FcMBL) can detect live tion anywhere in an organism. and dead microbes as well as microbial matter (including, For example, the use of one or more specific antibodies but not limited to, fragments of a microbe and endotoxins) can allow characterization of the nature and/or types of the in a biological sample (e.g., blood sample), and the detection 10 microbial material bound to the engineered microbe-binding results correlate with clinical symptoms or morbidity of an molecules or substrates. Specific detection of certain mol infection. ecules (e.g., proteins, carbohydrates, lipids) present on a Accordingly, in Some embodiments, the presence of intact microbe Surface, such as Lipid A on E. coli or any other microbes and/or microbial matter (including microbe cell molecules on a microbe of interest, can allow further dis fragments or matter derived from a microbe) bound on the 15 crimination of samples or identification of microbes present engineered microbe-binding molecules or Substrates can be in the samples. Without wishing to be limiting, as shown in used as a marker for infection or contamination. Current Example 16 and FIG. 33, in order to determine if the generic biomarkers for infection include molecules, for captured microbes and/or fragments thereof were associated example, cytokines; acute phase proteins such as CRP, with E. coli, FcMBL-coated magnetic beads with captured procalcitonin, and fibrinogen; erythrocyte sedimentation microbes and/or fragments thereof can be further contacted rate (ESR), and elevated or diminished leukocyte counts. with a specific antibody raised against Escherichia coli However, these generic biomarkers are not specific to infec lipopolysaccharide Lipid A (anti-LPS Lipid A antibody). As tion, but are also involved in non-infectious inflammation. shown in the bottom panel of FIG. 33, the microbes and/or In contrast, binding of microbes or fragments thereof fragments thereof captured on the FcMBL-coated magnetic (including matter derived from microbes) on an engineered 25 beads did not bind to anti-LPS antibodies, indicating that the microbe-binding molecule and/or Substrate can not only be microbes and/or microbial fragments bound to the FcMBL used for infection of a sampled organ or tissue or cell(s) coated microbeads were unlikely associated with E. coli. In (blood or otherwise) but also to any major infectious process contrast, the microbes and/or fragments thereof captured on ongoing anywhere in the body where sufficient microbial the FcMBL-coated magnetic microbeads bound to anti-LPS destruction or catabolism results in the presence of microbial 30 antibodies, indicating that the microbes and/or microbial matter in the bloodstream, urine or any other conveniently fragments bound to the FcMBL-coated microbeads were accessed fluid. There is currently no biological marker for likely associated with E. coli. Accordingly, the screening of infection that does not cross-react with generic non-infec a library of antibodies directed against a plurality of tious inflammation. Thus, this is a major breakthrough in the microbes (including pathogens) can allow direct diagnosis management of patients Suspected of infection. Without 35 of microbe-specific infections, e.g., anywhere in the body of wishing to be bound, not only can the engineered microbe a subject by a simple blood or urine test available in less than binding molecules and/or Substrates be used to detect an three hours in any microbiology laboratory equipped for infection in a Subject (e.g., a mammalian Subject), but they magnetic separation. can also be used to detect the presence or absence of a In a different embodiment, a rapid test can be performed microbe in any environment or on any device where a 40 using a "dipstick' format. For example, a membrane spotted microbe can be present, including but are not limited to, with lines of microbial species-specific antibodies (instead biomedical devices, clinics or hospitals, ponds or water of FcMBL molecules as shown in FIG. 13) can be incubated reservoirs, wastewater, water farms (including hydropon with the microbe-binding substrates (e.g., FcMBL-coated ics), and/or food processing plants or machines. microbeads) previously incubated with a fluid test sample. Indeed, the inventors have collected blood from de 45 The microbe-binding substrates (e.g., FcMBL-coated micro identified, hospitalized patients and demonstrated, in some beads) captured by proper antibodies on the membrane can embodiments, that the FcMBL assay is positive in patients form a detectable band (e.g., rust-colored for FcMBL-coated with negative blood cultures and correlates strongly with the magnetic microbeads) on the membrane, indicating the diagnosis of infection. Thus, in some embodiments, the species (one or many) of which microbial matter or FcMBL assay is more sensitive than conventional blood 50 microbes was captured. cultures for detection of an infection. In some embodiments, Other than antibody-based characterization methods, the FcMBL assay can be used for early diagnosis of an other known methods such as mass spectrometric charac infection. In some embodiments, the engineered microbe terization methods or PCR analysis can also be used to binding molecules and/or Substrates and/or diagnosis/detec characterize and/or identify the species of a microbe cap tion processes described herein can detect presence of a 55 tured on the engineered microbe-binding molecules and/or microbe and/or microbial matter in a test sample which Substrates. In some embodiments, the microbe-binding mol previously yielded a negative result in a traditional diagnosis ecules and/or substrates with captured microbes and/or method (e.g., a blood culture). Accordingly, the engineered microbial matter/fragments can be washed prior to any microbe-binding molecules and/or Substrates and/or diag further characterization methods such as mass spectrometric nosis/detection process described herein can enable a more 60 characterization methods. sensitive and faster diagnosis than the traditional diagnostic In some embodiments, the engineered microbe-binding method (e.g., a blood culture). molecules and/or substrates with captured microbes and/or Further, in some embodiments, the wide spectrum of the microbial matter/fragments can be subjected to direct analy engineered microbe-binding molecules or Substrates (e.g., sis for characterization and/or identification of species of FcMBL molecules or FcMBL-coated magnetic microbeads) 65 microbes and/or microbial matter bound thereon. For can enable the capture of most clinically relevant bacterial example, the engineered microbe-binding molecules and/or species. The presence of microbial matter or fragments of substrates with captured microbial materials can be directly US 9,593,160 B2 41 42 subjected to MALDI-TOF analysis (e.g., without separation Additionally or alternatively, another microbe/microbial of the captured microbial materials from the engineered fragment library can be established from in vitro analysis of microbe-binding molecules and/or substrates). microbes’ binding moieties to engineered microbe-binding Alternatively, any art-recognized protocols or methods molecule(s) described herein, wherein the microbes can be described herein can be applied on the engineered microbe Subjected to mechanical or chemical or antibiotic lysis or binding molecules and/or Substrates to isolate bound autolysis. The microbial material can be captured in different microbes and/or microbial compounds/fragments from the media, buffer, urine, blood or any appropriate medium. engineered microbe-binding molecules and/or Substrates The diagnostic profiles can be matched to any reference prior to any characterization analysis. Exemplary methods to profiles, e.g., specific in vivo or in situ derived microbe recover or isolate bound microbes and/or microbial com 10 profiles and/or specific in-vitro derived microbes profiles for pounds/fragments from the engineered microbe-binding identification with a probability score for generic infection, molecules and/or substrates, prior to any characterization clades level, family level, genus level or species level analysis, include, but are not limited to, Ca" chelation to identification. release captured materials from the engineered microbe Further, methods for detection of the presence or absence binding molecules and/or substrates; lowering pH to release 15 of a microbe and/or microbial matter on an environmental binding mediated by Fc-protein A interaction; protein Surface, food or water, a pharmaceutical product, or a extraction using formic acid and acetonitrile, and any com medical device by capture of a microbe and/or non-viable binations thereof. The control microbeads (e.g., microbeads microbial matter or particles present thereon are also within coated with molecules that do not react to microbes) can be the scope described herein. In some embodiments, the treated similarly for baseline determination. methods of any aspect described herein can be used to Screen In some embodiments, the extracted captured material pharmaceutical products (e.g., drugs, therapeutic agents or from the engineered microbe-binding molecules and/or Sub imaging agents), and/or medical devices (e.g., fluid delivery strates and/or non-specific control-bound material can be devices, or implantable devices) for the presence or absence subjected to PCR analysis. For example, the identity of the of a microbe and/or microbial matter (including but not extracted captured material can be determined by detecting 25 limited to endotoxin produced by a microbe, e.g., a gram the presence or absence of a gene encoding a protein specific negative microbe Such as E. coli and/or a gram-positive to a microbe species. Thus, the presence of one or more microbe Such as S. aureus). In one embodiment, the method microbe species-specific genes(s) can be indicative of the can be used to Screen pharmaceutical products (e.g., drugs, corresponding microbe species bound on the engineered therapeutic agents or imaging agents), and/or medical microbe-binding molecules. 30 devices (e.g., fluid delivery devices, or implantable devices) In some embodiments, extracted captured material from for the presence or absence of endotoxin produced by a the engineered microbe-binding molecules and/or sub microbe, e.g., a gram-negative microbe Such as E. coli strates, and/or non-specific control-bound material can be and/or a gram-positive microbe Such as S. aureus. Subjected to mass spectrometric analysis, including but not Exemplary Optimization or Modifications of Microbe-Tar limited to, MALDI-TOF or MALDI-TOF-TOF. The non 35 geting Substrates specific control-bound material can establish a baseline for In accordance with at least Some embodiments described the composition of the medium tested. This profile can be herein, engineered microbe-binding molecules and/or Sub used as reference for the analysis of the material bound to strates (e.g., FcMBL-bound paramagnetic microbeads) can the engineered microbe-binding molecules and/or Sub bind to a surface of a variety of microbes and/or microbial strates. Peaks present in the control-bound samples can be 40 matter described herein, e.g., but not limited to, bacterial, subtracted from the profile obtained from the material bound fungal, parasitic or viral. In some embodiments, a number of to the engineered microbe-binding molecules and/or Sub factors such as the orientation of engineered microbe-bind strates. The specific profile of the material that was bound to ing molecules (e.g., FcMBL) conjugated to or coated on a the microbe-binding molecules and/or Substrates (e.g., after Substrate (e.g., a paramagnetic microbead), size of a Sub subtraction of the reference profile) can constitute a 45 strate (e.g., a microbead), selection of linkers and microbe microbe/microbial fragment signature. Both positive and/or Surface-binding domains used in constructing an engineered negative charge analysis can be performed to identify infor microbe-targeting molecule, microbial assay condition, and mative peaks. any combinations thereof, can be optimized for binding of Recognition of a microbe signature can be analyzed by the microbe-targeting Substrates to microbes. any known methods in the art. For example, a microbe? 50 Optimization of Substrate Size and Densities of Engi microbial fragment signature can be recognized by compar neered Microbe-Targeting Molecules on the Substrate: ing the specific profile of the material that was bound to the Additionally or alternatively, the density of engineered microbe-binding molecules and/or Substrates to one or more microbe-binding molecules (e.g., FcMBL) conjugated to or microbe/microbial fragment signature libraries, e.g., using coated on a Substrate (e.g., a microbead) can be optimized to matching comparison algorithms based on the previously 55 capture microbes. accumulated profiles. In some embodiments where the engineered microbe For identification of microbe species, depending on ori binding molecule is FcMBL, the FcMBL differs from gins of microbes, a microbe/microbial fragment signature recombinant wild-type MBL in that the FcMBL is a dimeric library can be established by in vivo or in situ samples such protein with two Carbohydrate Recognition Domain (CRD) as clinical-trial derived samples and/or environment derived 60 heads whereas wild-type MBL has 9-18 heads in groups of samples (e.g., Samples collected from a clinical setting, 3. The affinity of the individual heads is 10 and MBL culture medium, food processing plant, water source). For binding to microbe Surfaces requires binding of multiple example, blood (or other biological fluids) of patients CRD heads to give high avidity binding. In order to achieve infected with known microbes, e.g., pathogens, can be this high avidity with the dimeric FcMBL protein, in some analyzed and a microbial material signature can be charac 65 embodiments, a plurality of (e.g., at least about 2, at least terized. Recognition of the signature in the same clinical about 5, at least about 10, at least about 25, at least about 50, context can establish the family/genus/species diagnosis. at least about 100, at least about 1000, at least about 10, at US 9,593,160 B2 43 44 least about 10, at least about 10, at least about 10) Additionally, the density of the engineered microbe-bind engineered microbe-binding molecules (e.g., FcMBL) can ing molecules on a Surface of the microbe-targeting Sub be multiplexed on a surface of a Substrate (e.g. magnetic strate can be optimized for microbial binding. In order to microbeads such as the MYONETM Streptavidin microbeads enhance binding of a specific microbe to the microbe from Life Technologies). The number of the engineered targeting Substrate, the distance between any two microbe microbe-binding molecules conjugated to a substrate can binding molecules on a Surface of the microbe-targeting vary with available surface area of a substrate. substrate can be less than the size of a microbe. Therefore, Accordingly, a number of factors, including density of a microbe can bind to more than one microbe-binding engineered microbe-binding molecules on a Substrate, size molecules (e.g., at least 2, at least 3, at least 4, at least 5, at 10 least 6, at least 7, at least 8, at least 9, at least 10, or more) of the substrate, and/or size of the engineered microbe present on the microbe-targeting Substrate with a greater binding molecules, can be varied to optimize binding of combined binding strength. microbes to the engineered microbe-binding Substrates (e.g., In some embodiments, the microbe-targeting Substrates but not limited to, FcMBL-coated beads). Some exemplary can comprise on their Surfaces a saturating amount of the optimizations/modifications can include, but are not limited engineered microbe-binding molecules described herein. As to, using a substrate (e.g., but not limited to, a microbead) of used herein, the term “saturating amount” refers to the different sizes; varying the density of engineered microbe maximum number or amount of engineered microbe-bind binding molecules (e.g., but not limited to, FcMBL) on the ing molecules that can be conjugated to and/or coated on a Substrate (e.g., but not limited to, a microbead) by binding Surface of a Substrate. The Saturating amount of the engi the engineered microbe-binding molecules (e.g., but not neered microbe-binding molecules that can be present on a limited to, FcMBL) to a substrate scaffold in various ori Surface of a Substrate is dependent on a number of factors ented arrays, e.g., but not limited to DNA, aptamers, or Such as size and/or structure of the engineered microbe extracellular matrix (e.g., fibronectin); producing fusion binding molecules, size and/or structure of the Substrate, proteins of microbe-binding domain(s) (e.g., but not limited orientation of the engineered microbe-binding molecules to, MBL CRD head and neck regions) bound to a linker 25 present on the Substrate, and any combinations thereof. described herein or fusion partner (or linker described Selection of Linkers and Microbe Surface-Binding herein) of different sizes (e.g., between about 100 kDa to Domain Used in Constructing an Engineered Microbe about 1000 kDa or between about 250 kDa to about 750 Targeting Molecule and Microbial Assay Condition: kDa. An exemplary fusion partner can include, but is not In some embodiments, a linker can be selected to provide limited to, the Fc portion of IgM, which is about 500 kDa); 30 binding sites of a microbe, wherein the binding interaction producing fusion proteins of microbe-binding domain(s) of the microbe to the linker is different from the interaction (e.g., but not limited to, MBL CRD head and neck regions) of the microbe to the microbe surface-binding domain. For with multimeric (e.g., at least dimeric, at least trimeric) example, in an engineered microbe-binding molecule where linkers described herein or fusion partners (or linkers the linker is a Fc molecule and the microbe surface-binding described herein); and any combinations thereof. As used 35 domain is derived from MBL or a fragment thereof, the Fc herein, the term “multimeric linker” or “multimeric fusion linker allows protein A or protein G binding, which is partner” refers to a linker or fusion partner comprising two calcium-independent, while the MBL binding domain or more identical linker units for providing attachment of requires calcium ions for binding with a microbe. Accord microbe-binding domains. By way of example only, a tri ingly, a protein A-expressing (e.g., S. aureus) or protein meric linker or fusion partner is a linker or fusion partner 40 G-expressing microbe can bind to both MBL binding comprising three identical linker units for attachment of domain and Fc linker in the presence of calcium ions, but microbe-binding domains. can bind to only Fc linker in the absence of calcium ions. In The binding of any microbe to a microbe-binding sub contrast, a protein A- and protein G-negative microbe (e.g., strate described herein can be determined by any methods E. coli) generally binds to neither MBL binding domain nor known in the art and/or described herein, such as by ELISA 45 Fc linker in the absence of calcium ions. In such embodi colorimetric assay or antibody-based imaging methods ments, by controlling the amount of calcium ions present in described in the Examples. Accordingly, the microbe-bind a microbial assay, one can distinguish protein A- or protein ing Substrate can be optimized for detection of a microbe, G-expressing microbes (e.g., S. aureus) from protein A- and e.g., by varying its density and/or size of engineered protein G-negative microbes (e.g., E. coli). Additional microbe-binding molecules, its Substrate structure and/or 50 details of such embodiments can be found in later sections size, and then determining their effects on the binding of the “Exemplary Process for Capture and/or Detection of a microbe to the microbe-binding substrate. Microbe and/or Microbial Matter in a Test Sample” and For example, Example 18 shows an exemplary method to “Exemplary Embodiments of Methods for Diagnosing a evaluate the microbe-capture efficiency of microbe-targeting Microbial Infection. magnetic microbeads (e.g., FcMBL-coated magnetic micro 55 Exemplary Process for Capture and/or Detection of a beads) having different sizes. In some embodiments, a Microbe and/or Microbial Matter in a Test Sample microbead (e.g., a magnetic microbead or a non-magnetic In one aspect, a process for detecting a microbe and/or microbead) as a Substrate for attachment of engineered microbial matter in a test sample is described herein. As microbe-binding molecules can have a size of about 10 nm shown in FIG. 17, the process 1200 comprises the optional to 10 um, about 20 nm to about 5um, about 40 nm to about 60 step 1202 (preprocessing of the sample), step 1204 (pro 1 um, about 50 nm to about 500 nm, or about 50 nm to about cessing of the sample), step 1206 comprising 1208 (microbe 200 nm. Without wishing to be bound by theory, the size of capture) and 1210 (microbe separation), and 1212 (microbe a microbead can be smaller than the size of a microbe so that detection). While these are discussed as discrete processes, more than one microbead (e.g., at least 2, at least 3, at least one or more of the preprocessing, processing, capture, 4, at least 5, at least 10 or more) can bind to the same 65 microbe separation, and detection can be performed in a microbe for enhanced capture and increased detection sen microfluidic device. Use of a microfluidic device can auto sitivity. mate the analysis process and/or allow analysis of multiple US 9,593,160 B2 45 46 samples at the same time. One of skill in the art is well aware at least four minutes, at least five minutes, at least ten of methods in the art for collecting, handling and processing minutes, at least fifteen minutes, at least thirty minutes, at biological fluids which can be used in the practice of the least forty-five minutes, or at least one hour. Such incubation present disclosure. The process described herein can allow can be at any appropriate temperature, e.g., room-tempera sample analysis at in short time periods. For example, the ture (e.g., about 16°C. to about 30°C.), a cold temperature process can be completed in less than 6 hours, less than 5 (e.g. about 0°C. to about 16°C.), or an elevated temperature hours, less than 4 hours, less than 3 hours, less than 2 hours, (e.g., about 30° C. to about 95°C.). In some embodiments, less than 1 hour, less than 30 minutes. In some embodiments, the sample is incubated for about fifteen minutes at room presence and identity of a microbe in the sample can be done temperature. In some embodiments, incubation is for about within 10 minutes to 60 minutes of starting the process. 10 5 seconds to about 60 seconds. In some embodiments, there In some embodiments, the sample can be a biological is no incubation and the sample mixture is used directly in fluid, e.g., blood, plasma, serum, lactation products, amni the sample processing step. otic fluids, sputum, saliva, urine, semen, cerebrospinal fluid, 1204 (Sample Processing): bronchial aspirate, perspiration, mucus, liquefied stool After the optional preprocessing step, the sample can be sample, synovial fluid, lymphatic fluid, tears, tracheal aspi 15 optionally processed by adding one or more processing rate, and any mixtures thereof. For example, the sample can reagents to the sample. These processing reagents can serve be a whole blood sample obtained from a subject. to lyse cells, degrade unwanted molecules present in the The process described herein can be utilized to detect the sample and/or dilute sample for further processing. These presence of a microbe in a sample of any given Volume. In processing reagents include, but are not limited to, Surfac some embodiments, sample volume is about 0.25 ml to tants and detergents, salts, cell lysing reagents, anticoagul about 50 ml, about 0.5 ml to about 25 ml, about 1 ml to about lants, degradative enzymes (e.g., proteases, lipases, nucle 15 ml, about 2 ml to about 10 ml. In some embodiments, ases, lipase, collagenase, cellulases, amylases and the like), sample Volume is about 5 ml. In one embodiment, sample and solvents, such as buffer Solutions. Amount of the pro volume is 8 ml. cessing reagent to be added can depend on the particular 1202 (Sample Preprocessing): 25 sample to be analyzed, the time required for the sample It can be necessary or desired that a test sample, such as analysis, identity of the microbe to be detected or the amount whole blood, be preprocessed prior to microbe detection as of microbe present in the sample to be analyzed. described herein, e.g., with a preprocessing reagent. Even in It is not necessary, but if one or more reagents are to be cases where pretreatment is not necessary, preprocessing can added they can present in a mixture (e.g., in a solution, be optionally done for mere convenience (e.g., as part of a 30 “processing buffer') in the appropriate concentrations. regimen on a commercial platform). A preprocessing reagent Amount of the various components of the processing buffer can be any reagent appropriate for use with the assays or can vary depending upon the sample, microbe to be processes described herein. detected, concentration of the microbe in the sample, or time The sample preprocessing step generally comprises add limitation for analysis. ing one or more reagent to the sample. This preprocessing 35 Generally, addition of the processing buffer can increase can serve a number of different purposes, including, but not the volume of the sample by 5%, 10%, 15%, 20% or more. limited to, hemolyzing blood cells, dilution of sample, etc. In some embodiments, about 50 ul to about 5000 ul of the The preprocessing reagents can be present in the sample processing buffer are added for each ml of the sample. In container before sample is added to the sample container or some embodiments, about 100 ul to about 250 ul of the the preprocessing reagents can be added to a sample already 40 processing buffer are added for each ml of the sample. In one present in the sample container. When the sample is a embodiment, about 800 ul of the processing buffer are added biological fluid, the sample container can be a VACU for each 200 ul of the sample. TAINER(R), e.g., a heparinized VACUTAINER(R). In some embodiments, a detergent or Surfactant comprises The preprocessing reagents include, but are not limited to, about 5% to about 20% of the processing buffer volume. In Surfactants and detergents, salts, cell lysing reagents, anti 45 Some embodiment, a detergent or Surfactant comprises about coagulants, degradative enzymes (e.g., proteases, lipases, 5% to about 15% of the processing buffer volume. In one nucleases, lipase, collagenase, cellulases, amylases and the embodiment, a detergent or Surfactant comprises about 10% like), and solvents, such as buffer Solutions. of the processing buffer volume. In some embodiments, a preprocessing reagent is a Sur Exemplary Surfactants and detergents include, but are not factant or a detergent. In one embodiment, the preprocessing 50 limited to, Sulfates, such as, ammonium lauryl Sulfate, reagent is Triton X 100. sodium dodecyl sulfate (SDS), and sodium lauryl ether Amount of preprocessing reagent to be added can depend sulfate (SLES) sodium myreth sulfate; sulfonates, such as, on a number of factors. Generally, the preprocessing reagent dioctyl Sodium SulfoSuccinate (Docusates), perfluorooctane is added to a final concentration of about 0.1 mM to about sulfonate (PFOS), perfluorobutanesulfonate, alkyl benzene 10 mM. If a liquid, the preprocessing reagent can be added 55 Sulfonates, and 3-(3-Cholamidopropyl)dimethylammonio so as to dilute the sample at least 10%, at least 20%, at least 1-propanesulfonate (CHAPS): 3-(3-cholamidopropyl)dim 30%, at least 40%, at least 50%, at least 60%, at least 60%, ethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO); at least 80%, at least 90%, at least 1-fold, at least 2-fold, at phosphates, such as alkyl aryl ether phosphate and alkyl least 3-fold, or at least 5-fold. ether phosphate; carboxylates, such as fatty acid salts, After addition of the preprocessing reagent, the reagent 60 Sodium Stearate, sodium lauroyl sarcosinate, perfluo can be mixed into the sample. This can be simply accom rononanoate, and perfluorooctanoate (PFOA or PFO); plished by agitating the sample, e.g., shaking or Vortexing octenidine dihydrochloride; alkyltrimethylammonium salts, the sample and/or moving the sample around, if it is in a such as cetyl trimethylammonium bromide (CTAB) and microfluidic device. cetyl trimethylammonium chloride (CTAC); cetylpyri After addition of the preprocessing reagent, the sample 65 dinium chloride (CPC); polyethoxylated tallow amine mixture can be incubated for a period of time, e.g., for at (POEA); benzalkonium chloride (BAC); benzethonium least one minute, at least two minutes, at least three minutes, chloride (BZT); 5-Bromo-5-nitro-1,3-dioxane; dimethyl US 9,593,160 B2 47 48 dioctadecylammonium chloride; dioctadecyldimethylam Examples of phosphodiesterase inhibitors suitable for use monium bromide (DODAB); Sultaines, such as cocami herein include, but are not limited to, anagrelide, dipyrida dopropyl hydroxysultaine; cetyl alcohol; Stearyl alcohol; mole, pentoxifyllin, and theophylline. cetostearyl alcohol (consisting predominantly of cetyl and Suitable dextrans include, but are not limited to, dex Stearyl alcohols); oleyl alcohol; polyoxyethylene glycol tran70, such as HYSKONTM (CooperSurgical, Inc., Shelton, alkyl ethers (Brij) such as, octaethylene glycol monododecyl Conn., U.S.A.) and MACRODEXTM (Pharmalink, Inc., ether and pentaethylene glycol monododecyl ether, poly Upplands Vasby, Sweden), and dextran 75, such as GEN oxypropylene glycol alkyl ethers; glucoside alkyl ethers, TRANTM 75 (Baxter Healthcare Corporation). Such as decyl glucoside, lauryl glucoside and octyl gluco Suitable thrombin antagonists include, but are not limited side; polyoxyethylene glycol octylphenol ethers, such as 10 to, hirudin, bivalirudin, lepirudin, desirudin, argatroban, Triton X-100; polyoxyethylene glycol alkylphenol ethers, melagatran, Ximelagatran and dabigatran. Such as Nonoxynol-9; glycerol alkyl esters, such as glyceryl As used herein, anticoagulants can also include factor Xa laurate; polyoxyethylene glycol sorbitan alkyl esters, such as inhibitors, factor Ha inhibitors, and mixtures thereof. Vari Polysorbate 20 (Polyoxyethylene (20) sorbitan monolau ous direct factor Xa inhibitors are known in the art includ rate), Polysorbate 40 (Polyoxyethylene (20) sorbitan mono 15 ing, those described in Hirsh and Weitz, Lancet, 93:203-241, palmitate), Polysorbate 60 (Polyoxyethylene (20) sorbitan (1999); Nagahara et al. Drugs of the Future, 20: 564-566, monostearate), and Polysorbate 80 (Polyoxyethylene (20) (1995); Pinto et al., 44: 566-578, (2001); Pruitt et al, Biorg. sorbitan monooleate); cocamide ME; cocamide DEA; dode Med. Chem. Lett., 10: 685-689, (2000); Quan et al., J. Med. cyldimethylamine oxide; poloxamers: DOC: nonyl phe Chem. 42: 2752-2759, (1999); Sato et al., Eur: J. Pharmacol, noxypolyethoxylethanol NP-40 (Tergitol-type NP-40); octyl 347: 231-236, (1998); Wong et al., J. Pharmacol. Exp. phenoxypolyethoxylethanol (Noidet P-40); cetyltrimethyl Therapy, 292:351-357, (2000). Exemplary factor Xa inhibi ammonium bromide; and any mixtures thereof. tors include, but are not limited to, DX-9065a, RPR-120844, In some embodiments, one ml of the processing buffer can BX-807834 and SEL Series Xa inhibitors. DX-9065a is a comprise about 1U to about 100 U of a degradative enzyme. synthetic, non-peptide, propanoic acid derivative, 571 D In some embodiments, one ml of the processing buffer 25 selective factor Xa inhibitor. It directly inhibits factor Xa in comprises about 5U to about 50 U of a degradative enzyme. a competitive manner with an inhibition constant in the In one embodiment, one ml of the processing buffer com nanomolar range. See for example, Herbert et al., J. Phar prises about 10 U of a degradative enzyme. Enzyme unit (U) macol. Exp. Ther: 276:1030-1038 (1996) and Nagahara etal, is an art known term for the amount of a particular enzyme Eur: J. Med. Chem. 30(suppl): 140s-143s (1995). As a non that catalyzes the conversion of 1 umol of Substrate per 30 peptide, synthetic factor Xa inhibitor, RPR-120844 (Rhone minute. Poulenc Rorer), is one of a series of novel inhibitors which In some embodiments, one ml of the processing buffer can incorporate 3-(S)-amino-2-pyrrolidinone as a central tem comprise about 1 lug to about 10 ug of an anti-coagulant. In plate. The SEL series of novel factor Xa inhibitors Some embodiment, one ml of the processing buffer can (SEL1915, SEL-2219, SEL-2489, SEL-2711: Selectide) are comprise about 1 lug to about 5 ug of an anti-coagulant. In 35 pentapeptides based on L-amino acids produced by combi one embodiment, one ml of the processing buffer comprises natorial chemistry. They are highly selective for factor Xa about 4.6 ug of an anti-coagulant. and potency in the pM range. In some embodiments, one ml of the processing buffer can Factor Ha inhibitors include DUP714, hirulog, hirudin, comprise about 1 mg to about 10 mg of anti-coagulant. In melgatran and combinations thereof. Melagatran, the active Some embodiment, one ml of the processing buffer can 40 form of pro-drug Ximelagatran as described in Hirsh and comprise about 1 mg to about 5 mg of anti-coagulant. In one Weitz, Lancet, 93:203-241, (1999) and Fareed et al. Current embodiment, one ml of the processing buffer comprises Opinion in Cardiovascular, pulmonary and renal investiga about 4.6 mg of anti-coagulant. tional drugs, 1:40-55, (1999). Exemplary anti-coagulants include, but are not limited to, Generally, salt concentration of the processing buffer can heparin, heparin Substitutes, Salicylic acid, D-phenylalanyl 45 range from about 10 mM to about 100 mM. In some L-prolyl-L-arginine chloromethyl ketone (PPACK), Hiru embodiments, the processing buffer comprises a salt at a din, Ancrod (Snake Venom, Vipronax), tissue plasminogen concentration of about 25 mM to about 75 mM. In some activator (tPA), urokinase, Streptokinase, plasmin, pro embodiment, the processing buffer comprises a salt at a thrombopenic anticoagulants, platelet phosphodiesterase concentration of about 45 mM to about 55 mM. In one inhibitors, dextrans, thrombin antagonists/inhibitors, ethyl 50 embodiment, the processing buffer comprises a salt at a ene diamine tetraacetic acid (EDTA), acid citrate dextrose concentration of about 43 mM to about 45 mM. (ACD), sodium citrate, citrate phosphate dextrose (CPD). The processing buffer can be made in any suitable buffer Sodium fluoride, Sodium oxalate, potassium oxalate, lithium solution known the skilled artisan. Such buffer solutions oxalate, sodium iodoacetate, lithium iodoacetate and mix include, but are not limited to, TBS, PBS, BIS-TRIS, tures thereof. 55 BIS-TRIS Propane, HEPES, HEPES Sodium Salt, MES, Suitable heparinic anticoagulants include heparins or MES Sodium Salt, MOPS, MOPS Sodium Salt, Sodium active fragments and fractions thereof from natural, Syn Chloride, Ammonium acetate solution, Ammonium formate thetic, or biosynthetic sources. Examples of heparin and Solution, Ammonium phosphate monobasic solution, heparin Substitutes include, but are not limited to, heparin Ammonium tartrate dibasic solution, BICINE buffer Solu calcium, Such as calciparin; heparin low-molecular weight, 60 tion, Bicarbonate buffer solution, Citrate Concentrated Solu Such as enoxaparin and lovenox; heparin sodium, Such as tion, Formic acid solution, Imidazole buffer Solution, MES heparin, lipo-hepin, liquaemin Sodium, and panheprin; hepa Solution, Magnesium acetate solution, Magnesium formate rin sodium dihydroergotamine mesylate; lithium heparin; Solution, Potassium acetate Solution, Potassium acetate solu and ammonium heparin. tion, Potassium acetate solution, Potassium citrate tribasic Suitable prothrombopenic anticoagulants include, but are 65 Solution, Potassium formate Solution, Potassium phosphate not limited to, anisindione, dicumarol, warfarin Sodium, and dibasic solution, Potassium phosphate dibasic solution, the like. Potassium Sodium tartrate solution, Propionic acid solution, US 9,593,160 B2 49 50 STE buffer solution, STET buffer solution, Sodium acetate capture process, a microbe-targeting Substrate added into a solution, Sodium formate solution, Sodium phosphate diba test sample can capture one or more microbes present in the sic solution, Sodium phosphate monobasic Solution, Sodium test sample. In some embodiments, the microbe capture tartrate dibasic solution, TNT buffer solution, TRIS Glycine process can be repeated and/or performed for a Sufficient buffer solution, TRIS acetate-EDTA buffer solution, Trieth amount of time to allow for concentrating and/or cleaning up ylammonium phosphate solution, Trimethylammonium the test sample before microbe detection. Thus, microbe acetate solution, Trimethylammonium phosphate solution, capture and separation process described herein can be used Tris-EDTA buffer solution, TRIZMAR Base, and for concentrating and/or cleaning up a sample before analy TRIZMAR) HCL. Alternatively, the processing buffer can be sis for a target component in the sample. made in water. 10 In some embodiments, the processing buffer comprises a In some embodiments, the microbe capture process can mixture of Triton-X, DNAse I, human plasmin, CaCl and comprise mixing nano- and/or micron-sized beads or par Tween-20. In one embodiment, the processing buffer con ticles coated with affinity molecules (e.g., FcMBL or engi sists of a mixture of Trirton-X, DNAse I, human plasmin, neered microbe-binding molecules described herein) which CaCl and Tween-20 in a TBS buffer. 15 can bind to a microbe in the sample. These affinity molecule In one embodiment, one ml of the processing buffer coated nano- and/or micron-sized beads or particles are also comprises 100 ul of Triton-X100, 10ul of DNAse (1 U/1 ul), referred to as "coated-microbeads' herein. These coated 10 ul of human plasmin at 4.6 mg/ml and 870 ul of a mixture microbeads can be magnetic microbeads or non-magnetic of TBS, 0.1% Tween-20 and 50 mM CaCl. microbeads (e.g., fluorescent microbeads). Reagents and treatments for processing blood before In some embodiments, the coated-microbeads can be assaying are also well known in the art, e.g., as used for microbe-targeting magnetic microbeads described herein. assays on Abbott TDx, AXSYMR, and ARCHITECTR) Amount of coated-microbeads added to the sample can be analyzers (Abbott Laboratories), as described in the litera dependent on a number of different factors, such as, number ture (see, e.g., Yatscoff et al., Abbott TDx Monoclonal of affinity molecules on each microbead, size of the micro Antibody Assay Evaluated for Measuring Cyclosporine in 25 bead, binding affinity of the affinity molecule to the microbe, Whole Blood, Clin. Chem. 36: 1969-1973 (1990), and and concentration of the microbe in the sample. Addition Wallemacq et al., Evaluation of the New AXSYM ally, amount of coated-microbeads added to the sample can Cyclosporine Assay: Comparison with TDx Monoclonal be adjusted to optimize the capture of microbes. In some Whole Blood and EMIT Cyclosporine Assays, Clin. Chem. embodiments, amount of coated-microbeads added to the 45: 432-435 (1999)), and/or as commercially available. 30 sample is such that a microbead binds with one microbe. Additionally, pretreatment can be done as described in However, each microbe can be bound to more than one Abbott’s U.S. Pat. No. 5,135,875, European Pat. Pub. No. 0 coated-microbeads. This can reduce cross-linking of mul 471 293, and U.S. Pat. App. Pub. No. 2008/0020401, content tiple microbes together which can lead to coagulation and/or of all of which is incorporated herein by reference. It is to precipitation of such cross-linked microbes from the sample. be understood that one or more of these known reagents 35 Generally, about 100 to about 10 coated-microbeads can be and/or treatments can be used in addition to or alternatively added to each ml of the sample. In some embodiments, about to the sample treatment described herein. 10 to about 5x10' coated-microbeads can be added for each In some embodiments, after addition of the processing ml of sample. Stated another way, in Some embodiments, the buffer, the sample comprises 1% Triton-X, 10 U of DNase, total amount of the microbe-binding molecules contacted 4.6 mg/ml of plasmin, 5 mM Calcium, 0.01% of Tween 20, 40 with the test sample can range from about 0.01 ug to about 2.5 mM of Tris, 150 mM of NaCl and 0.2 mM of KCl in 1 mg, about 0.1 ug to about 500 ug, about 0.5 g to about addition to the components already present in the sample. 250 ug, about 1 Jug to about 100 ug, or about 3 Lig to about After addition of the processing buffer, the sample can 60 g. In some embodiments, the total amount of the undergo mixing. This can be simply accomplished by agi microbe-binding molecules contacted with the test sample tating the sample, e.g., shaking or Vortexing the sample 45 can range from about 500 ug to about 1000 mg, about 1 mg and/or moving the sample around, if it is in a microfluidic to about 750 mg, about 5 mg to about 500 mg, about 10 mg device. In other embodiments where the microbe-targeting to about 250 mg. or about 25 mg to about 100 mg. Substrate is in a form of a dipstick or a membrane, the In some embodiments, a plurality of coated-microbeads microbe-targeting dipstick or membrane can be dipped in a can be contacted with a test sample. The plurality of Volume of a test sample and gently agitated with a rocking 50 coated-microbeads can comprise at least two Subsets (e.g., 2. motion. 3, 4, 5, or more subsets), wherein each subset of coated After addition of the processing reagents, the sample can microbeads have a pre-determined dimension. In some be incubated for a period of time, e.g., for at least one embodiments, the plurality of coated-microbeads can com minute, at least two minutes, at least three minutes, at least prise a first Subset of the coated-microbeads and a second four minutes, at least five minutes, at least ten minutes, at 55 subset of the coated-microbeads. In such embodiments, the least fifteen minutes, at least thirty minutes, at least forty first subset of the coated-microbeads each has a first pre five minutes, or at least one hour. Such incubation can be at determined dimension; and the second Subset of the coated any appropriate temperature, e.g., room-temperature (e.g., microbeads each has a second pre-determined dimension. about 16°C. to about 30°C.), a cold temperature (e.g. about The pre-determined dimension of a coated-microbead 0° C. to about 16° C.), or an elevated temperature (e.g., 60 depends, in part, on the dimension of a microbead described about 30° C. to about 95° C.). In some embodiments, the herein to which the engineered microbe-binding molecules sample is incubated for about fifteen minutes at room are conjugated. For example, the microbead can have a size temperature. of about 10 nm to 10 um, about 20 nm to about 5um, about 1206 (1208 (Microbe Capture) and 1210 (Microbe Sepa 40 nm to about 1 um, about 50 nm to about 500 nm, or about ration)): 65 50 nm to about 200 nm. After processing of the sample, the sample can be Sub Additionally, each subset of the coated-microbeads can jected to a microbe capture process. During the microbe comprise on their Surfaces Substantially the same density or US 9,593,160 B2 51 52 different densities of the affinity molecules (e.g., FcMBL or microbe-targeting Substrate, e.g., incubation for at least one engineered microbe-binding molecules described herein). minute, at least two minutes, at least three minutes, at least Different subsets of the plurality of the coated-microbeads four minutes, at least five minutes, at least ten minutes, at can be brought into contact with a test sample in any manner. least fifteen minutes, at least about twenty minutes, at least For example, in some embodiments, the plurality of the thirty minutes, at least forty-five minutes, or at least one coated-microbeads can be provided as a single mixture hour. In one embodiment, the sample mixture can be incu comprising at least two Subsets of the coated-microbeads to bated for a period of about 10-20 minutes. Such incubation be added into a test sample. In some embodiments, in order can be performed at any appropriate temperature, e.g., to distinguish among different Subsets of the coated-micro room-temperature (e.g., about 16°C. to about 30°C.), a cold beads, the coated-microbeads in each Subset can have a 10 temperature (e.g. about 0°C. to about 16°C.), or an elevated distinct detection label, e.g., a distinctly-fluorescent label temperature (e.g., about 30° C. to about 95° C.). In some that can be sorted afterward, for example, by flow cytometry. embodiments, the incubation can be performed at a tem In other embodiments, the plurality of the coated-micro perature ranging from about room temperature to about 37° beads can be brought into contact with a test sample in a C. In some embodiments, the sample can be incubated for sequential manner. For example, a test sample can be 15 about 10 mins to about 20 mins at room temperature. In contacted with a first subset of the coated-microbeads, some embodiments, the sample is incubated for about fifteen followed by a contact with at least one more subsets of the minutes at room temperature. coated-microbeads. The previous subset of the coated-mi To prevent or reduce agglutination (or non-specific bind crobeads can be removed from the test sample before ing) during separation of the microbes from the sample, addition of another subset of the coated-microbeads into the additional reagents can be added to the sample mixture. test sample. Such reagents are also referred to as blocking reagents In some embodiments, the coated-microbeads are or a herein. For example, these blocking reagents can comprise microbe-targeting Substrate is present in the processing a ligand of the affinity molecules on the coated-microbeads. buffer. In one embodiment, one ml of the processing buffer Addition of Such blocking reagents can reduce agglutination comprises 100 ul of Triton-X100, 10 ul of a solution 25 by binding with any empty ligand binding sites on the comprising about 25 million microbeads (AKT-FC-MEBL on affinity molecules. Accordingly, when microbe-targeting 1 um MYONETM C1 streptavidin microbeads), 10 ul of magnetic microbeads are used for capturing the microbes, DNAse (1 U/1 ul), 10ul of human plasmin at 4.6 mg/ml and the blocking reagent can be a carbohydrate, such as man 870 ul of a mixture of TBS, 0.1% Tween-20. In some nose. Amount of additional reagent can depend on the embodiments, the processing buffer can include a calcium 30 amount of microbeads added to the sample. Generally, about salt, e.g., CaCl (e.g., ~50 mM CaCl). In some embodi the reagent is added to a final concentration of about 0.1 mM ments, the processing or capture buffer can include no to about 10 mM. The amount of the blocking agent required calcium salt, e.g., CaCl2. can vary, at least partly, with the amount and/or Surface area After addition of the coated-microbeads, the coated-mi of the microbe-targeting Substrate that is in contact with a crobeads can be mixed in the sample to allow microbes to 35 test sample. In some embodiments, the blocking reagent can bind with the microbeads. This can be simply accomplished be added to a final concentration of about 0.1% (w/v) to by agitating the sample, e.g., shaking or Vortexing the about 10% (w/v), about 0.5% (w/v) to about 7.5% (w/v), or sample and/or moving the sample around in a microfluidic about 1% (w/v) to about 5% (w/v). In some embodiments, device. In other embodiments where the microbe-targeting about 1% casein can be used as a blocking agent in the assay Substrate is in a form of a dipstick or a membrane, the 40 described herein. microbe-targeting dipstick or membrane can be dipped in a After addition of the blocking reagent, the sample mixture Volume of a test sample and gently agitated with a rocking can be incubated for a period of time to allow the blocking motion. reagent to bind to with the affinity molecules, e.g., for at The Volume of a test sample required for contacting the least one minute, at least two minutes, at least three minutes, microbe-targeting Substrate can vary with, e.g., the selection 45 at least four minutes, at least five minutes, at least ten of the microbe-targeting Substrate (e.g., microbeads, fibers, minutes, at least fifteen minutes, at least thirty minutes, at filters, filters, fibers, screens, mesh, tubes, hollow fibers), the least forty-five minutes, or at least one hour. Such incubation concentration of microbes present in a test sample, and/or can be at any appropriate temperature, e.g., room-tempera the platform used to carry out the assay (e.g., a microfluidic ture (e.g., about 16°C. to about 30°C.), a cold temperature device or a blood collection tube, a microtiter plate). In some 50 (e.g. about 0°C. to about 16°C.), or an elevated temperature embodiments, the test sample volume used to perform the (e.g., about 30° C. to about 95°C.). In some embodiments, assay described herein, e.g., in a microfluidic platform, can the sample is incubated for about fifteen minutes at room range from about 1 uL to about 500 u, from about 5 uL to temperature. In some embodiments, incubation is for about about 250 uL, or from about 10 uL to about 100 uL. In other 5 seconds to about 60 seconds. In some embodiments, the embodiments, the test sample volume used to perform the 55 incubation can be performed at a temperature ranging from assay described herein, e.g., in a tube platform, can range about room temperature to about 37° C. In some embodi from about 0.05 mL to about 50 mL, from about 0.25 ml to ments, the sample is incubated for about fifteen minutes at about 50 ml, about 0.5 ml to about 25 ml, about 1 ml to about room temperature. 15 ml, or about 2 ml to about 10 ml. In some embodiments, To prevent or reduce non-specific binding during the the test sample Volume used to perform the assay described 60 contact between a microbe-targeting Substrate and a test herein can be about 1 mL to about 5 ml. In one embodiment, sample, in some embodiments, the microbe-targeting Sub the test sample Volume used to perform the assay described strate (e.g., coated-microbeads) and/or the test sample can be herein is about 5 ml to about 10 mL. pre-treated with a blocking agent that does not react with After addition of the microbe-targeting Substrate (e.g., microbes, e.g., casein, normal serum, BSA, non-fat dry milk coated-microbeads) into a test sample (containing a process 65 powder and any art-recognized block agent, before contact ing buffer), the sample mixture can be incubated for a period ing each other. Optionally, microbe-targeting Substrate after of time to allow the microbe of interest to bind onto the blocking can be washed with any art-recognized buffer to US 9,593,160 B2 53 54 remove any leftover blocking agent. The number of wash In some embodiments, the concentration of a chelating steps can range from 1 to many, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9. agent present in the test sample containing the microbe 10 or more wash steps. In one embodiment, the microbe targeting substrate can be sufficient to reduce the likelihood targeting Substrate after blocking can be washed with a of a protein A- and protein G-negative microbe (e.g., E. buffer, e.g., TBST, for about at least 1-3 times. coli), if present in the test sample, to bind with at least one Exemplary Optional Modifications to 1208 (Microbe microbe-binding molecule. For example, the concentration Capture): of a chelating agent present in the test sample with the In accordance with one aspect described herein, the test microbe-targeting Substrate can be sufficient to reduce the sample can be contacted with a microbe-targeting Substrate number of protein A- and protein G-negative microbes (e.g., in the presence of a chelating agent. Without wishing to be 10 E. coli), if present in the test sample, to bind with at least one bound by theory, the addition of a chelating agent to a test microbe-binding molecule, by at least about 30%, at least sample and/or processing buffer can reduce the likelihood of about 40%, at least about 50%, at least about 60%, at least any protein A- and protein G-negative microbe (e.g., E. about 70%, at least 80% or higher, as compared to the coli), but not protein A- or protein G-expressing microbe number of protein A- and protein G-negative microbes (e.g., (e.g., S. aureus) in the test sample, to bind with at least one 15 E. coli) bound on the microbe binding molecules in the microbe-binding molecule. Accordingly, detection of any absence of the chelating agent. In some embodiments, the microbes bound on the microbe-targeting Substrate concentration of a chelating agent present in the test sample described herein in the presence of a chelating agent can with the microbe-targeting Substrate can be sufficient to determine the presence or absence of a protein A- or protein reduce the number of protein A- and protein G-negative G-expressing microbe in a test sample. microbes (e.g., E. coli), if present in the test sample, to bind The chelating agent can be added into the processing with at least one microbe-binding molecule, by at least about buffer comprising the test sample. The amount of the chelat 85%, at least about 90%, at least about 95%, at least about ing agent is sufficient to chelate free calcium ions and thus 98%, at least about 99%, up to and including 100%, or any prevent or reduce calcium-dependent carbohydrate recogni values between about 85% and about 100%, as compared to tion domain binding (e.g., mannose-binding lectin) with a 25 the number of protein A- and protein G-negative microbes microbe. The amount of the chelating agent needed to (e.g., E. coli) bound on the microbe-binding molecules in the prevent or reduce calcium-dependent carbohydrate recogni absence of the chelating agent. tion domain binding (e.g., mannose-binding lectin) with a The protein A-expressing and protein G-expressing microbe can depend on, e.g., the concentration of free microbes can generally bind to microbe-binding molecules calcium ions present in a test sample and optionally a 30 via two independent (but additive) mechanisms: Fc-medi capture buffer, e.g., used to dilute a chelating agent and/or a ated binding and microbe Surface-binding domain (e.g., test sample. Thus, in some embodiments, the concentration MBL)-mediated binding. Without wishing to be bound by of the chelating agent can be higher than the total concen theory, while the protein A-expressing and protein G-ex tration of free calcium ions present in the combined solution pressing microbes can still be captured on the microbe of a test sample and a capture buffer. For example, in some 35 targeting Substrate in the presence of a chelating agent, the embodiments, the concentration of the chelating agent can presence of free calcium ions can further increase the be at least about 30% higher, including at least about 40%, number of protein A-expressing and protein G-expressing at least about 50%, at least about 60%, at least about 70%, microbes bound to the microbe-targeting Substrate, because at least about 80%, at least about 90%, at least about 95%, the overall binding in the presence of calcium ions can be at least about 98%, up to and including 100%, or any percent 40 almost twice as strong as in the absence of calcium ions. between about 30% and about 100%, higher than the total Accordingly, in Some embodiments, the concentration of concentration of free calcium ions present in the combined a chelating agent present in the test sample containing the solution of a test sample and a capture buffer. In other microbe-targeting Substrate can reduce the number of pro embodiments, the concentration of the chelating agent can tein A-expressing microbes and/or protein G-expressing be at least about 1.5-fold, at least about 2-fold, at least about 45 microbes bound onto the microbe-targeting Substrate, but 3-fold, at least about 4-fold, at least about 5-fold, at least Such effect as compared to that on the protein A- and protein about 6-fold, at least about 7-fold, at least about 8-fold, at G-negative microbes (e.g., E. coli) is much smaller, e.g., at least about 9-fold, at least about 10-fold, at least about least about 30% smaller, at least about 40% smaller, at least 15-fold, at least about 20-fold, at least about 30-fold, at least about 50%, at least about 60% smaller, at least about 70% about 40-fold, at least about 50-fold, at least about 75-fold, 50 smaller, or at least about 80% smaller. For example, as at least about 100-fold or more, higher than the total shown in FIG. 29, while the concentration of a chelating concentration of free calcium ions present in the combined agent (e.g., 100 mM EDTA) is sufficient to reduce the Solution of a test sample and a capture buffer. In one binding of protein A- and protein G-negative microbes (e.g., embodiment, the concentration of the chelating agent can be E. coli) with a microbe-targeting Substrate (e.g., a microbe at least about 5-fold to about 50-fold, or at least about 7-fold 55 targeting membrane) to an undetectable level, there is still a to about 25-fold, higher than the total concentration of free detectable level of protein A-expressing microbes (e.g., S. calcium ions present in the combined solution of a test aureus) binding to the microbe-targeting membrane. There sample and a capture buffer. fore, in some embodiments, the concentration of a chelating In some embodiments, the concentration of a chelating agent used in the assay described herein should be high agent present in the test sample and optionally a processing 60 enough to prevent at least about 80% or higher, including at or capture buffer, e.g., used to dilute the chelating agent least about 90%, at least about 95%, up to and including and/or the test sample, can range from about 0.1 mM to 100%, of the protein A- and protein G-negative microbes about 1 M, about 10 mM to about 500 mM, about 20 mM (e.g., E. coli) from binding to be microbe-targeting Substrate, to about 250 mM, or about 25 mM to about 125 mM. In one but low enough to allow at least about 30% or higher, embodiment, the concentration of a chelating agent present 65 including at least about 40%, at least about 50%, at least in the test sample and optionally a capture buffer can be about 60%, at least about 70% or higher, of the protein about 25 mM to about 125 mM. A-expressing microbes (e.g., S. aureus) or protein G-ex US 9,593,160 B2 55 56 pressing microbes to bind with the microbe-targeting Sub reaction with other molecules or ions to mediate binding of strate. In one embodiment, the concentration of a chelating carbohydrate patterns on a microbial cell Surface to a agent used in the assay described herein should be high microbe Surface-binding domain (e.g., MBL) of the engi enough to prevent at least about 90% or higher, of the neered microbe-binding molecule. Accordingly, in some protein A- and protein G-negative microbes (e.g., E. coli), if 5 embodiments, free calcium ions can be present in the any present in the test sample, from binding to be microbe absence of chelating agent. In some embodiments, free targeting Substrate, but low enough to allow at least about calcium ions can be present in a solution comprising a 50% of the protein A-expressing microbes (e.g., S. aureus) chelating agent and calcium ions, wherein the amount of or protein G-expressing microbes, if any present in the test calcium ions present in the solution is at least about 30% sample, to bind with the microbe-targeting Substrate. 10 more than an amount Sufficient to interact with Substantially Examples of calcium ion-chelating agents can include, all the chelating agent molecules present in the Solution to but are not limited to, 1.2-bis(2-aminophenoxy)ethane-N,N. form chelate complexes. For example, in some embodi N',N'-tetraacetic acid, ethylenediaminetetraacetic acid ments, in order to obtain free calcium ions, the amount of (EDTA); ethylene glycol-bis(2-aminoethylether)-N.N.N',N'- calcium ions present in the Solution can be at least about tetraacetic acid; ethylene glycol-bis(B-aminoethyl ether)-N, 15 30%, including at least about 40%, at least about 50%, at N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy) least about 60%, at least about 70%, at least about 80%, at ethane-N,N,N',N'-tetraacetic acid (BAPTA), a buffer least about 90%, at least about 95%, at least about 98%, up containing citrate, N.N-Bis(2-(bis-(carboxymethyl)amino) to and including 100% and any percent between 30% and ethyl)-glycine (DTPA), nitrilo-2,2'2"-triacetic acid (NTA), a 100%, more than an amount sufficient to interact with buffer that precipitates a calcium ion from the test sample, Substantially all the chelating agent molecules present in the including, e.g., a phosphate buffer, a carbonate buffer and a Solution to form chelate complexes. In some embodiments, bicarbonate buffer, a low pH buffer (e.g., a pH buffer less in order to obtain free calcium ions, the amount of calcium than pH 7 or less than pH 6), citric acids and its salts, ions present in the Solution can be at least about 1-fold, at gluconic acid and its salts, alkali metal pyrophosphates, least about 2-fold, at least about 3-fold, at least about 4-fold, alkali metal polyphosphates, sodium hexametaphosphate, 25 at least about 5-fold, at least about 6-fold, at least about triethylene tetramine, diethylene triamine, o-phenanthroline, 7-fold, at least about 8-fold, at least about 9-fold, at least oxalic acid and any combinations thereof. about 10-fold, at least about 15-fold, at least about 20-fold, The chelating agent can be directly added to the test at least about 25-fold, at least about 50-fold, at least about sample or prepared in a processing or capture buffer, which 100-fold, at least about 500-fold, at least about 1000-fold, is then added to the test sample in contact with the microbe 30 more than an amount Sufficient to interact with Substantially targeting Substrate. The processing or capture buffer can be all the chelating agent molecules present in the Solution to any buffered solutions, e.g., with a pH ranging from about 6 form chelate complexes. In some embodiments, free calcium to about 10. In some embodiments, the processing or capture ions can be present in a solution when the concentration of buffer can include, but is not limited to, a tris-buffered saline, calcium ions in the solution is at least about 1.5-fold, at least a phosphate buffered saline or a combination thereof. In 35 about 2-fold, at least about 3-fold, at least about 4-fold, at Some embodiments, the processing or capture buffer can least about 5-fold, at least about 6-fold, at least about 7-fold, include a surfactant, e.g., to prevent non-specific binding of at least about 8-fold, at least about 9-fold, at least about a microbe to a microbe-Surface-binding domain of the 10-fold, at least about 20-fold, or higher than the concen microbe-targeting Substrate, and/or to Saturate non-specific tration of a chelating agent present in the same solution. binding sites, if any, present in the microbe-targeting Sub 40 In some embodiments, calcium ions can be obtained from strate. A Surfactant or detergent, e.g., as described earlier, a water-soluble calcium salt. By the term “water-soluble can be dissolved in a buffered solution in any amount, e.g., calcium salt' is meant a calcium salt which has significant ranging from about 0.001% (v/v) to about 5% (v/v), from solubility in water at room temperature, for example at least about 0.01% (v/v) to about 2.5% (v/v), or from about 0.05% 1 gram per 100 ml water, at least 10 grams per 100 ml water, (v/v) to about 1% (v/v). In some embodiments, the surfac 45 or at least 25 grams per 100 ml water or higher. Examples tant added to the processing or capture buffer can include of calcium salts include, without limitations, calcium chlo Tween 80 or polysorbate 80 at a concentration of about ride, calcium fluoride, calcium bromide, calcium iodide, 0.01% to about 0.1%. In one embodiment, the surfactant calcium nitrate, calcium citrate, calcium formate, calcium added to the processing or capture buffer can include Tween acetate, calcium gluconate, calcium ascorbate, calcium lac 80 or polysorbates 80 at a concentration of about 0.05%. 50 tate, calcium glycinate and mixtures thereof. In some After incubation, the microbe-targeting Substrate can then embodiments, calcium chloride can be used as a source of be analyzed, as described below, for the presence or absence calcium ions. of a bound microbe. In the absence of a microbe-targeting Free calcium ions can be present at a concentration or an Substrate-bound microbe, in Some embodiments, the previ amount Sufficient to mediate binding of calcium-dependent ous volume of the test sample or a new fresh volume of the 55 carbohydrate recognition domain with a microbe Surface. In test sample can be contacted with a fresh microbe-targeting Some embodiments, free calcium ions can be present at a Substrate in the presence of free calcium ions, e.g., to concentration of at least about 1 uM, at least about 10 uM, determine the presence or absence of protein A- and protein at least about 25uM, at least about 50 uM, at least about 100 G-negative microbes (e.g., E. coli). In some embodiments, uM, at least about 250 uM, at least about 500 LM, or at least the free calcium ions can be produced adding a Sufficient 60 about 1 mM or higher. In some embodiments, the free amount of calcium salts in the test sample. If there has been calcium ions can be present at a concentration of at least a chelating agent present in the test sample, a higher amount about 1 mM, at least about 2.5 mM, at least about 5 mM, at of calcium salts is generally needed in order to obtain free least about 10 mM, at least about 25 mM, at least about 50 calcium ions. mM, at least about 75 mM, at least about 100 mM or higher. As used herein, the term “free calcium ions' refers to 65 In other embodiments, the free calcium ions can be present calcium ions that are not complexed with any molecule or at a concentration of at least about 100 mM, at least about compound, e.g., a chelating agent, which can hinder its 150 mM, at least about 200 mM, at least about 300 mM, at US 9,593,160 B2 57 58 least about 400 mM, at least about 500 mM, at least about embodiments, the concentration of a chelating agent used in 600 mM, at least about 700 mM, at least about 800 mM, at the assay described herein is sufficient to elute off or remove least about 900 mM, at least about 1 M or higher. In one from the microbe-targeting substrate at least about 85% of embodiment, the free calcium ions can be present at a the bound protein A- and protein G-negative microbes (e.g., concentration of about 1 mM to about 10 mM. In one 5 E. coli), including at least about 85%, at least about 90%, at embodiment, the free calcium ions can be present at a least about 95%, at least about 98%, up to and including concentration of at least about 5 mM. 100%, or any values between about 85% and about 100%, While a chelating agent can be added during an initial of the bound protein A- and protein G-negative microbes capture of a microbe on a microbe-targeting Substrate, the (e.g., E. coli). chelating agent can also be first excluded to allow the initial 10 As noted above, the protein A-expressing and protein capture of any microbe, including protein A- and protein G-expressing microbes can bind to microbe-binding mol G-negative microbes, on a microbe-targeting Substrate in the ecules via Fc-mediated and calcium ion-dependent MBL presence of free calcium ions, but added after the capture to mediated binding. Without wishing to be bound by theory, remove any captured protein A- or protein G-negative the concentration of a chelating agent used in the assay microbes from the microbe-targeting Substrate. 15 described herein can also elute off or remove at least a Accordingly, in Some embodiments, the microbe capture portion of the protein A-expressing and/or protein G-ex can comprise (i) contacting at least a first volume of a test pressing microbes from the microbe-targeting Substrate. For sample with a microbe-targeting Substrate described herein example, the concentration of a chelating agent used to elute in the presence of free calcium ions, and (ii) contacting the off or remove protein A- and protein G-negative microbes microbe-binding molecule of the microbe-targeting Sub- 20 from the microbe-targeting Substrate can be sufficient to strate described herein, upon the contact with the test elute off or remove no more than 60%, no more than 50%, sample, with a solution comprising a chelating agent. no more than 40%, no more than 30%, no more than 20%, When the microbe-targeting substrate is contacted with a no more than 10% or lower, of the bound protein A-express test sample in the presence of free calcium ions as described ing and/or protein G-expressing microbes. In some embodi herein, microbes that primarily depend on calcium-depen- 25 ments, the concentration of a chelating agent used to elute dent MBL-mediated binding such as protein A- and protein off or remove from the microbe-targeting Substrate at least G-negative microbes, e.g., E. coli can bind to the microbe about 80% or more, including at least about 90% or more, target Substrate, in addition to microbes associated with of the bound protein A- and protein G-negative microbes can Fc-mediated binding such as protein A-expressing microbes be sufficient to elute off or remove no more than 50%, or (e.g., S. aureus), and protein G-expressing microbes. 30 more than 40% of the bound protein A-expressing and/or To elute off or remove from the microbe-targeting sub protein G-expressing microbes. As shown in FIG. 29, while strate the captured microbes that primarily depend on cal the concentration of a chelating agent (e.g., 100 mM EDTA) cium-dependent MBL-mediated binding Such as protein A is sufficient to elute off or remove substantially all protein A and protein G-negative microbes, e.g., E. coli, the microbe and protein G-negative microbes (e.g., E. coli) from a binding molecules on the microbe-targeting Substrates can 35 microbe-targeting Substrate to an undetectable level, there is be contacted with a solution comprising a sufficient amount still a detectable level of protein A-expressing microbes of a chelating agent as described herein. The Solution (e.g., S. aureus) remained bound to the microbe-targeting comprising the chelating agent can be same as a capture membrane. buffer described above. In such embodiments, the microbe As a person having ordinary skill in the art can appreciate, targeting Substrate can be incubated with the Solution com- 40 the assay described herein can further comprise isolating the prising a chelating agent for a period of time to allow microbe-targeting Substrate from the test sample, e.g., as microbes that primarily bind to microbe-binding molecules described below, before contacting microbe-binding mol via calcium-dependent MBL-mediated binding to elute off ecules on its Substrate Surface with the solution comprising the microbe-targeting Substrate, e.g., incubation for at least the chelating agent described herein. one minute, at least two minutes, at least three minutes, at 45 1210 (Microbe Separation from Sample): least four minutes, at least five minutes, at least ten minutes, The sample mixture is then subjected to a microbe sepa at least fifteen minutes, at least thirty minutes, at least ration process. In some embodiments, because microbes are forty-five minutes, or at least one hour. Such incubation can bound with one or more magnetic microbeads, a magnet can be performed at any appropriate temperature, e.g., room be employed to separate the bound microbes from the test temperature (e.g., about 16° C. to about 30° C.), a cold 50 sample. The skilled artisan is well aware of methods for temperature (e.g. about 0°C. to about 16°C.), or an elevated carrying out magnetic separations. Generally, a magnetic temperature (e.g., about 30° C. to about 95° C.). In some field gradient can be applied to direct the capture of mag embodiments, the microbe-targeting Substrate can be incu netic microbeads. Optionally, the bound microbe can be bated with the solution comprising a chelating agent for at washed with a buffer to remove any leftover sample and least about 5 mins to about 15 mins at room temperature. 55 unbound components. Number of wash steps can range from In these embodiments, the concentration of a chelating 1 to many, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more wash steps. agent used in the assay described herein is sufficient to elute Without wishing to be bound by a theory, capture and off or remove from the microbe-targeting Substrate at least separation of the bound microbes from the sample can about 30% of the bound protein A- and protein G-negative concentrate the microbes and also remove components, microbes (e.g., E. coli). For example, the concentration of a 60 which can interfere with the assay or process, from the test chelating agent used in the assay described herein is Sufi sample. cient to elute off or remove from the microbe-targeting The magnetic field source can be any magnet device substrate at least about 30% of the bound protein A- and positioned to generate the magnetic field gradient that is protein G-negative microbes (e.g., E. coli), including at least used to pull the captured microbe out from the sample. An about 40%, at least about 50%, at least about 60%, at least 65 electromagnetic controller can be used to control and adjust about 70%, at least 80% or higher, of the bound protein A the magnetic field and gradients thereof, and to control the and protein G-negative microbes (e.g., E. coli). In some migration, separation and orientation of the magnetically US 9,593,160 B2 59 60 bound microbes. The magnetic field gradient can be gener and/or the test sample can be washed with a buffer (e.g., ated by a permanent magnet or by an electromagnetic signal TBST) for about at least 1-3 times. generator. The electromagnetic signal generator can include 1212 (Microbe Detection/Analysis): an electromagnet or electrically-polarizable element, or at A detection component, device or system can be used to least one permanent magnet. The magnetic field gradient can detect and/or analyze the presence of the separated microbe, be produced at least in part according to a pre-programmed for example, by spectroscopy, electrochemical detection, pattern. The magnetic field gradient can have a defined polynucleotide detection, fluorescence anisotropy, fluores magnetic field strength and/or spatial orientation. In some cence resonance energy transfer, electron transfer, enzyme embodiments, the magnetic field gradient has a defined assay, magnetism, electrical conductivity, isoelectric focus 10 ing, chromatography, immunoprecipitation, immunosepara magnetic field strength. The term “magnetic field gradient’ tion, aptamer binding, filtration, electrophoresis, use of a as used herein refers to a variation in the magnetic field with CCD camera, immunoassay, ELISA, Gram staining, immu respect to position. By way of example only, a one-dimen nostaining, microscopy, immunofluorescence, western blot, sional magnetic field gradient is a variation in the magnetic polymerase chain reaction (PCR), RT-PCR, fluorescence in field with respect to one direction, while a two-dimensional 15 situ hybridization, sequencing, mass spectroscopy, or Sub magnetic field gradient is a variation in the magnetic field stantially any combination thereof. The separated microbe with respect to two directions. can remain bound on the microbe-targeting Substrate during As used herein, the term “magnetic field’ refers to mag detection and/or analysis, or be isolated form the microbe netic influences which create a local magnetic flux that flows targeting Substrate prior to detection and/or analysis. through a composition and can refer to field amplitude, In some embodiments, labeling molecules that can bind squared-amplitude, or time-averaged squared-amplitude. It with the microbe can also be used to label the microbes for is to be understood that magnetic field can be a direct-current detection. As used herein, a “labeling molecule' refers to a (DC) magnetic field or alternating-current (AC) magnetic molecule that comprises a detectable label and can bind with field. The magnetic field strength can range from about a target microbe. Labeling molecules can include, but are not 0.00001 Tesla per meter (T/m) to about 10 T/m. In some 25 limited to, MBL or a portion thereof, FcMBL, AKT-FcMBL, embodiments, the magnetic field strength can range from wheat germ agglutinin, lectins, antibodies (e.g., gram-nega about 0.0001 T/m to about 10 T/m. In some other embodi tive antibodies or gram-positive antibodies, antibiotics to ments, the magnetic field strength can range from about specific microbial strains or species), antigen binding frag 0.001 T/m to about 10 T/m. ments of antibodies, aptamers, ligands (agonists or antago In some embodiments, microbe capture and/or microbe 30 nists) of cell-surface receptors and the like. The labeling targeting Substrate separation can be performed by a rapid molecule can also be a non-specific labeling molecule that microbe diagnostic device as described in Int. Pat. App. No. non-specifically stains all viable cells in a sample. WO 2011/091037, filed Jan. 19, 2011, the content of which As used herein, the term “detectable label” refers to a is incorporated herein by reference. A rapid microbe diag composition capable of producing a detectable signal indica nostic device as described in Int. Pat. App. No. WO 2011/ 35 tive of the presence of a target. Detectable labels include any 091037, filed Jan. 19, 2011, can be modified to replace the composition detectable by spectroscopic, photochemical, capture chamber or capture and visualization chamber with biochemical, immunochemical, electrical, optical or chemi an S-shaped flow path. A magnet can then be used to capture cal means. Suitable labels include fluorescent molecules, bound microbe against the flow path wall; separating the radioisotopes, nucleotide chromophores, enzymes, Sub bound microbe from rest of the sample. 40 strates, chemiluminescent moieties, bioluminescent moi In some embodiments, microbe capture and/or separation eties, and the like. As such, a label is any composition is by a device or method as described in U.S. Pat. App. Pub. detectable by spectroscopic, photochemical, biochemical, No. 2009/0220932, No. 2009/007861, No. 2010/0044232, immunochemical, electrical, optical or chemical means No. 2007/0184463, No. 2004/0018611, No. 2008/0056949, needed for the methods and devices described herein. No. 2008/0014576, No. 2007/0031819, No. 2008/0108120, 45 A wide variety of fluorescent reporter dyes are known in and No. 2010/0323342, the contents of which are all incor the art. Typically, the fluorophore is an aromatic or het porated herein by reference. eroaromatic compound and can be a pyrene, anthracene, Without limitations, if a microbe-targeting substrate does naphthalene, acridine, stilbene, indole, benzindole, oxazole, not possess a magnetic property, isolation of a microbe thiazole, benzothiazole, cyanine, carbocyanine, Salicylate, targeting Substrate (e.g., particles, posts, fibers, dipsticks, 50 anthranilate, coumarin, fluorescein, rhodamine or other like membrane, filters, capillary tubes, etc.) from the test sample compound. can be carried out by non-magnetic means, e.g., centrifuga Exemplary fluorophores include, but are not limited to, tion, and filtration. In some embodiments where the 1.5 IAEDANS: 1,8-ANS: 4-Methylumbelliferone; 5-car microbe-targeting Substrate is in a form a dipstick or mem boxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein brane, the microbe-targeting dipstick or membrane can be 55 (5-FAM); 5-Carboxynapthofluorescein (pH 10): 5-Car simply removed from the test sample, where microbes, if boxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Car any, in the test sample, remained bound to the engineered boxyfluorescein); 5-Hydroxy Tryptamine (HAT); 5-ROX microbe-binding molecules conjugated to the dipstick or (carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethyl membrane Substrate. rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G: 6-JOE: Optionally, the microbe-targeting Substrate after isolated 60 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D from the test sample or processing buffer can be washed with (7-AAD): 7-Hydroxy-4-methylcoumarin; 9-Amino-6- a buffer (e.g., TBST) to remove any residues of test sample, chloro-2-methoxyacridine; ABQ: Acid Fuchsin; ACMA Solution comprising the chelating agent or any unbound (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; microbes. The number of wash steps can range from 1 to Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feul many, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more wash steps. 65 gen SITSA: Aequorin (Photoprotein); Alexa Fluor 350TM: In one embodiments, the microbe-targeting Substrate after Alexa Fluor 430TM; Alexa Fluor 488TM; Alexa Fluor 532TM; isolated from the solution comprising the chelating agent Alexa Fluor 546TM; Alexa Fluor 568TM; Alexa Fluor 594TM; US 9,593,160 B2 61 62 Alexa Fluor 633TM; Alexa Fluor 647TM; Alexa Fluor 660TM; NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Nora Alexa Fluor 680TM; Alizarin Complexon: Alizarin Red; drenaline; Nuclear Fast Red; Nuclear Yellow: Nylosan Bril Allophycocyanin (APC): AMC, AMCA-S; AMCA (Amin liant lavin E8G: Oregon GreenTM: Oregon Green 488-X: omethylcoumarin); AMCA-X; Aminoactinomycin D; Oregon GreenTM 488; Oregon GreenTM 500; Oregon Aminocoumarin; Anilin Blue: Anthrocyl stearate: APC-Cy7; GreenTM 514; Pacific Blue; Pararosaniline (Feulgen); PE APTS: Astrazon Brilliant Red 4G, Astrazon Orange R: Cy5; PE-Cy7; PerCP: PerCP-Cy5.5; PE-TexasRed (Red Astrazon Red 6B. Astrazon Yellow 7 GLL; Atabrine: ATTO 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite TAGTM CBQCA: ATTO-TAGTM FO: Auramine: Aurophos BKL: Phorwite Rev. Phorwite RPA; Phosphine 3R; Photo phine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiaz Resist; Phycoerythrin B PE; Phycoerythrin R PE: ole); BCECF (high pH); BCECF (low pH); Berberine 10 PKH26; PKH67; PMIA: Pontochrome Blue Black; POPO Sulphate: Beta Lactamase; BFP blue shifted GFP (Y66H): 1; POPO-3; PO-PRO-1; PO-PRO-3: Primuline: Procion BG-647; Bimane; Bisbenzamide: Blancophor FFG; Blanco Yellow; Propidium Iodid (PI); PyMPO; Pyrene: Pyronine: phor SV: BOBOTM-1: BOBOTM-3; Bodipy 492/515; Bodipy Pyronine B: Pyrozal Brilliant Flavin 7GF: QSY 7: Quina 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/ crine Mustard; Resorufin; RH 414, Rhod-2; Rhodamine; 550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; 15 Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rho Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; damine 6G.; Rhodamine B 540; Rhodamine B 200; Rhod Bodipy 650/665-X: Bodipy 665/676; Bodipy Fl; Bodipy FL amine B extra; Rhodamine BB; Rhodamine BG; Rhodamine ATP: Bodipy Fl-Ceramide: Bodipy R6G SE: Bodipy TMR: Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Bodipy TMR-X conjugate; Bodipy TMR-X, SE: Bodipy Rhodamine Red; Rhodamine WT: Rose Bengal; R-phyco TR; Bodipy TR ATP; Bodipy TR-X SE: BO-PROTM-1: erythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; BO-PROTM-3; Brilliant Sulphoflavin FF: Calcein: Calcein S65L: S65T: Sapphire GFP, Serotonin; Sevron Brilliant Red Blue; Calcium CrimsonTM: Calcium Green; Calcium 2B: Sevron Brilliant Red 4G: Sevron Brilliant Red B; Green-1 Ca' Dye, Calcium Green-2 Ca"; Calcium Green Sevron Orange; Sevron Yellow L: sgBFPTM; sgBFPTM (su 5N Ca"; Calcium Green-C18 Ca"; Calcium Orange: Cal per glow BFP); sgGFPTM; sgGFPTM (super glow GFP): cofluor White; Carboxy-X-rhodamine (5-ROX); Cascade 25 SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic BlueTM: Cascade Yellow; Catecholamine; CFDA; CFP Acid); SPQ (6-methoxy-N-(3-sulfopropyl)-quinolinium); Cyan Fluorescent Protein; Chlorophyll; Chromomycin A; Stilbene: Sulphorhodamine B can C; Sulphorhodamine G Chromomycin A; CMFDA; Coelenterazine: Coelenterazine Extra; Tetracycline; Tetramethylrhodamine; Texas RedTM: cp: Coelenterazine f: Coelenterazine fep: Coelenterazine h; Texas Red-XTM conjugate; Thiadicarbocyanine (DiSC3): Coelenterazine hcp: Coelenterazine ip: Coelenterazine O: 30 Thiazine Red R. Thiazole Orange; Thioflavin 5: Thioflavin Coumarin Phalloidin: CPM Methylcoumarin: CTC: Cy2TM: S: Thioflavin TCN. Thiolyte: Thiozole Orange; Tinopol Cy3.1 8: Cy3.5TM Cy3TM; Cy5.1 8: Cy5.5TM; Cy5 TM; CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3: Cy7TM; Cyan GFP, cyclic AMP Fluorosensor (FiCRhR); d2: TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl (TetramethylRodamineIsoThioCyanate); True Blue: Tru Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl: 35 Red: Ultralite; Uranine B: Uvitex SFC; wt GFP; WW 781; Dapoxyl 2: Dapoxyl 3: DCFDA; DCFH (Dichlorodihydro XL665; X-Rhodamine; XRITC; Xylene Orange; Y66F: fluorescein Diacetate); DDAO: DHR (Dihydorhodamine Y66H; Y66W.; Yellow GFP; YFP: YO-PRO-1: YO-PRO-3; 123); Di-4-ANEPPS: Di-8-ANEPPS (non-ratio); DiA (4-Di YOYO-1; and YOYO-3. Many suitable forms of these 16-ASP); DIDS; Dihydorhodamine 123 (DHR); DiO fluorescent compounds are available and can be used. (DiOC18(3)); DiR: DiR (DilC18(7)); Dopamine; DSRed; 40 Other exemplary detectable labels include luminescent DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; and bioluminescent markers (e.g., biotin, luciferase (e.g., ELF 97: Eosin; Erythrosin; Erythrosin ITC: Ethidium bacterial, firefly, click beetle and the like), luciferin, and homodimer-1 (Eth)-1); Euchrysin; Europium (III) chloride; aequorin), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), Europium: EYFP; Fast Blue; FDA; Feulgen (Pararosani enzymes (e.g., galactosidases, glucorinidases, phosphatases line): FITC; FL-645; Flazo Orange: Fluo-3; Fluo-4; Fluo 45 (e.g., alkaline phosphatase), peroxidases (e.g., horseradish rescein Diacetate: Fluoro-Emerald; Fluoro-Gold (Hydrox peroxidase), and cholinesterases), and calorimetric labels yStilbamidine); Fluor-Ruby: Fluorx: FM 1-43TM: FM 4-46; Such as colloidal gold or colored glass or plastic (e.g., Fura RedTM (high pH); Fura-2, high calcium; Fura-2, low polystyrene, polypropylene, and latex) beads. Patents teach calcium; Genacryl Brilliant Red B; Genacryl Brilliant Yel ing the use of such labels include U.S. Pat. Nos. 3,817,837. low 10GF: Genacryl Pink 3G: Genacryl Yellow 5GF; GFP 50 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV 4.366,241, each of which is incorporated herein by refer excitation (witGFP); GFP wild type, UV excitation (witGFP): CCC. GFPuv; Gloxalic Acid; Granular Blue: Haematoporphyrin; Means of detecting such labels are well known to those of Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS: skill in the art. Thus, for example, radiolabels can be Hydroxycoumarin: Hydroxystilbamidine (FluoroGold); 55 detected using photographic film or Scintillation counters, Hydroxytryptamine; Indodicarbocyanine (DiD); Indotricar fluorescent markers can be detected using a photo-detector bocyanine (DiR); Intrawhite Cf. JC-1: JO-JO-1: JO-PRO-1: to detect emitted light. Enzymatic labels are typically LaserPro; Laurodan; LDS 751; Leucophor PAF. Leucophor detected by providing the enzyme with an enzyme substrate SF; Leucophor WS; Lissamine Rhodamine: Lissamine Rho and detecting the reaction product produced by the action of damine B: LOLO-1; LO-PRO-1; Lucifer Yellow; Mag 60 the enzyme on the enzyme Substrate, and calorimetric labels Green; Magdala Red (Phloxin B); Magnesium Green; Mag can be detected by visualizing the colored label. nesium Orange; Malachite Green; Marina Blue: Maxilon In some embodiments, the detectable label is a fluoro Brilliant Flavin 10 GFF: Maxilon Brilliant Flavin 8 GFF: phore or a quantum dot. Without wishing to be bound by a Merocyanin; Methoxycoumarin; Mitotracker Green FM: theory, using a fluorescent reagent can reduce signal-to Mitotracker Orange; Mitotracker Red; Mitramycin; Mono 65 noise in the imaging/readout, thus maintaining sensitivity. bromobimane: Monobromobimane (mEBBr-GSH); Mono Accordingly, in Some embodiments, prior to detection, the chlorobimane: MPS (Methyl Green Pyronine Stilbene); microbes isolated from or remained bound on the microbe US 9,593,160 B2 63 64 targeting Substrate can be stained with at least one stain, e.g., Sigma-Aldrich, St. Louis, Mo.). In some embodiments, at least one fluorescent staining reagent comprising a enzyme assays can be configured as binding assays that microbe-binding molecule, wherein the microbe-binding provide for detection of microbe. For example, in some molecule comprises a fluorophore or a quantum dot. embodiments, a labeling molecule can be conjugated with Examples of fluorescent stains include, but are not limited an enzyme for use in the enzyme assay. An enzyme substrate to, any microbe-targeting element (e.g., microbe-specific can then be introduced to the one or more immobilized antibodies or any microbe-binding proteins or peptides or enzymes Such that the enzymes are able to catalyze a oligonucleotides) typically conjugated with a fluorophore or reaction involving the enzyme Substrate to produce a detect quantum dot, and any fluorescent stains used for detection as able signal. described herein. 10 In some embodiments, an enzyme-linked assay (ELISA) In some embodiments, a labeling molecule can be con can be used to detect signals from the labeling molecule. In figured to include a “smart label, which is undetectable ELISA, the labeling molecule can comprise an enzyme as when conjugated to the microbe-binding molecules, but the detectable label. Each labeling molecule can comprise produces a color change when released from the engineered one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) molecules in the presence of a microbe enzyme. Thus, when 15 enzymes. Additionally, each labeling molecule can comprise a microbe binds to the engineered microbe-binding mol one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) sites ecules, the microbe releases enzymes that release the detect for binding with a microbe. Without wishing to be bound by able label from the engineered molecules. An observation of a theory, presence of multimeric probe molecules can a color change indicates presence of the microbe in the enhance ELISA signal. sample. For ELISA, any labeling molecule conjugated to an In some embodiments, the microbe-targeting Substrate enzyme can be used. Exemplary labeling molecule include can be conjugated with a label. Such as a detectable label or those comprising MBL, FcMBL, AKT-FcMBL, wheat germ a biotin label. agglutinin, lectins, antibodies (e.g., gram-negative antibod In some embodiments, the labeling molecule can com ies or gram-positive antibodies), antigen binding fragments prise MBL or a microbe-binding molecule described herein. 25 of antibodies, aptamers, ligands (agonists or antagonists) of In one embodiment, the labeling molecule comprises cell-surface receptors and the like. FcMBL. Without wishing to be bound by a theory, labeling In some embodiments, the labeling molecule can com molecules based on MBL, and FcMBL in particular, attach prise MBL or FcMBL labeled with a detectable label. selectively to a broad range of microbes, and so they enable Similarly, a variety of enzymes can be used, with either the method described herein to detect the majority of blood 30 colorimetric or fluorogenic Substrates. In some embodi borne microbes with high sensitivity and specificity. ments, the reporter-enzyme produces a calorimetric change Any method known in the art for detecting the particular which can be measured as light absorption at a particular label can be used for detection. Exemplary methods include, wavelength. Exemplary enzymes include, but are not limited but are not limited to, spectrometry, fluorometry, micros to, beta-galactosidases, peroxidases, catalases, alkaline copy imaging, immunoassay, and the like. While the 35 phosphatases, and the like. microbe capture step can specifically capture microbes, it In some embodiments, the enzyme is a horseradish per can be beneficial to use a labeling molecule that can enhance oxidase (HRP). this specificity. If imaging, e.g., microscopic imaging, is to In some embodiments, the enzyme is an alkaline peroxi be used for detecting the label, the staining can be done dase (AP). either prior to or after the microbes have been laid out for 40 A microbe-binding molecule and the enzyme can be microscopic imaging. Additionally, imaging analysis can be linked to each other by a linker. In some embodiments, the performed via automated image acquisition and analysis. linker between the microbe-binding molecule and the For optical detection, including fluorescent detection, enzyme is an amide bond. In some embodiments, the linker more than one stain or dye can be used to enhance the between the microbe-binding molecule and the enzyme is a detection or identification of the microbe. For example, a 45 disulfide (S S) bond. first dye or stain can be used that can bind with a genus of When the microbe-binding molecule is a peptide, poly microbes, and a second dye or strain can be used that can peptide or a protein, the enzyme can be linked at the bind with a specific microbe. Colocalization of the two dyes N-terminus, the C-terminus, or at an internal position of the then provides enhanced detection or identification of the microbe-binding molecule. Similarly, the enzyme can be microbe by reducing false positive detection of microbes. 50 linked by its N-terminus, C-terminus, or an internal position. In some embodiments, microscopic imaging can be used In one embodiment, the ELISA probe molecule can to detect signals from label on the labeling agent. Generally, comprise a MBL or a portion there of or a FcMBL molecule the microbes in the Subsample are stained with a staining linked to a HRP. Conjugation of HRP to any proteins and reagent and one or more images taken from which an artisan antibodies are known in the art. In one embodiment, can easily count the number of cells present in a field of 55 FcMBL-HRP construct is generated by direct coupling HRP view. to FcMBL using any commercially-available HRP conjuga In particular embodiments, microbe can be detected tion kit. In some embodiments, the microbes isolated from through use of one or more enzyme assays, e.g., enzyme or remained bound on the microbe-targeting Substrate can be linked assay (ELISA). Numerous enzyme assays can be incubated with the HRP-labeled microbe-binding mol used to provide for detection. Examples of Such enzyme 60 ecules, e.g., MBL or a portion thereof, or a FcMBL molecule assays include, but are not limited to, beta-galactosidase linked to a HRP for a period of time, e.g., at least about 5 assays, peroxidase assays, catalase assays, alkaline phos mins, at least about 10 mins, at least about 15 mins, at least phatase assays, and the like. In some embodiments, enzyme about 20 mins, at least about 25 mins, at least about 30 mins. assays can be configured Such that an enzyme will catalyze The typical concentrations of HRP-labeled molecules used a reaction involving an enzyme Substrate that produces a 65 in the ELISA assay can range from about 1:500 to about fluorescent product. Enzymes and fluorescent enzyme Sub 1:20,000 dilutions. In one embodiment, the concentration of strates are known and are commercially available (e.g., HRP-labeled microbe-binding molecules, e.g., MBL or a US 9,593,160 B2 65 66 portion thereof, or a FcMBL molecule linked to a HRP Further amplification of the ELISA signal can be obtained molecule, can be about 1:1000 to about 1:10000 dilutions. by multimerizing the recognition molecule (e.g., the In one embodiment, the ELISA probe molecule can microbe-binding molecule) or by multimerizing the detec comprise a MBL or a portion thereof, or a FcMBL molecule tion enzyme (HRP, etc.). For instance, phage expression can linked to a AP. Conjugation of AP to any proteins and be used to yield multimerized MBL and provide a scaffold antibodies are known in the art. In one embodiment, to increase the concentration of HRP (either through direct FcMBL-AP construct is generated by direct coupling AP to coupling of HRP to the phage particles or using an HRP FcMBL using any commercially-available AP conjugation antiM13 conjugated antibody). kit. In some embodiments, the microbes isolated from or In some embodiments, microbe can be detected through remained bound on the microbe-targeting Substrate can be 10 use of immunoassay. Numerous types of detection methods incubated with the AP-labeled microbe-binding molecule, may be used in combination with immunoassay based e.g., MBL or a portion thereof, or a FcMBL molecule linked methods. to a AP for a period of time, e.g., at least about 5 mins, at Without limitations, detection of microbes in a sample can least about 10 mins, at least about 15 mins, at least about 20 also be carried out using light microscopy with phase mins, at least about 25 mins, at least about 30 mins. The 15 contrast imaging based on the characteristic size (5 um typical concentrations of AP-labeled molecules used in the diameter), shape (spherical to elliptical) and refractile char ELISA assay can range from about 1:1000 to about 1:20,000 acteristics of target components such as microbes that are dilutions. In one embodiment, the concentration of AP distinct from all normal blood cells. Greater specificity can labeled microbe-binding molecules, e.g., MBL or a portion be obtained using optical imaging with fluorescent or cyto thereof, or a FcMBL molecule linked to a AP molecule, can chemical stains that are specific for all microbes or specific be about 1:5000 to about 1:10000 dilutions. subclasses (e.g. calcofluor (1 uM to 100 uM) for chitin in Following incubation with the ELISA probe molecules, fungi, fluorescent antibodies directed against fungal Surface the sample can be washed with a wash buffer one or more molecules, gram stains, acid-fast stains, fluorescent MBL, (e.g., 1, 2, 3, 4, 5 or more) times to remove any unbound fluorescent Fc-MBL, etc.). probes. An appropriate substrate for the enzyme (e.g., HRP 25 Microbe detection can also be carried out using an epif or AP) can be added to develop the assay. Chromogenic luorescent microscope to identify the characteristic size (5 substrates for the enzymes (e.g., HRP or AP) are known to um diameter), shape (spherical to elliptical) and staining one of skill in the art. A skilled artisan can select appropriate characteristics of microbes. For example, fungi stain differ chromogenic Substrates for the enzyme, e.g., TMB Substrate ently from all normal blood cells, strongly binding calco for the HRP enzyme, or BCIP/NBT for the AP enzyme. In 30 fluor (1 M to 100LLM) and having a rigid ellipsoid shape not some embodiments, the wash buffer used after incubation found in any other normal blood cells. with an ELISA probe molecule can contain calcium ions at In some embodiments, a microbe can be detected through a concentration of about at least about 0.01 mM, at least use of spectroscopy. Numerous types of spectroscopic meth about 0.05 mM, at least about 0.1 mM, at least about 0.5 ods can be used. Examples of Such methods include, but are mM, at least about 1 mM, at least about 2.5 mM, at least 35 not limited to, ultraviolet spectroscopy, visible light spec about 5 mM, at least about 10 mM, at least about 20 mM, troscopy, infrared spectroscopy, X-ray spectroscopy, fluores at least about 30 mM, at least about 40 mM, at least about cence spectroscopy, mass spectroscopy, plasmon resonance 50 mM or more. In alternative embodiments, the wash buffer (e.g., Cherif et al., Clinical Chemistry, 52:255-262 (2006) used after incubation with an ELISA probe molecule can and U.S. Pat. No. 7,030,989; herein incorporated by refer contain no calcium ions. In some embodiments, the wash 40 ence), nuclear magnetic resonance spectroscopy, Raman buffer used after incubation with an ELISA probe molecule spectroscopy, fluorescence quenching, fluorescence reso can contain a chelating agent. A wash buffer can be any nance energy transfer, intrinsic fluorescence, ligand fluores art-recognized buffer used for washing between incubations cence, and the like. with antibodies and/or labeling molecules. An exemplary In some embodiments, a microbe can be detected through wash buffer can include, but is not limited to, TBST. 45 use of fluorescence anisotropy. Fluorescence anisotropy is In some embodiments, without wishing to be bound by based on measuring the steady state polarization of sample theory, it can be desirable to use a wash buffer without a fluorescence imaged in a confocal arrangement. A linearly surfactant or a detergent for the last wash before addition of polarized laser excitation source preferentially excites fluo a chromogenic Substrate, because a Surfactant or detergent rescent target molecules with transition moments aligned may have adverse effect to the enzymatic reaction with a 50 parallel to the incident polarization vector. The resultant chromogenic Substrate. fluorescence is collected and directed into two channels that One advantage of the ELISA-based approach is that the measure the intensity of the fluorescence polarized both solid substrate does not need to be dispersed or dissociated parallel and perpendicular to that of the excitation beam. from the microbe before binding the secondary reagents. With these two measurements, the fluorescence anisotropy, This is in contrast to microscopic techniques, in which 55 r, can be determined from the equation: r=(Intensity paral excess residual solid substrate may obscure the microbe lel-Intensity perpendicular)/(Intensity parallel--2(Intensity during imaging. Furthermore, the optical readout compo perpendicular)) where the I terms indicate intensity mea nents for ELISA are likely cheaper than in the microscopy Surements parallel and perpendicular to the incident polar case, and there is no need for focusing or for demanding that ization. Fluorescence anisotropy detection of fluorescent the sample be on the same focal plane. A further advantage 60 molecules has been described. Accordingly, fluorescence of the ELISA-based approach is that it can take advantage of anisotropy can be coupled to numerous fluorescent labels as commercially available laboratory equipment. In particular, have been described herein and as have been described in the when the Solid Substrate is magnetic, magnetic separation art. can be automated using the KINGFISHER(R) system, the In some embodiments, microbe can be detected through brief culture can be performed using an airlift fermenter, and 65 use of fluorescence resonance energy transfer (FRET). Fluo the colorimetric/fluorescent readout can be attained using a rescence resonance energy transfer refers to an energy standard plate reader. transfer mechanism between two fluorescent molecules. A US 9,593,160 B2 67 68 fluorescent donor is excited at its fluorescence excitation mate Oxidase Assay Kit, AMPLEXR Red Hydrogen Per wavelength. This excited State is then nonradiatively trans oxide/Peroxidase Assay Kit, AMPLEXR Red Monoamine ferred to a second molecule, the fluorescent acceptor. Fluo Oxidase Assay Kit, AMPLEXR Red Neuraminidase (Siali rescence resonance energy transfer may be used within dase) Assay Kit, AMPLEXR Red Phosphatidylcholine-Spe numerous configurations to detect captured microbe. For cific Phospholipase C Assay Kit, AMPLEX(R) Red Sphin example, in Some embodiments, a first labeling molecule gomyelinase Assay kit, AMPLEXR Red Uric Acid/Uricase can be labeled with a fluorescent donor and second labeling Assay Kit, AMPLEX(R) Red Xanthine/Xanthine Oxidase molecule can be labeled with a fluorescent acceptor. Accord Assay Kit, THIOLTRACKERTM Violet (Glutathione Detec ingly, such labeled first and second labeling molecules can tion Reagent), THIOLTRACKERTM Violet (Glutathione be used within competition assays to detect the presence 10 Detection Reagent), and VYBRANTR Cell Metabolic and/or concentration of microbe in a sample. Numerous Assay Kit from Invitrogen; Adenosine 5'-triphosphate (ATP) combinations of fluorescent donors and fluorescent accep Luminescence Assay Kit (ENLITENR) from Promega; tors can be used for detection. ATPLITETM from PerkinElmer Life Sciences; ATP Biolu In some embodiments, a microbe can be detected through minescence Assay kit HS II from Boehringer Mannheim, use of polynucleotide analysis. Examples of Such methods 15 Germany; Adenosine 5'-triphosphate (ATP) Luminescence include, but are not limited to, those based on polynucleotide Assay Kit from EMD Millipore; Reactive Oxygen Species hybridization, polynucleotide ligation, polynucleotide (ROS) Assays from Cell BioLabs, Inc.; Cellular Reactive amplification, polynucleotide degradation, and the like. Oxygen Species Detection Assay Kit from ABCAMR); Methods that utilize intercalation dyes, fluorescence reso hROS Detection Kit from Cell Technology, Inc.; and ABTS nance energy transfer, capacitive deoxyribonucleic acid Antioxidant Assay Kit, ORAC Antioxidant Assay Kit, detection, and nucleic acid amplification have been OxiSelect HORAC Activity Assay Kit, OxiSelect In vitro described, for example, in U.S. Pat. No. 7,118,910 and U.S. ROS/RNS Assay Kit (Green Fluorescence), OxiSelect Intra Pat. No. 6,960,437; herein incorporated by reference). Such cellular ROS Assay Kit (Green Fluorescence), OxiSelect methods can be adapted to provide for detection of one or ORAC Activity Assay Kit, OxiSelect Total Antioxidant more microbe nucleic acids. In some embodiments, fluores 25 Capacity (TAC) Assay Kit, and Total Antioxidant Capacity cence quenching, molecular beacons, electron transfer, elec Assay Kit from BioCat. trical conductivity, and the like can be used to analyze In some embodiments, microbes isolated from or polynucleotide interaction. Such methods are known and remained bound on microbe-targeting Substrate can be have been described, for example, in Jarvius, DNA Tools and labeled with nucleic acid barcodes for subsequent detection Microfluidic Systems for Molecular Analysis, Digital Com 30 and/or multiplexing detection. Nucleic acid barcoding meth prehensive Summaries of Uppsala Dissertations from the ods for detection of one or more analytes in a sample are Faculty of Medicine 161, ACTA UNIVERSITATIS UPSA well known in the art. LIENSIS UPPSALA 2006, ISBN: 91-554-6616-8: Singh In other embodiments, the captured microbe can be ana Zocchi et al. Proc. Natl. Acad. Sci, 100:7605-7610 (2003); lyzed and/or detected in the capture chamber or capture and Wang et al. Anal. Chem, 75:3941-3945 (2003); and Fanetal, 35 visualization chamber of a rapid microbe diagnostic device Proc. Natl. Acad. Sci, 100:9134-9137 (2003) and in U.S. Pat. described in the Int. Pat. App. No. Int. Pat. App. No. WO No. 6,958,216; U.S. Pat. No. 5,093,268; and U.S. Pat. No. 2011/091037, filed Jan. 19, 2011. Alternatively, the captured 6,090,545, the content of all of which is incorporated herein microbe can be recovered (i.e., removed) and analyzed by reference. In some embodiments, the polynucleotide and/or detected. analysis is by polymerase chain reaction (PCR). The fun 40 In some embodiments, the captured microbe is recovered damentals of PCR are well-known to the skilled artisan, see, and analyzed and/or detected using a particle on membrane e.g. McPherson, et al., PCR, A Practical Approach, IRL assay as described in U.S. Pat. No. 7,781,226, content of Press, Oxford, Eng. (1991), hereby incorporated by refer which is incorporated herein by reference. A particle on CCC. membrane assay as described in U.S. Pat. No. 7,781,226 can In some embodiments, a metabolic assay is used to 45 be operably linked with a rapid microbe diagnostic device of determine the relative number of microbes in a sample the Int. Pat. App. No. Int. Pat. App. No. WO 2011/091037 to compared to a control. As will be apparent to one of ordinary reduce the number of sample handling steps, automate the skill in the art any metabolic indicator that can be associated process and/or integrate the capture, separation and analysis/ with cells can be used, such as but not limited to, turbidity, detection steps into a microfluidic device. fluorescent dyes, and redox indicators such as, but not 50 In some embodiments, microbe capture, separation and limited to, Alamar Blue, MTT, XTT, MTS, and WST. analysis can be done using a hybrid microfluidic SPR and Metabolic indicators can be components inherent to the cells molecular imagining device as described in U.S. Pat. App. or components added to the environment of the cells. In Pub. No. US 2011 FOO3928O. Some embodiments, changes in or the State of the metabolic In some embodiments, the processes or assays described indicator can result in alteration of ability of the media 55 herein can detect the presence or absence of a microbe containing the sample to absorb or reflect particular wave and/or identify a microbe in a test sample in less than 24 lengths of radiation. hours, less than 12 hours, less than 10 hours, less than 8 Exemplary metabolic assays include, but are not limited hours, less than 6 hours, less than 4 hours, less than 3 hours, to, ATP Luminescence, reactive oxygen species (ROS) less than 2 hours, less than 1 hour, or lower. In some assays, ResaZurin assays, Luminol, MTT-metabolic assays, 60 embodiments, the processes or assays described herein can and the like. Further, as one of skill in the art is well aware, detect the presence or absence of a microbe and/or identify kits and methods for carrying out metabolic assays are a microbe in a test sample in less than 6 hours, less than 4 commercially available. For example, 2-(N-(7-Nitrobenz-2- hours, less than 3 hours, less than 2 hours, less than 1 hour, oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG). or lower. ATP Determination Kit, AMPLEXR Red Galactose/Galac 65 Optional Additional Analyses or Treatment Culturing: tose Oxidase Assay Kit, AMPLEXR Red Glucose/Glucose In some embodiments of any aspects described herein, the Oxidase Assay Kit, AMPLEXR Red Glutamic Acid/Gluta assay or process can further comprise culturing any microbe US 9,593,160 B2 69 70 bound on the microbe-targeting Substrate (e.g., microbe strate with the low pH buffer and the chelating agent can be targeting magnetic microbeads) for a period of time. In Such concurrent or sequentially. In one embodiment, the microbe embodiments, the microbe bound on the microbe-targeting binding molecules of the microbe-targeting Substrate can be substrate can expand in population by at least about 10% further contacted with arginine (e.g., 2 M) with EDTA or after culturing for a period of time. EGTA at pH 4.4. In some embodiments, the microbe bound on the The isolated microbes can then be used for analyses microbe-targeting Substrate (e.g., microbe-targeting mag described earlier or additional treatment, e.g., expansion in netic microbeads) can be cultured for a period of time, e.g., culture, antibiotic sensitivity testing, sequencing and/or at least about 15 mins, at least about 30 mins, at least about DNA or RNA analysis. 1 hour, at least about 2 hours, at least about 3 hours, at least 10 Optional Additional Analyses or Treatment Antibiotic about 6 hours, at least about 9 hours, at least about 12 hours, Sensitivity or Susceptibility Testing: at least about 18 hours, at least about 24 hours or longer. In some embodiments, the microbe bound on the microbe In some embodiments of any aspects described herein, the targeting Substrate (e.g., microbe-targeting magnetic micro process or assay described herein can further comprise beads) can be cultured for at least about 30 mins to at least 15 Subjecting the microbes bound on the microbe-targeting about 3 hours. Substrate (e.g., microbe-targeting magnetic microbeads) In some embodiments, the number of microbes bound on and/or the expanded cultures of microbes isolated from the the microbe-targeting Substrate (e.g., microbe-targeting microbe-targeting Substrate (e.g., microbe-targeting mag magnetic microbeads) after culturing for a certain period of netic microbeads) to one or more antibiotics. The response time can be increased or expanded by at least about 30%, at of the microbe to an antibiotic can then be evaluated with least about 40%, at least about 50%, at least about 60%, at any known methods in the art, e.g., by measuring the least about 70%, at least about 80%, at least about 90%, at viability of microbes. Thus, an appropriate antibiotic can be least about 100%, as compared to the number of the identified for treatment of an infection caused by a microbe, microbes originally bound on the microbe-targeting Sub even though the specific species of the microbe bound onto strate. In some embodiments, the number of microbes bound 25 the microbe-targeting Substrate is initially unknown. Addi on the microbe-targeting Substrate (e.g., microbe-targeting tional details for use of engineered microbe-targeting mol magnetic microbeads) after culturing for a certain period of ecules described herein in antibiotic sensitivity testings can time can be increased or expanded by at least about 1.5-fold, be found, e.g., in U.S. Prov. App. Nos. 61/604.878 filed Feb. at least about 2-fold, at least about 3-fold, at least about 29, 2012 and 61/647,860 filed May 16, 2012. 4-fold, at least about 5-fold, at least about 10-fold, at least 30 Any processes or steps described herein can be performed about 50-fold, at least about 100-fold, at least about 500 by a module or device. While these are discussed as discrete fold, at least about 1000-fold, at least about 10000-fold, at processes, one or more of the processes or steps described least about 100000-fold, as compared to the number of the herein can be combined into one system for carrying out the microbes originally bound on the microbe-targeting Sub assays of any aspects described herein. Strate. 35 In some embodiments, the microbes bound on the Exemplary Embodiments of Methods for microbe-targeting Substrates (e.g., microbe-targeting mag Diagnosing or Locating a Microbial Infection or netic microbeads) can be cultured on a microbe-compatible Contamination culture medium, e.g., plated on an agar plate or cultured in LB broth. One of skill in the art will readily recognize 40 In general, embodiments of the assays or processes of any microbial culture techniques, including, but not limited to, aspects described herein can be used to detect the presence the use of incubators and/or equipment used to provide a or absence of a microbe and/or microbial matter in a test gentle agitation, e.g., rotator platforms, and shakers, if sample or in situ (e.g., where the microbe actually resides, necessary, e.g., to prevent the cells from aggregation without e.g., in a water reservoir or on a working Surface). For Subjecting them to a significant shear stress and provide 45 example, in Some embodiments, a test sample, e.g., obtained aerial agitation. from a Subject or an environmental source, or an environ The microbes can remain bound on the microbe-targeting mental Surface can be contacted with engineered microbe Substrate (e.g., microbe-targeting magnetic microbeads) dur binding molecules or engineered microbe-binding Substrates ing detection and/or additional analyses described herein or described herein, such that any microbes, if present, in the they can be detached, eluted off or removed from a microbe 50 test sample or environmental Surface can be captured by the targeting Substrate prior to detection or additional analyses engineered microbe-binding molecules or engineered described herein. In some embodiments where the bound microbe-binding Substrates e.g., using any embodiments of microbes are desired to be detached, eluted off or removed the exemplary process described above. In some embodi from a microbe-targeting Substrate, the microbe-binding ments, the captured microbes bound on the engineered molecules of the microbe-targeting substrate can be further 55 microbe-binding molecules and/or microbe-binding Sub contacted with a low pH buffer, e.g., a pH buffer less than 6, strates can then be subjected to different analyses as less than 5, less than 4, less than 3, less than 2, less than 1 described above, e.g., for identifying a microbe genus or or lower. In some embodiments, a low pH buffer that does species such as by immunoassay (e.g., using antibodies to a not cause precipitation of a chelating agent, if present, can specific microbe), mass spectrometry, PCR, etc. In alterna be used. In one embodiment, a low pH buffer can be 60 tive embodiments where the engineered microbe-binding arginine. In another embodiment, a low pH buffer can be molecules comprise an imaging agent (e.g., a bubble, a pyrophosphate. liposome, a sphere, a diagnostic contrast agent or a detect In some embodiments of any aspects described herein, the able label described herein), the binding of the microbes to microbe-binding molecules of the microbe-targeting Sub the engineered microbe-binding molecules can be detected strate can be further contacted with a low pH buffer and a 65 in situ for identification of localized microbial infection or chelating agent. In some embodiments, the contact of the contamination, and also allow localized treatment of the microbe-binding molecules of the microbe-targeting Sub infection or contamination. US 9,593,160 B2 71 72 In some embodiments, the assays or processes described herein in the presence of free calcium ions, and (ii) contact herein can be used to diagnose or locate a microbial infec ing the microbe-binding molecule of the microbe-targeting tion in situ in a subject. For example, engineered microbe substrate described herein, upon the contact with the test targeting microbeads comprising an imaging agent (e.g., the sample, with a solution comprising a chelating agent. In engineered microbe-targeting microbeads can be linked to some embodiments described herein, the method can further an imaging agent, e.g., a bubble, a liposome, a sphere, a comprise analyzing the microbe-targeting Substrate for the diagnostic contrast agent or a detectable label described presence or absence of a bound microbe. The presence of a herein) can be administered to a subject, either systemically microbe bound onto the microbe-targeting Substrate indi (e.g., by injection), or locally. In such embodiments, the cates the presence of a protein-A expressing microbe or a engineered microbe-targeting microbeads comprising an 10 protein G-expressing microbe in the test sample; and the imaging agent can be used to identify and/or localize pock absence of a microbe bound onto the microbe-targeting ets of localized microbial infection (e.g., in a tissue) in the Substrate indicates the absence of a protein-A expressing or subject and optionally allow localized treatment of the a protein G-expressing microbe in the test sample. microbial infection, which is described in the section In some embodiments, the method can further comprise “Exemplary Compositions and Methods for Treating and/or 15 administering or prescribing to the Subject an antimicrobial Preventing a Microbial Infection' below. agent when the subject is detected with S. aureus. Non While an engineered microbe-binding molecule described limiting examples of an antimicrobial agent can include any herein (e.g., FcMBL) can bind to a broad spectrum of therapeutic agent for treatment of S. aureus. In some microbes, in certain embodiments, a microbe species (e.g., embodiments, an antimicrobial agent can be an antibiotic S. aureus) can be isolated and/or differentiated from another commonly indicated for treatment of S. aureus, including, species (E. coli) based on their distinct abilities of binding but not limited to, penicillin, methicillin, nafcillin, oxacillin, to the engineered microbe-binding molecules or Substrates cloxacillin, dicloxacillin, flucloxacillin, Vancomycin, and described herein. For example, the inventors have demon any combinations thereof. strated that S. aureus can bind to FcMBL via both calcium In some embodiments where a microbe is absent on the dependent MBL-mediated interaction and calcium-indepen 25 microbe-targeting Substrate, the method can further com dent Fc-mediated interaction, while E. coli can bind to prise analyzing the test sample or the solution comprising FcMBL primarily via calcium-dependent MBL-mediated the chelating agent after removal of the microbe-targeting interaction. Without wishing to be limiting, an exemplary Substrate to determine the presence or absence of a protein method for diagnosing an infection caused by S. aureus A- and protein G-negative microbe. For example, additional based on Such unique ability of S. aureus binding to an 30 calcium ions (e.g., calcium salts) can be added to the test engineered microbe-binding molecule (e.g., FcMBL) is sample or the Solution comprising the chelating agent in an described below for illustration purposes. One of skill in the amount more than what is needed to react with substantially art can readily make any necessary modifications to the all of the chelating agent molecules Such that there are free exemplary illustration and/or adopt any embodiments of the calcium ions available to mediate carbohydrate recognition assays or processes described herein to detect the presence 35 domain (e.g., MBL)-mediated binding between a microbe or absence of any microbe in a test sample or in situ and/or and the microbe-targeting Substrate. A fresh microbe-target diagnosing different kinds of microbial infections in a Sub ing substrate can then be contacted with the treated test ject. sample or the solution comprising the chelating agent in the For example, there is a strong need for more rapid and/or presence of free calcium ions to detect the presence or effective diagnostic methods for distinguishing at least S. 40 absence of a protein A- and protein G-negative microbe aureus from other bacteria, e.g., E. coli, which can permit (e.g., E. coli). Alternatively, a fresh microbe-targeting Sub physicians to initiate an appropriate drug therapy early on, strate can be contacted with a fresh volume of the test rather than starting with a sub-optimal or a completely sample in the presence of free calcium ions (e.g., addition of ineffective antibiotic. A delay in treatment of a microbial a calcium salt at a concentration, e.g., of at least about 1 mM, infection, e.g., S. aureus, can significantly affect the treat 45 at least about 5 mM, or higher) to detect the presence or ment outcome, and can be sometimes fatal. absence of a protein A- and protein G-negative microbe Accordingly, in Some embodiments, the assays or pro (e.g., E. coli). Detection methods described above for a cesses described herein can be used to distinguish a protein protein A-expressing or protein G-expressing microbe A-expressing microbe or a protein G-expressing microbe bound on a microbe-targeting Substrate can be used for Such from a protein A- and protein G-negative microbe (e.g., E. 50 purposes as well. Detection methods described in the Inter coli) in a test sample. In particular, the inventors have national Application No. WO 2011/090954, the content of demonstrated that S. aureus can be differentiated from E. which is incorporated herein by reference, can also be coli using some embodiments of the assays or processes employed herein to determine the presence or absence of described herein. In some embodiments, a microbe-targeting protein A- and protein G-negative microbes (e.g., E. coli). Substrate comprises a Substrate coupled to a fusion protein 55 In those embodiments, when a microbe (e.g., protein A between the Fc portion of human IgG1 and the neck and and protein G-negative microbe (e.g., E. coli)) is detected in carbohydrate recognition domain (CRD) of human Mannose a Subject, the method can further comprise administering or Binding Lectin (MBL) can be used for such microbial prescribing to the Subject an appropriate antimicrobial agent differentiation. described herein to treat the corresponding microbe (e.g., the Accordingly, exemplary methods of determining the pres 60 protein A- and protein G-negative microbe, e.g., E. coli). ence or absence of Staphylococcus aureus infection in a Without wishing to be bound by theory, some embodi subject are also provided herein. For example, the method ments of the engineered microbe-binding molecules can be can comprise contacting at least a first volume of a test used to opsonize a microbe, which is then cleared out by an sample with a microbe-targeting Substrate described herein innate immune response. In some embodiments, FcMBL in the presence of a chelating agent. Alternatively, the 65 protein can be a more potent opsonin of a microbe. g., S. method can comprise (i) contacting at least a first volume of aureus than Fc or wild-type MBL. Accordingly, in some a test sample with a microbe-targeting Substrate described embodiments, when the Subject is diagnosed with a micro US 9,593,160 B2 73 74 bial infection using the methods described herein, the sub ments, the microbead can be labeled for specific imaging of ject can be administered or prescribed with a composition infected sites. For SPECT imaging the tracer radioisotopes comprising at least one engineered microbe-binding mol typically used such as iodine-123, technetium-99m, xenon ecule described herein. 133, thallium-201, and fluorine-18 can be used. Technetium Without limitations, the methods of any aspects described 99m can be used for scintigraphic assay. Iodine-derived or herein can be used to diagnose a microbe that is resistant to other radioopaque contrast agents can also be incorporated at least one, at least two, at least three, at least four or more in the beads for radiographic or CT-Scan imaging. The use antibiotics. For example, in one embodiment, the methods of paramagnetic or Superparamagnetic microbeads can be described herein can be used to diagnose methicillin-resis used for magnetic resonance imaging as contrast agents to tant S. aureus. In another embodiment, the methods 10 alter the relaxation times of atoms within a nidus of infec described herein can be used to diagnose Vancomycin tion. In another embodiment, the microspheres can be fluo resistant S. aureus. rescently dyed and applied to a Surgical wound to determine Exemplary Compositions and Methods for Treating and/or the extension of an infectious process. This can be useful for Preventing a Microbial Infection assisting the Surgeon in distinguishing between infected and The binding of microbes to engineered microbe-targeting 15 healthy tissues during debridment Surgeries for osteomyeli molecules can facilitate isolation and removal of microbes tis, cellulitis or fasciitis. and/or microbial matter from an infected area. Accordingly, Accordingly, another aspect provided herein related to another aspect provided herein relate to compositions for compositions for treating and/or preventing a microbial treating and/or preventing a microbial infection or microbial infection in a tissue of a subject. In some embodiments, the contamination comprising one or more engineered microbe composition comprises at least one engineered microbe targeting molecules or microbe-targeting Substrates (e.g., targeting molecule as described herein. In some embodi microbe-targeting magnetic microbeads) described herein. ments, the amount of the engineered microbe-targeting In some embodiments, the composition can be formulated molecules and/or microbe-targeting Substrates present in the for treating and/or preventing a microbial infection or a composition is sufficient to reduce the growth and/or spread microbial contamination present in an environmental Sur 25 of the microbe in the tissue of the subject. The phrase face. The term “environmental surface' as used herein refers “reducing the growth and/or spread of the microbe in the to any surface and/or body of an environment or an object. tissue' as used herein refers to reducing the number of The environmental object can be a non-living object or a colonies of the microbe and/or movement of the microbe in living object, e.g., a botanical plant. Examples of an envi the tissue. In some embodiments, the engineered microbe ronmental Surface can include, but is not limited to, a 30 targeting molecule can capture and localize a microbe pres medical device, an implantable device, a Surface in a hos ent in a tissue such that the number of colonies of the pital or clinic (e.g., an operating room or an intensive-care microbe in the tissue can be reduced by at least about 30%, unit), a machine or working Surface for manufacturing or at least about 40%, at least about 50%, at least about 60%, processing food or pharmaceutical products (e.g., drugs, at least about 70%, at least about 80%, at least about 90%, therapeutic agents or imaging agents), a cell culture, a water 35 at least about 95%, at least about 98%, up to and including treatment plant, a water reservoir and a botanical plant. 100%, as compared to in the absence of the engineered In some embodiments, the composition can be formulated microbe-targeting molecule. In some embodiments, the for treating and/or preventing microbial infection in a body engineered microbe-targeting molecule can capture and fluid of a subject, e.g., blood. While in some embodiments, localize a microbe present in a tissue Such that the number the engineered microbe-targeting molecules of the compo 40 of colonies of the microbe in the tissue can be reduced by at sition described herein can capture microbes and/or micro least about 1.5-fold, at least about 2-fold, at least about bial matter in a circulating body fluid, e.g., blood, in other 3-fold, at least about 4-fold, at least about 5-fold, at least embodiments, the engineered microbe-targeting molecules about 6-fold, at least about 7-fold, at least about 8-fold, at can opsonize a microbe and/or microbial matter Such that the least about 9-fold, at least about 10-fold, at least about microbe and/or microbial matter can be recognized by an 45 15-fold, at least about 20-fold or more, as compared to in the innate immune system for clearance. absence of the engineered microbe-targeting molecules. In Unlike wild-type MBL that can induce systemic comple one embodiment, the binding of the engineered microbe ment activation (see, e.g., Sprong T. (2009) Clin Infect Dis. targeting molecules with a microbe (e.g., S. aureus) reduces 49: 1380-1386), in some embodiments, the engineered the number of colonies by at least about 4-fold to at least microbe-targeting molecules can act as dominant negative 50 about 6-fold (e.g., at least about 5-fold), as compared to in molecules by binding microbes and/or microbial matter the absence of the engineered microbe-targeting molecules, without stimulating downstream inflammatory cascades, after a period of at least about 12 hours, at least about 16 and thus reduce system inflammatory syndromes and/or hours or at least about 24 hours. sepsis symptoms in Vivo, e.g., reduction of disseminated In other embodiments, the engineered microbe-targeting intravascular coagulation (DIC). 55 molecule can capture and localize a microbe present in a Alternatively, the engineered microbe-targeting mol tissue such that the movement of the microbe within the ecules can localize a microbe and can thus prevent it from tissue (e.g., in terms of a distance traveled deeper into the spreading, e.g., deeper into a wound. In particular, the tissue and/or area of spread from the infected site) can be inventors have demonstrated that S. aureus can strongly bind reduced by at least about 30%, at least about 40%, at least to Some embodiments of the engineered microbe-targeting 60 about 50%, at least about 60%, at least about 70%, at least molecules (e.g., microbe-binding magnetic microbeads) due about 80%, at least about 90%, at least about 95%, at least to the presence of both carbohydrate patterns and protein A about 98%, up to and including 100%, as compared to in the on its microbial Surface capable of independent binding to absence of the engineered microbe-targeting molecule. In the engineered microbe-targeting molecules. Thus, in some Some embodiments, the engineered microbe-targeting mol embodiments, the engineered microbe-targeting molecules 65 ecule can capture and localize a microbe present in a tissue can be used to localize a microbe load, which can then be such that the movement of the microbe within the tissue easily removed from an infected area. In some embodi (e.g., in terms of a distance traveled deeper into the tissue US 9,593,160 B2 75 76 and/or area of spread from the infected site) can be reduced lated from Such natural source. Substances of similar struc by at least about 1.5-fold, at least about 2-fold, at least about ture and mode of action can be synthesized chemically, or 3-fold, at least about 4-fold, at least about 5-fold, at least natural compounds can be modified to produce semi-syn about 6-fold, at least about 7-fold, at least about 8-fold, at thetic antibiotics. Exemplary classes of antibiotics include, least about 9-fold, at least about 10-fold, at least about but are not limited to, (1) B-lactams, including the penicil 15-fold, at least about 20-fold or more, as compared to in the lins, cephalosporins monobactams, methicillin, and car absence of the engineered microbe-targeting molecule. bapenems; (2) aminoglycosides, e.g., gentamicin, kanamy In some embodiments, the composition can further com cin, neomycin, tobramycin, netilmycin, paromomycin, and prise at least one of an antimicrobial agent and a drug amikacin; (3) tetracyclines, e.g., doxycycline, minocycline, delivery vehicle. For example, in some embodiments, the 10 oxytetracycline, tetracycline, and demeclocycline; (4) Sul composition can further comprise at least 1, at least 2, at least 3, at least 4, at least 5 or more antimicrobial agents. In fonamides (e.g., mafenide, Sulfacetamide, Sulfadiazine and Some embodiments, the composition can further comprise SulfaSalazine) and trimethoprim; (5) quinolones, e.g., cipro one or a plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30. floxacin, norfloxacin, and ofloxacin; (6) glycopeptides (e.g., 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more) delivery 15 Vancomycin, telavancin, teicoplanin); (7) macrollides, which vehicles. In some embodiments, the composition can further include for example, erythromycin, azithromycin, and comprise a combination of at least one (including at least 2, clarithromycin; (8) carbapenems (e.g., ertapenem, dorip at least 3, at least 4, at least 5 or more) antimicrobial agent enem, meropenem, and imipenem); (9) cephalosporins (e.g., and at least one (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30. cefadroxil, cefepime, and ceftobiprole); (10) lincosamides 40, 50, 60, 70, 80,90, 100, 500, 1000 or more) drug delivery (e.g., clindamycin, and lincomycin); (11) monobactams vehicle. As used herein, the term “drug delivery vehicle' (e.g., aztreonam); (12) nitrofurans (e.g., furazolidone, and generally refers to any material that can be used to carry an nitrofurantoin); (13) Penicillins (e.g., amoxicillin, and Peni active agent to a target site. Examples of drug delivery cillin G); (14) polypeptides (e.g., bacitracin, colistin, and vehicles includes, but are not limited to, a cell, a peptide polymyxin B); and (15) other antibiotics, e.g., ansamycins, particle, a polymeric particle, a dendrimer, a vesicle, a 25 polymycins, carbacephem, chloramphenicol, lipopeptide, liposome, a hydrogel, a nucleic acid scaffold, an aptamer, and drugs against mycobacteria (e.g., the ones causing and any combinations thereof, diseases in mammals, including tuberculosis (Mycobacte In some embodiments where a drug delivery vehicle is rium tuberculosis) and leprosy (Mycobacterium leprae), and included, an engineered microbe-targeting molecule and/or any combinations thereof. an antimicrobial agent can be dispersed within (e.g., encap 30 Additional exemplary antimicrobial agent can include, sulated or embedded in) a drug delivery vehicle and/or but are not limited to, antibacterial agents, antifungal agents, coated on a surface of the drug delivery vehicle. antiprotozoal agents, antiviral agents, and any mixtures In some embodiments where the composition includes at thereof. least one antimicrobial agent, the antimicrobial agent can be Exemplary antibacterial agents include, but are not lim present as a separate entity from the engineered microbe 35 ited to, Acrosoxacin, Amifioxacin, Amoxycillin, Ampicillin, targeting molecule and/or it can be fused with at least one Aspoxicillin, AZidocillin, Azithromycin, Aztreonam, Balo engineered microbe-targeting molecule, e.g., by genetic floxacin, lc Benzylpenicillin, Biapenem, Brodimoprim, modification and/or chemical conjugation. Cefaclor, Cefadroxil, Cefatrizine, Cefcapene, Cefdinir, The term “antimicrobial agent” as used herein refers to Cefetamet, Cefnmetazole, Cefprozil, Cefroxadine, Ceftib any entity with antimicrobial activity, i.e. the ability to 40 uten, Cefuroxime, Cephalexin, Cephalonium, Cephalori inhibit or reduce the growth and/or kill a microbe, e.g., by dine, Cephamandole, Cephazolin, Cephradine, Chlorquinal at least about 30%, at least about 40%, at least about 50%, dol, Chlortetracycline, Ciclacillin, Cinoxacin, at least about 75%, at least about 90% or more, as compared Ciprofloxacin, Clarithromycin, Clavulanic Acid, Clindamy to in the absence of an antimicrobial agent. An antimicrobial cin, Clofazimine, Cloxacillin, Danofloxacin, Dapsone, agent can be, for example, but not limited to, a silver 45 Demeclocycline, Dicloxacillin, Difloxacin, Doxycycline, nanoparticle, a small molecule, a peptide, a peptidomimet Enoxacin, Enrofloxacin, Erythromycin, Fleroxacin, Flo ics, an antibody or a fragment thereof, a nucleic acid, an moxef, Flucloxacillin, Flumequine, Fosfomycin, Isoniazid, enzyme (e.g., an antimicrobial metalloendopeptidase such as Levofloxacin, Mandelic Acid, Mecillinam, Metronidazole, lysostaphin), an aptamer, a drug, an antibiotic, a chemical or Minocycline, Mupirocin, Nadifloxacin, Nalidixic Acid, any entity that can inhibit the growth and/or kill a microbe. 50 Nifuirtoinol, Nitrofurantoin, Nitroxoline, Norfloxacin, Examples of an antimicrobial peptide that can be included in Ofloxacin, Oxytetracycline, Panipenem, Pefloxacin, Phe the composition described herein, include, but are not lim noxymethylpenicillin, Pipemidic Acid, Piromidic Acid, Piv ited to, mefloquine, Venturicidin A, antimycin, myxothiazol. ampicillin, Pivmecillinam, Prulifloxacin, Rufloxacin, Spar Stigmatellin, diuron, iodoacetamide, potassium tellurite floxacin, Sulbactam, Sulfabenzamide, Sulfacytine, hydrate, al L-vinylglycine, N-ethylmaleimide, L-allygly 55 Sulfametopyrazine, Sulphacetamide, Sulphadiazine, Sulph cine, diary quinoline, betaine aldehyde chloride, acivcin, adimidine, Sulphamethizole, Sulphamethoxazole, Sulpha psicofuraine, buthionine Sulfoximine, diaminopemelic acid, nilamide, Sulphasomidine, Sulphathiazole, Temafioxacin, 4-phospho-D-erythronhydroxamic acid, motexafin gado Tetracycline, Tetroxoprim, Tinidazole, Tosufloxacin, linium and/or Xycitrin or modified versions or analogues Trimethoprim, and pharmaceutically acceptable salts or thereof. 60 esters thereof. In some embodiments, an antimicrobial agent included in Exemplary antifungal agents include, but are not limited the composition can be an antibiotic. As used herein, the to, Bifonazole, Butoconazole, Chlordantoin, Chlorphenesin, term “antibiotic' is art recognized and includes antimicro Ciclopirox Olamine, Clotrimazole, Eberconazole, Econ bial agents naturally produced by microorganisms such as azole, Fluconazole, Flutrimazole, Isoconazole, Itraconazole, bacteria (including Bacillus species), actinomycetes (includ 65 Ketoconazole, Miconazole, Nifuroxime, Tioconazole, Ter ing Streptomyces) or fungi that inhibit growth of or destroy conazole, Undecenoic Acid, and pharmaceutically accept other microbes, or genetically-engineered thereof and iso able salts or esters thereof. US 9,593,160 B2 77 78 Exemplary antiprotozoal agents include, but are not lim or microbe-targeting magnetic microbeads) can be formu ited to, Acetarsol, AZanidazole, Chloroquine, Metronida lated into a wound dressing spray, which can be handy and Zole, Nifuratel, Nimorazole, Omidazole, Propenidazole, used anywhere, e.g., during a transportation on an emer Secnidazole, Sineflingin, Tenonitrozole, Temidazole, Tinida gency vehicle. When the wound dressing spray containing Zole, and pharmaceutically acceptable salts or esters thereof. the microbe-targeting magnetic microbeads, the microbe Exemplary antiviral agents include, but are not limited to, targeting magnetic microbeads with bound microbes (e.g., S. Acyclovir, Brivudine, Cidofovir, Curcumin, Desciclovir, aureus) can be removed from the wound with a magnetic 1-Docosanol, Edoxudine, gO Fameyclovir, Fiacitabine, field gradient before re-application of the spray. Ibacitabine, Imiquimod, Lamivudine, Penciclovir, Valacy Debridement Fluids or Sprays: clovir, Valganciclovir, and pharmaceutically acceptable salts 10 In some embodiments, the composition described herein or esters thereof. can be formulated as part of a debridement fluid (optionally In some embodiments, the antimicrobial agent can with Suspended particulates that are abrasive to a lesion include silver present in any form, e.g., a nanoparticle, a area). In some embodiments, the composition described colloid, a suspension, powder, and any combinations herein can be formulated as part of a debridement spray. As thereof. 15 used herein, the term “debridement generally refers to In some embodiments, the composition can be used to complete or partial removal of a subjects dead, damaged, treat and/or prevent an infection caused by any microbe and/or infected tissue to improve the healing potential of the described herein. In one embodiment, the composition can remaining healthy and/or non-infected tissue. By way of be used to treat and/or prevent an infection caused by S. example only, a plurality of engineered microbe-targeting Ca,S. molecules (e.g., microbe-targeting microparticles or mag In some embodiments, the composition can be used to netic microbeads) can be suspended in a debridement fluid treat and/or prevent an infection caused by a microbe that is or spray, e.g., for use in an orthopedic procedure. The resistant to at least one, at least two, at least three, at least debridement fluid or spray containing the engineered four or more antimicrobial agents described herein. In one microbe-targeting molecules can be applied to a lesion, an embodiment, the composition can be used to treat and/or 25 abscess or a wound, where the engineered microbe-targeting prevent an infection caused by a microbe that is resistant to microparticles or magnetic microbeads can capture a at least one, at least two, at least three, at least four or more microbe (e.g., S. aureus) and/or microbial matter from the antibiotics described herein. For example, in one embodi lesion, abscess or wound. The debridement fluid or spray can ment, the composition can be used to treat and/or prevent an then be removed from the applied site by vacuum, or infection caused by methicillin-resistant S. aureus. In 30 suction. In some embodiments, the debridement fluid or another embodiment, the composition can be used to treat spray containing the engineered microbe-targeting magnetic and/or prevent an infection caused by Vancomycin-resistant microbeads can be also removed from the applied site by S. aureus. exposing the applied site to a magnetic field gradient, which Exemplary antimicrobial applications and/or products: can pull or attract the applied microbe-targeting magnetic The compositions described herein can be formulated or 35 microbeads out from the applied site. configured for different applications and/or products Such Medical Device Coating: antimicrobial products. In some embodiments, the compo In some embodiments, the composition described herein sition described herein can be formulated as pharmaceutical can be coated on a Surface of a medical device, e.g., a fluid compositions as described below, e.g., for therapeutic treat delivery device such as hollow fibers, tubing or a spiral ment as an antibiotic or antiseptic. 40 mixer in an extracorporeal device, or an implantable device Wound Dressings: Such as an indwelling catheter, chip or scaffold. By way of In some embodiments, the composition described herein example only, a plurality of engineered microbe-targeting can be formulated for topical application, e.g., in wounds, molecules can be coated or conjugated to a surface of a fluid lesions or abscesses. By way of example only, in some delivery device such that when a fluid (e.g., blood) flows embodiments, a plurality of engineered microbe-targeting 45 through the fluid delivery device coated with engineered molecules can be blended with, attached to or coated on a microbe-targeting molecules, any microbe (e.g., S. aureus) wound dressing, for example, but not limited to, a bandage, and/or microbial matter present in the fluid (e.g., blood) can an adhesive, a gauze, a film, a gel, foam, hydrocolloid, be extracted therefrom, thus reducing the chance of a alginate, hydrogel, paste (e.g., polysaccharide paste), a microbial infection. In another embodiment, a plurality of spray, a granule and a bead. 50 engineered microbe-targeting molecules coated on a medical In some embodiments, the wound dressing can include an device can comprise a detectable label, e.g., a “smart label' additional antimicrobial agent described herein and/or an described herein, which can provide a detectable signal antiseptic chemical, e.g., boracic lint and/or medicinal castor when any microbe (e.g., S. aureus) binds to a Surface of the oil. medical device, indicating that the medical device has been In one embodiment, a plurality of engineered microbe 55 contaminated and/or infected, and thus is not appropriate for targeting molecules (e.g., microbe-targeting microparticles use or implantation. or microbe-targeting magnetic microbeads) can be attached Provided herein are also methods for removing a microbe or coated onto a wound dressing Such as a bandage or an and/or microbial matter from a target area comprising con adhesive. When Such wound dressing is applied to a wound tacting the target area with at least one composition or a lesion, any microbe (e.g., S. aureus) and/or microbial 60 described herein. As removal of a microbe and/or microbial matter present in the wound or lesion can bind and localized matter from an infected area can treat and/or prevent a to the wound dressing. Thus, regular replacement of the microbial infection or microbial contamination, provided wound dressing can remove the microbe from the wound or herein also include methods for treating and/or preventing a lesion and thus prevent the microbe from moving deeper microbial infection or microbial contamination in a target into the wound or lesion for further infection. 65 area. An exemplary method comprises contacting the target In one embodiment, a plurality of engineered microbe area with a composition. The target area can be anywhere, targeting molecules (e.g., microbe-targeting microparticles e.g., an environmental Surface or in a body of a Subject (e.g., US 9,593,160 B2 79 80 body fluid, and/or tissue). In some embodiments, the method composition comprising at least one engineered microbe comprises contacting the tissue of the Subject with any targeting molecule described herein, and a pharmaceutically embodiments of the composition described herein. In some acceptable carrier. embodiments, the tissue can have an open wound, a lesion Depending on the selected administration route, the com or an abscess. positions or preparations can be in any form, e.g., a tablet, In one embodiment, the composition can be formulated a lozenge, a suspension, a free-flowing powder, an aerosol, for use as a wound dressing described herein. and a capsule. The term “pharmaceutically acceptable,” as As the engineered microbe-targeting molecules can local used herein, refers to those compounds, materials, compo ize a microbe (e.g., S. aureus) for easier removal of the sitions, and/or dosage forms which are, within the scope of 10 Sound medical judgment, Suitable for use in contact with the microbe from the tissue, in some embodiments, the method tissues of human beings and animals without excessive can further comprise replacing the previously-applied com toxicity, irritation, allergic response, or other problem or position in contact with the tissue with a fresh composition complication, commensurate with a reasonable benefit/risk after a period of time. For example, depending on the ratio. condition of the microbial infection and/or specific compo 15 As used herein, the term “pharmaceutically acceptable sitions, the previously-applied composition can be replaced carrier refers to a pharmaceutically-acceptable material, every 1 hour, every 2 hours, every 3 hours, every 4 hours, composition or vehicle for administration of an active agent every 5 hours, every 6 hours, every 8 hours, every 10 hours, described herein. Pharmaceutically acceptable carriers every 12 hours, every 16 hours, every 24 hours or longer. include any and all solvents, dispersion media, coatings, In some embodiments, the method can further comprise antibacterial and antifungal agents, isotonic and absorption administering an additional treatment to the tissue. Exem delaying agents, and the like which are compatible with the plary additional treatments can include, but are not limited activity of the active agent and are physiologically accept to, a negative-pressure treatment, a vacuum-assisted able to the subject. Some examples of materials which can debridement, administration of an antimicrobial agent, or serve as pharmaceutically-acceptable carriers include: (i) any combinations thereof. 25 Sugars, such as lactose, glucose and Sucrose; (ii) starches, Without limitations, the compositions and/or methods of Such as corn starch and potato starch; (iii) cellulose, and its any aspects described herein can be used to treat and/or derivatives, such as Sodium carboxymethyl cellulose, meth prevent a microbial infection or contamination in vitro, in ylcellulose, ethyl cellulose, microcrystalline cellulose and situ or in vivo. In some embodiments, the compositions cellulose acetate; (iv) powdered tragacanth; (V) malt, (vi) 30 gelatin; (vii) lubricating agents, such as magnesium Stearate, and/or methods of any aspects described herein can be used Sodium lauryl Sulfate and talc, (viii) excipients, such as to treat and/or prevent a microbial infection or contamina cocoa butter and suppository waxes; (ix) oils, such as peanut tion in a fluid or on any surface, including, but not limited oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn to, a tissue surface, a Solid Substrate Surface, e.g., a medical oil and Soybean oil; (X) glycols, such as propylene glycol, device Surface, an environmental Surface, or food. 35 (xi) polyols, such as glycerin, Sorbitol, mannitol and poly Additionally, in Some embodiments where the composi ethylene glycol (PEG); (xii) esters, such as ethyl oleate and tion comprises at least one engineered microbe-targeting ethyl laurate; (xiii) agar, (xiv) buffering agents, such as molecule conjugated to a detectable label described herein magnesium hydroxide and aluminum hydroxide: (XV) alg or an imaging agent, can be used to image an infection in inic acid; (Xvi) pyrogen-free water, (Xvii) isotonic saline; situ, e.g., in a subject or on an environmental Surface. 40 (xviii) Ringer's solution; (xix) ethyl alcohol; (XX) pH buff S. aureus infections can sometimes be difficult to treat as ered solutions; (XXi) polyesters, polycarbonates and/or poly S. aureus has protein A on its cell Surface. Protein A is a anhydrides; (XXii) bulking agents, such as polypeptides and wall-anchored protein with either four or five domains, each amino acids (XXiii) serum component, such as serum albu of which can bind to the Fc region of IgG. The X-ray min, HDL and LDL; (xxiv) C2-C12 alcohols, such as structure of protein AIgG-binding domains in complex with 45 ethanol; and (XXV) other non-toxic compatible Substances the Fc region of IgG has been reported, and residues from employed in pharmaceutical formulations. Wetting agents, helix I that are involved in the interaction have been iden coloring agents, release agents, coating agents, Sweetening tified and evaluated by site directed mutagenesis. The inter agents, flavoring agents, perfuming agents, preservative and action between protein A and IgG can coat the Surface of the antioxidants can also be present in the formulation. For cell with IgG molecules that are in an orientation incorrect 50 compositions or preparations described herein to be admin to be recognized by the neutrophil Fc receptor (FIG. 22). istered orally, pharmaceutically acceptable carriers include, This can indicate the anti-phagocytic effect of protein A and but are not limited to pharmaceutically acceptable excipients its role in pathogenesis of S. aureus infections. Protein-A- Such as inert diluents, disintegrating agents, binding agents, deficient mutants of S. aureus are reported to be phagocy lubricating agents, Sweetening agents, flavoring agents, col tosed more efficiently by neutrophils in vitro and show 55 oring agents and preservatives. Suitable inert diluents decreased virulence in several animal infection models (See, include Sodium and calcium carbonate, sodium and calcium e.g., Fraser T., Nature Reviews Microbiology 2005: 3 (12): phosphate, and lactose, while corn starch and alginic acid 948-58). In accordance with some aspects provided herein, are Suitable disintegrating agents. Binding agents may the compositions and/or methods described herein can be include starch and gelatin, while the lubricating agent, if used to treat or prevent S. aureus microbial infection. 60 present, will generally be magnesium Stearate, Stearic acid or Pharmaceutical Compositions talc. If desired, the tablets may be coated with a material Some embodiments of the engineered microbe-targeting Such as glyceryl monostearate or glyceryl distearate, to delay molecules can be used for therapeutic purposes. For admin absorption in the gastrointestinal tract. istration to a Subject in need thereof, engineered microbe Pharmaceutically acceptable carriers can vary in a prepa targeting molecules described herein can be provided in 65 ration described herein, depending on the administration pharmaceutically acceptable compositions. Accordingly, in route and formulation. The compositions and preparations yet another aspect, provided herein is a pharmaceutical described herein can be delivered via any administration US 9,593,160 B2 81 82 mode known to a skilled practitioner. For example, the comprise an actuator to control release of the composition compositions and preparations described herein can be through the outlet. Such delivery device can be any device delivered in a systemic manner, via administration routes to facilitate the administration of any composition described Such as, but not limited to, oral, and parenteral including herein to a Subject, e.g., a syringe, a dry powder injector, a intravenous, intramuscular, intraperitoneal, intradermal, and nasal spray, a nebulizer, or an implant such as a microchip, Subcutaneous. In some embodiments, the compositions and e.g., for Sustained-release or controlled release of any com preparations described herein are in a form that is suitable position described herein. for injection. In other embodiments, the compositions and In some embodiments of the products described herein, preparations described herein are formulated for oral admin the microbe-targeting microparticles described herein itself istration. 10 can be modified to control its degradation and thus the When administering parenterally, a composition and release of active agents. In some embodiments, the engi preparation described herein can be generally formulated in neered microbe-targeting molecules, microbe-targeting a unit dosage injectable form (solution, Suspension, emul microparticles and/or microbe-targeting cells described sion). The compositions and preparations suitable for injec herein can be combined with other types of delivery systems tion include sterile aqueous Solutions or dispersions. The 15 available and known to those of ordinary skill in the art. carrier can be a solvent or dispersing medium containing, for They include, for example, polymer-based systems such as example, water, cell culture medium, buffers (e.g., phos polylactic and/or polyglycolic acids, polyanhydrides, poly phate buffered saline), polyol (for example, glycerol, pro caprolactones, copolyoxalates, polyesteramides, poly pylene glycol, liquid polyethylene glycol, and the like), orthoesters, polyhydroxybutyric acid, and/or combinations suitable mixtures thereof. In some embodiments, the phar thereof. Microcapsules of the foregoing polymers containing maceutical carrier can be a buffered solution (e.g. PBS). drugs are described in, for example, U.S. Pat. No. 5,075,109. An oral composition can be prepared in any orally accept Other examples include nonpolymer Systems that are lipid able dosage form including, but not limited to, tablets, based including sterols such as cholesterol, cholesterol capsules, emulsions and aqueous Suspensions, dispersions esters, and fatty acids or neukal fats such as mono-, di- and and solutions. Commonly used carriers for tablets include 25 triglycerides; hydrogel release systems; liposome-based sys lactose and corn starch. Lubricating agents, such as magne tems; phospholipid based-systems; Silastic systems; peptide sium Stearate, are also typically added to tablets. For oral based systems; or partially fused implants. Specific administration in a capsule form, useful diluents include examples include, but are not limited to, erosional systems lactose and dried corn starch. When aqueous Suspensions or in which the composition is contained in a form within a emulsions are administered orally, the active ingredient can 30 matrix (for example, as described in U.S. Pat. Nos. 4,452, be suspended or dissolved in an oily phase combined with 775, 4,675,189, 5,736,152, 4,667,014, 4,748,034 and -29 emulsifying or suspending agents. If desired, certain Sweet 5,239,660), or diffusional systems in which an active com ening, flavoring, or coloring agents can be added. Liquid ponent controls the release rate (for example, as described in preparations for oral administration can also be prepared in U.S. Pat. Nos. 3,832,253, 3,854,480, 5,133,974 and 5,407, the form of a dry powder to be reconstituted with a suitable 35 686). The formulation may be as, for example, micro Solvent prior to use. spheres, hydrogels, polymeric reservoirs, cholesterol matri The compositions can also contain auxiliary Substances ces, or polymeric systems. In some embodiments, the Such as wetting or emulsifying agents, pH buffering agents, system may allow Sustained or controlled release of the gelling or viscosity enhancing additives, preservatives, col composition to occur, for example, through control of the ors, and the like, depending upon the route of administration 40 diffusion or erosion/degradation rate of the formulation and the preparation desired. Standard texts, such as “REM containing the composition. In addition, a pump-based hard INGTON'S PHARMACEUTICAL SCIENCE, 17th edi ware delivery system can be used to deliver one or more tion, 1985, incorporated herein by reference, may be con embodiments of the compositions or preparations described Sulted to prepare suitable preparations, without undue herein. Use of a long-term Sustained release formulations or experimentation. With respect to compositions described 45 implants can be particularly Suitable for treatment of some herein, however, any vehicle, diluent, or additive used infections. Long-term release, as used herein, means that a should have to be biocompatible with the active agents formulation or an implant is made and arranged to deliver described herein. Those skilled in the art will recognize that compositions or preparations described herein at a therapeu the components of the compositions should be selected to be tic level for at least 30 days, or at least 60 days. In some biocompatible with respect to the active agent. This will 50 embodiments, the long-term release refers to a formulation present no problem to those skilled in chemical and phar or an implant being configured to deliver an active agent at maceutical principles, or problems can be readily avoided by a therapeutic level over several months. reference to standard texts or by simple experiments (not Regeneration of Microbe-Binding Substrates (e.g., Microbe involving undue experimentation). Binding Microbeads) In some embodiments, the compositions and preparations 55 In some applications, an artisan may want to detach or described herein can be formulated in an emulsion or a gel. release a pathogen captured by or bound to an engineered Such gel compositions and preparations can be implanted microbe-targeting molecule. As discussed herein, calcium locally to a diseased tissue region of a Subject. ions are involved in binding interactions of the engineered For in vivo administration, the compositions or prepara microbe-targeting molecules described herein with microbe tions described herein can be administered with a delivery 60 Surface. A skilled artisan will appreciate that detaching the device, e.g., a Syringe. Accordingly, an additional aspect pathogen from Support bound microbe-targeting molecule described herein provides for delivery devices comprising at also regenerates the Support bound microbe-targeting mol least one chamber with an outlet, wherein the at least one ecule. chamber comprises a pre-determined amount of any com Accordingly, disclosed herein are methods for inhibiting position described herein and the outlet provides an exit for 65 Ca' assisted interactions between two components, e.g., in the composition enclosed inside the chamber. In some a complex, by reducing the amount of Ca" ions available embodiments, a delivery device described herein can further for the interactions. This can be accomplished by contacting US 9,593,160 B2 83 84 or incubating the complex with a buffer or solution com If the targeting molecule with the bound pathogen is prising a chelating agent which chelates calcium ions. attached to a Support Surface, e.g., a microparticle or a Exemplary chelating agents include, but are not limited to, magnetic microparticle, the pathogen can be detached by 1.2-Bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic incubating or contacting the Support with a low pH buffer or acid; Ethylenediaminetetraacetic acid (EDTA); Ethylene a buffer in which calcium is not soluble. Thus provided glycol-bis(2-aminoethylether)-N.N.N',N'-tetraacetic acid; herein is also a method for detaching a microbe from a and Ethylene glycol-bis(B-aminoethyl ether)-N.N.N',N'-tet Support bound microbe-targeting molecule. The method raacetic acid. comprising contacting, washing, or incubating the Support For some uses, chelating agents can be problematic. For bound pathogen with a low pH buffer or a buffer in which example, chelating agents such as EDTA and EGTA can be 10 calcium in insoluble. After a predetermined time (e.g., 5 harsh or dangerous to biological samples. Accordingly, the mins, 10 mins, 15 mins, 30 mins, 25 mins, 30 mins, 35 mins, inventors have also discovered alternative methods for 40 mins, 45 mins, 50 mins, 55 mins, 1 hour, 1.25 hours, 1.5 reducing the amount of Ca" ions available for assisting in hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours or more) complex formation. In one example, the complex can be has passed, the buffer can be removed and the support contacted or incubated with a low pH buffer. Without 15 optionally washed one or more times. Without wishing to be wishing to be bound by a theory, low pH buffer protonates bound by a theory, this regenerates the Support bound the negatively charged carboxyl groups (glutamate side targeting molecules for binding with pathogens in Sample. In chains) on the engineered microbe-targeting molecules that other words, detaching the microbes allows one to re-use the are responsible for binding calcium. Protonating these side Support bound targeting molecules. The detached pathogens chains can remove their negative charge, can remove their can be used for analysis, detection or for any other use. ability to bind to positively charged calcium ions. In some Kits embodiments, the low pH buffer is of about pH 6.75, about Kits for capturing, detecting and/or determining the pres pH 6.5, about pH 6.25, about pH 6, about pH 5.75, about pH ence or absence of a microbe and/or microbial matter in a 5.5, about pH 5.25, about pH 5, about pH 4.5, about pH 4. sample are also provided herein. In some embodiments, the about pH 3.5, about pH 3, about pH 2.5 or lower. In one 25 kit can comprise: (a) one or more containers containing a embodiment, buffer is of pH about 2.8. In some embodi population of engineered microbe-targeting molecules ments, the low pH buffer can further comprise a chelating described herein; and (b) at least one reagent. In these agent. embodiments, a user can generate their own microbe-target Alternatively or in addition to a low pH buffer, one can ing Substrates by conjugating the provided engineered also use a buffer in which calcium is not soluble. For 30 microbe-targeting molecules to their desired Substrate, e.g., example, calcium can interact with one or more components using any art-recognized conjugation chemistry and/or of the buffer and can precipitate out of the buffer solution. methods described herein. In such embodiments, the reagent Thus, contacting or incubating the complex in Such a buffer can include, but is not limited to, a coupling agent for can lead to precipitation of the calcium ions making them conjugation of engineered microbe-targeting molecules to a unavailable for the necessary interaction with the targeting 35 Substrate. In some embodiments, the kit can further com molecule-microbe interface. Generally, buffers in which prise one or more Substrates (e.g., microbeads such as calcium is not soluble include an anion which forms a salt magnetic microbeads) to which the engineered microbe with the Ca" ion. Thus formed salt is less soluble in the targeting molecules described herein are conjugated. In Such solvent of the buffer. Exemplary anion which produce embodiments, a user can further modify the Surface chem insoluble salts with Ca" include, but are not limited to, 40 istry of the provided substrate prior to conjugation of the phosphates, oxalates, carbonates, sulfates, fluorides, glu engineered microbe-targeting molecules to the Substrate. conic acid, oxido-trioxo-manganese, Stearic acid, and the In other embodiments, the kit can provide microbe like. In some embodiments, the buffer can further comprise targeting Substrates that are ready for use. Accordingly, in a chelating agent. these embodiments, the kit can comprise: (a) one or more In some embodiments, the buffer is a 0.2M glycine buffer 45 microbe-targeting Substrates described herein; and (b) at of pH 2.8. In some embodiments, the buffer is a 0.1M least one reagent. In some embodiments, the microbe sodium phosphate buffer of pH 6.0. targeting Substrate can include one or more microbe-binding Many of the calcium salts become more insoluble at dipsticks, e.g., as described herein. In other embodiments, elevated temperature. Accordingly, during detachment of the the microbe-targeting Substrate can include a population of pathogen, temperature of the buffer can be increased or 50 microbe-targeting microbeads (including, but not limited to, decrease. In some embodiments, the buffer is heated during polymeric microbeads and magnetic microbeads). In some detachment of the pathogen. In some other embodiments, embodiments, the microbe-targeting Substrate can include a the buffer is cooled during detachment of the pathogen. population of microbe-targeting magnetic microbeads. The Temperature of the buffer can be increased or decreased by microbe-targeting microbeads or microbe-targeting mag at least 5°C., at least 10°C., at least 15° C., at least 20°C., 55 netic microbeads can be provided in one or more separate at least 25° C. or more relative to room temperature. containers, if desired. In some embodiments, the population The method described herein for inhibiting Ca" assisted of the microbe-targeting microbeads or magnetic micro interactions between two components can also be used for beads contained in one or more containers can be detaching a pathogen from an engineered microbe-targeting lyophilized. molecule. For example, the pathogen-targeting molecule 60 In some embodiments of any aspects of the kits described complex can be contacted or incubated with a low pH buffer herein, the population of the microbeads or microbe-target or with a buffer in which calcium is not soluble. ing microbeads can comprise at least one distinct Subset of In one embodiment, a bound pathogen can be detached the microbeads or microbe-targeting microbeads, respec from a targeting molecule using a 0.2M glycine buffer at pH tively. For example, each distinct subset of the microbeads 2.8. In another embodiment, a bound pathogen can be 65 or microbe-targeting microbeads can be provided in a sepa detached from a targeting molecule using a 0.1M sodium rate container. In some embodiments, the distinct Subset of phosphate buffer at pH 6.0. the microbeads or microbe-targeting microbeads can have a US 9,593,160 B2 85 86 size. In some embodiments, the distinct subset of microbe the microbe-targeting molecules described herein, or an targeting microbeads can comprise on their Surfaces a dif antibody specific to at least one type of carbohydrate rec ferent density of engineered microbe-targeting molecules ognition domain (e.g., C-type lectins vs. S-type lectins) from the rest of the population. In these embodiments, two employed in the microbe-targeting molecules described or more Subsets of the microbe-targeting microbes having herein. However, the antibody can also be a common different sizes and/or different coating density of the engi antibody that binds to all the microbes or pathogens recog neered microbe-binding molecules can be used to detect and nized by the microbe-targeting molecules provided in the differentiate microbes of different classes and/or sizes, e.g., kit. Without limitations, a molecule attached to a detectable employing the methods described herein. In some embodi label can also include any ligand targeting microbial cell ments, the distinct Subset of microbe-targeting Substrates, 10 Surface proteins or receptors, including carbohydrates, lip e.g., microbe-targeting microbeads, can comprise a different ids, lectins, aptamers, protein, peptides, nucleic acid, poly carbohydrate recognition domain from the others. nucleotides, antibody or a portion thereof, an antibody-like In some embodiments of any aspects of the kits described molecule, peptidomimetic, and any combinations thereof. herein, the Substrates (e.g., microbeads) or microbe-target In some embodiments, at least one of the containers can ing Substrates (e.g., microbe-targeting microbeads) can fur 15 contain a distinct population of the molecule-detectable ther comprise a detection label. By way of example only, label conjugate as described earlier. The distinct population depending on the choice of detection methods, each distinct of the molecule-detectable label conjugate can contain a Subset of the microbeads can comprise a unique detection unique molecule with the detectable label same as others, or label or the same detection label. For example, if each a conjugate comprising a distinct detectable label (e.g., a distinct Subset of the microbe-targeting microbeads is used unique fluorescent molecule) and a distinct molecule. As in a different sampling well, the same detection label can be each distinct detectable label can identify the associated used on the microbe-targeting microbeads. However, if it is protein, conjugates comprising a distinct detectable label desirable to detect multiple different microbe-targeting associated with a distinct molecule can allow detecting in a microbeads in the same well, it is preferably to have each single sample at least two or more distinct populations of the distinct Subset of microbe-targeting microbeads comprising 25 engineered microbe-targeting Substrates (e.g., microbe-tar a distinct detection label. geting magnetic microbeads); for example, each distinct Detectable labels suitable for use in any kits provided population of the engineered microbe-targeting magnetic herein include any composition detectable by spectroscopic, microbeads can bind to a distinct genus or species or photochemical, biochemical, immunochemical, electrical, type/size of a microbe. In alternative embodiments, the optical or chemical means. Any art-recognized detectable 30 molecule-detectable label conjugates in each of the contain labels or the ones described herein can be included in the kits ers can comprise the same detectable label. For example, the described herein. detectable label can comprise an enzyme (e.g., horseradish Means of detecting such labels are well known to those of peroxidase or alkaline phosphatase) that produces a color skill in the art and exemplary detection methods are change in the presence of an enzyme substrate. In Such described herein. For example, radiolabels can be detected 35 embodiments, the kit can further comprise one or more using photographic film or Scintillation counters, fluorescent containers containing an enzyme Substrate that changes markers can be detected using a photo-detector to detect color in the presence of the enzyme. emitted light. Enzymatic labels are typically detected by In one embodiment, the microbe-targeting Substrate pro providing the enzyme with an enzyme Substrate and detect vided in the kit can include a dipstick or test strip or ing the reaction product produced by the action of the 40 membrane containing one or more engineered microbe enzyme on the enzyme Substrate, and calorimetric labels can targeting molecules, e.g., microbe-binding dipstick or mem be detected by visualizing the colored label. brane described herein. In this embodiment, the kit can In some embodiments of any aspects described herein, the comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, kits can further comprise one or more containers containing 150, 200 or more microbe-binding dipsticks or test strips a population of detectable labels, wherein the detectable 45 described herein. These kits comprising the microbe-binding label is conjugated to a molecule. In some embodiments, at dipsticks or test Strips can be used as a diagnostic or probe least one of the containers can contain a distinct population for a microbe anywhere, e.g., at home, in clinics or hospitals, of detectable labels. on emergency vehicles, in outdoor environments, in food The molecule conjugated to a detectable label can be any processing plants, and anywhere in need of microbe capture molecule that binds to a microbe of interest. For example, in 50 and/or detection. Some embodiments, the molecule conjugated to a detectable In some embodiments, each microbe-targeting Substrate label can comprise the same carbohydrate recognition or product described herein, e.g., each microbe-binding domains as used in the microbe-targeting Substrates (e.g., dipstick or membrane, can be individually packaged to microbe-targeting magnetic microbeads). In Such embodi maintain their sterility. In some embodiments, two or more ments, at least one population of the molecule-detectable 55 products (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, or more label conjugate can comprise at least one carbohydrate products Such as microbe-binding dipsticks or membranes) recognition domain or a fragment thereof, e.g., derived from can be packaged into one single unit. In Such embodiments, mannose-binding lectin or at least a portion of the CRD users can sterilize any unused products after opening, e.g., domain, e.g., encoded by SEQ ID NO. 4, or a fragment with UV radiation, high temperature, gamma-radiation, eth thereof. In some embodiments, the molecule conjugated to 60 ylene oxide sterilization or any other known methods that a detectable label can further comprise a Fc region of an would not significantly affect the activity of the engineered immunoglobulin. In alternative embodiments, the molecule microbe-targeting molecules for microbe detection. conjugated to a detectable label can comprise an antibody In other embodiments, the microbe-targeting Substrate specific to at least one genus, species, or type/class of provided in the kit can include a population of microbe microbes (e.g., gram-positive VS. gram-negative microbes; 65 targeting microbeads or magnetic microbeads. In some protein A-expressing or protein G-expressing microbes vs. embodiments, the microbe-targeting microbeads or mag protein A- or protein G-negative microbes) recognized by netic microbeads can be lyophilized. US 9,593,160 B2 87 88 Depending on the configuration/combination of the mol tainers containing a population of detectable labels ecule-detectable label conjugates provided in the kit, differ described earlier, each of which is conjugated to a targeting ent populations of the microbe-targeting microbeads or agent specific for a microbe, e.g., without limitations, one or magnetic microbeads can be mixed together with a test more embodiments of an engineered microbe-targeting mol sample in a single reaction, or different populations each can ecule or a fragment thereof, an antibody specific for at least be applied separately to different aliquots of the same test one microbe (e.g., antibodies specific for Gram-positive sample. After contacting the test sample with the microbe microbes such as anti-LTA antibodies, antibodies specific for targeting microbeads or magnetic microbeads, any microbes Gram-negative microbes such as anti-LPS antibodies, or or pathogens recognized by the microbe-targeting molecules antibodies specific for fungus, and any combinations will bind to the microbe-targeting microbeads or magnetic 10 thereof). The use of an additional targeting agent specific for microbeads. a microbe conjugated to a detectable label can not only In some embodiments, the kit can further comprise at least facilitate the detection of microbes or pathogens, but can one blood collection container or any equivalent sample also increase the specificity of the detection for a microbe or container or chamber, including at least 1, at least 2, at least a pathogen. 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 15 In any aspects of the kits provided herein, when the 9, at least 10, at least 15, at least 20 blood collection detection label includes an enzyme (e.g., horseradish per containers or equivalent sample containers or chambers. In oxidase, alkaline phosphatase and any others commonly Some embodiments, the population of the microbe-targeting used for colorimetric detection), the kits can further com microbeads or magnetic microbeads can be pre-loaded in at prise one or more containers containing an enzyme substrate least one blood collection container. In some embodiments, that produces a color change in the presence of the enzyme. the blood collection container can further comprise an One of skill in the art can readily recognize an appropriate anti-coagulant agent described herein. In some embodi enzyme Substrate for any art-recognized enzymes used for ments, a blood sample can be directly added to such blood colorimetric detection. By way of example only, an exem collection container containing a population of the microbe plary substrate for alkaline phosphatase can include BCIP/ targeting and/or microbe-binding microbeads or magnetic 25 NBT or PNPP (p-Nitrophenyl Phosphate, Disodium Salt); an microbeads for carrying out a microbe detection assay, e.g., exemplary Substrate for horseradish peroxidase can include as described in Example 10 and FIG. 14. While Example 10 TMB. and FIG. 14 illustrates the use of microbe-targeting magnetic In any aspects of the kits provided herein, the at least one microbeads for capture of microbes, an ordinary artisan will reagent can be a wash buffer, a dilution buffer, a stop buffer, readily appreciate that some embodiments of the microbe 30 e.g., to stop the color development, a buffer Solution con targeting microbeads (without magnetic properties) taining a chelating agent described herein, or any combina described herein can also be applicable for the assay. For tions thereof. In one embodiment, at least one of the reagents example, instead of using a magnet to collect the microbe provided in the kit can include at least one buffered solution targeting magnetic microbeads after contact with a test containing a chelating agent. The chelating agent can be sample (e.g., a blood sample), the microbe-targeting micro 35 used to chelate any ions (e.g., divalent ions) present in the beads (without magnetic properties) can also be collected, test samples or assay buffer, e.g., for inhibiting calcium e.g., by filtration, centrifugation or any other methods dependent binding of certain microbes, but not others, to known in the art. Some embodiments of the microbe-binding molecules In some embodiments where the kits comprise microbe described herein. Accordingly, Such kit can be used to targeting magnetic microbeads, the kits can further comprise 40 distinguish one microbe (e.g., S. aureus) from another (e.g., a magnet adapted for use with the assay for isolation of the E. coli) in a test sample, e.g. employing some embodiments microbe-targeting magnetic microbeads from a test sample. of the method described herein. For example, if the assay is carried out in a blood collection In any aspects of the kits provided herein, the kits can tube, the magnet can be adapted for use with the blood further comprise at least one microtiter plate, e.g., for collection tube, e.g., a magnet can be designed to be a 45 performing the reaction and the detection. magnet collar Surrounding the blood collection tube to In addition to the above mentioned components, any immobilize or isolate the microbe-targeting magnetic micro embodiments of the kits described herein can include infor beads from a test sample or an assay buffer. mational material. The informational material can be In any aspects of the kits provided herein, the kits can descriptive, instructional, marketing or other material that further comprise a portable readout machine or device, e.g., 50 relates to the methods described herein and/or the use of the to determine and display the signal produced from the assay aggregates for the methods described herein. For example, performed with the kit. For example, the readout machine or the informational material can describe methods for using device can detect a colorimetric signal and/or a fluorescent the kits provided herein to perform an assay for pathogen or signal produced from the assay of pathogen detection per microbe capture and/or detection. The kit can also include an formed with the kits described herein. 55 empty container and/or a delivery device, e.g., which can be In any aspects of the kits described herein, the kits can used to deliver a test sample to a test container. further include a reference for comparison with a readout The informational material of the kits is not limited in its determined from a test sample. An exemplary reference can form. In many cases, the informational material, e.g., be a strip or a chart showing different colors corresponding instructions, is provided in printed matter, e.g., a printed to various extents or degrees of a microbial infection. 60 text, drawing, and/or photograph, e.g., a label or printed Depending on different embodiments of the engineered sheet. However, the informational material can also be microbe-targeting molecules and/or products provided in the provided in other formats, such as Braille, computer read kits, some embodiments of any aspects of the kits described able material, video recording, or audio recording. In herein can further comprise an additional agent. For another embodiment, the informational material of the kit is example, in Some embodiments where the engineered 65 a link or contact information, e.g., a physical address, email microbe-targeting molecules present on the Substrate are address, hyperlink, website, or telephone number, where a unlabeled, the kit can further comprise one or more con user of the kit can obtain substantive information about the US 9,593,160 B2 89 90 formulation and/or its use in the methods described herein. material. Such as non-cellular fractions of blood, saliva, or Of course, the informational material can also be provided urine, which can be used to measure plasma/serum bio in any combination of formats. marker expression levels. In some embodiments, the kit can contain separate con The biological fluid sample can be freshly collected from tainers, dividers or compartments for each component and a subject or a previously collected Sample. In some embodi informational material. For example, each different compo ments, the biological fluid sample used in the assays and/or nent can be contained in a bottle, vial, or syringe, and the methods described herein can be collected from a subject no informational material can be contained in a plastic sleeve or more than 24 hours, no more than 12 hours, no more than 6 packet. In other embodiments, the separate elements of the hours, no more than 3 hours, no more than 2 hours, no more kit are contained within a single, undivided container. For 10 than 1 hour, no more than 30 mins or shorter. example, a collection of the magnetic microbeads is con In some embodiments, the biological fluid sample or any tained in a bottle, vial or syringe that has attached thereto the fluid sample described herein can be treated with a chemical informational material in the form of a label. and/or biological reagent described herein prior to use with In general, the kits described herein can be used to the assays and/or methods described herein. In some separate, remove, and/or detect a microbe present in a test 15 embodiments, at least one of the chemical and/or biological sample. In some embodiments, the kits can be used to reagents can be present in the sample container before a fluid differentiate between different microbe species, classes, and/ sample is added to the sample container. For example, blood or sizes, by employing the methods and/or assays described can be collected into a blood collection tube such as VACU herein. By way of example only, some embodiments of the TAINER(R), which has already contained heparin. Examples kits can be used to detect the presence or absence of any of the chemical and/or biological reagents can include, protein A-expressing microbe or any protein G-expressing without limitations, Surfactants and detergents, salts, cell microbe in a test sample. Accordingly, Some embodiments lysing reagents, anticoagulants, degradative enzymes (e.g., of the kits described herein can be used to detect or deter proteases, lipases, nucleases, collagenases, cellulases, amy mine the presence or absence of at least one staphylococcus lases), and solvents such as buffer Solutions. species, excluding S. epidermidis, in a test sample. In one 25 In some embodiments, the test sample can include a fluid embodiment, the assays, methods, and kits described herein or specimen obtained from an environmental source, e.g., can be used to detect or determine the presence or absence but not limited to, water Supplies (including wastewater), of S. aureus in a test sample. In some embodiments, the ponds, rivers, reservoirs, Swimming pools, soils, food pro assays, methods, and kits described herein can be used to cessing and/or packaging plants, agricultural places, hydro detect or determine the presence or absence of at least one 30 cultures (including hydroponic food farms), pharmaceutical streptococci species in a test sample. manufacturing plants, animal colony facilities, and any In some embodiments, the kits described herein can be combinations thereof. used to screen a pharmaceutical product (e.g., a drug, a In some embodiments, the test sample can include a fluid therapeutic agent, or an imaging agent), and/or a medical (e.g., culture medium) from a biological culture. Examples device (including, but not limited to, implantable devices) 35 of a fluid (e.g., culture medium) obtained from a biological for the presence or absence of microbial matter (including, culture includes the one obtained from culturing or fermen but not limited to, endotoxins secreted by a microbe). tation, for example, of single- or multi-cell organisms, Test Sample including prokaryotes (e.g., bacteria) and eukaryotes (e.g., In accordance with various embodiments described animal cells, plant cells, yeasts, fungi), and including frac herein, a test sample or sample, including any fluid or 40 tions thereof. In some embodiments, the test sample can specimen (processed or unprocessed), that is suspected of include a fluid from a blood culture. In some embodiments, comprising a microbe and/or microbial matter can be Sub the culture medium can be obtained from any source, e.g., jected to an assay or method, kit and system described without limitations, research laboratories, pharmaceutical herein. The test sample or fluid can be liquid, supercritical manufacturing plants, hydrocultures (e.g., hydroponic food fluid, Solutions, Suspensions, gases, gels, slurries, and com 45 farms), diagnostic testing facilities, clinical settings, and any binations thereof. The test sample or fluid can be aqueous or combinations thereof. non-aqueous. In some embodiments, the test sample can include a In some embodiments, the test sample can be an aqueous media or reagent Solution used in a laboratory or clinical fluid. As used herein, the term “aqueous fluid refers to any setting, such as for biomedical and molecular biology appli flowable water-containing material that is Suspected of com 50 cations. As used herein, the term “media' refers to a medium prising a microbe and/or microbial matter. for maintaining a tissue, an organism, or a cell population, In some embodiments, the test sample can include a or refers to a medium for culturing a tissue, an organism, or biological fluid obtained from a subject. Exemplary biologi a cell population, which contains nutrients that maintain cal fluids obtained from a subject can include, but are not viability of the tissue, organism, or cell population, and limited to, blood (including whole blood, plasma, cord blood 55 Support proliferation and growth. and serum), lactation products (e.g., milk), amniotic fluids, As used herein, the term “reagent” refers to any solution sputum, saliva, urine, semen, cerebrospinal fluid, bronchial used in a laboratory or clinical setting for biomedical and aspirate, perspiration, mucus, liquefied feces, synovial fluid, molecular biology applications. Reagents include, but are lymphatic fluid, tears, tracheal aspirate, and fractions not limited to, saline solutions, PBS solutions, buffered thereof. In some embodiments, a biological fluid can include 60 solutions, such as phosphate buffers, EDTA, Tris solutions, a homogenate of a tissue specimen (e.g., biopsy) from a and any combinations thereof. Reagent solutions can be Subject. used to create other reagent solutions. For example, Tris In some embodiments, the biological fluid sample solutions and EDTA solutions are combined in specific obtained from a subject, e.g., a mammalian Subject such as ratios to create “TE' reagents for use in molecular biology a human Subject or a domestic pet Such as a cat or dog, can 65 applications. contain cells from the subject. In other embodiments, the In Some embodiments, the test sample can be a non biological fluid sample can contain non-cellular biological biological fluid. As used herein, the term “non-biological US 9,593,160 B2 91 92 fluid refers to any fluid that is not a biological fluid as the Nile virus, Yellow Fever virus, causative agents of trans term is defined herein. Exemplary non-biological fluids missible spongiform encephalopathies, Creutzfeldt-Jakob include, but are not limited to, water, salt water, brine, disease agent, variant Creutzfeldt-Jakob disease agent, Can buffered solutions, saline solutions, Sugar Solutions, carbo dida, CryptcoOccus, Cryptosporidium, Giardia lamblia, hydrate solutions, lipid solutions, nucleic acid solutions, Microsporidia, Plasmodium vivax, Pneumocystis carinii, hydrocarbons (e.g. liquid hydrocarbons), acids, gasoline, Toxoplasma gondii, Trichophyton mentagrophytes, Entero petroleum, liquefied samples (e.g., liquefied samples), and cytozoon bieneusi, Cyclospora Cayetanensis, Encephalito mixtures thereof. zoon hellen, Encephalitozoon cuniculi, among other Exemplary Microbes or Pathogens viruses, bacteria, archaea, protozoa, and fungi). As used herein, the term “microbes’ or “microbe' gen 10 In some embodiments, the engineered microbe-targeting erally refers to microorganism(s), including bacteria, fungi, molecules or Substrates, products and kits described herein protozoan, archaea, protists, e.g., algae, and a combination can be used to differentiate a protein A-expressing or protein thereof. The term “microbes’ encompasses both live and G-expressing microbe from protein A- and protein G-nega dead microbes. The term “microbes’ also includes patho tive microbes (e.g., E. coli) by employing the methods or genic microbes or pathogens, e.g., bacteria causing diseases 15 assays described herein. Such as plague, tuberculosis and anthrax; protozoa causing In some embodiments, a protein A-expressing microbe diseases such as malaria, sleeping sickness and toxoplasmo includes Staphylococcus species. Examples of Staphylococ sis; fungi causing diseases such as ringworm, candidiasis or cus species include, but are not limited to, S. aureus group histoplasmosis; and bacteria causing diseases such as sepsis. (e.g., S. aureus, S. Simiae), S. auricularis group (e.g., S. Microbe-Induced Diseases: auricularis), S. carnosus group (e.g., S. carnosus, S. condi In some other embodiments, the engineered microbe menti, S. massiliensis, S. piscifermentans, S. simulans), S. targeting molecules or Substrates, products and kits epidermidis group (e.g., S. capitis, S. caprae, S. epidermidis, described herein can be used to detect or bind to the S. Saccharolyticus), S. haemolyticus group (e.g., S. devriesei, following microbes that causes diseases and/or associated S. haemolyticus, S. hominis), S. hyicus-intermedius group microbial matter: Bartonella hemselae, Borrelia burgdorferi, 25 (e.g., S. chromogenes, S. fells, S. delphini, S. hyicus, S. Campylobacter jejuni, Campylobacterfetus, Chlamydia tra intermedius, S. lutrae, S. microti, S. muscae, S. pseudinter chomatis, Chlamydia pneumoniae, Chlamydia psittaci, Sim medius, S. rostri, S. Schleiferi), S. lugdunensis group (e.g., S. Kania negevensis, Escherichia coli (e.g., O157:H7 and K88), lugdunensis), S. Saprophyticus group (e.g., S. arlettae, S. Ehrlichia chafeensis, Clostridium botulinum, Clostridium cohnii, S. equorum, S. gallinarum, S. kloosii, S. leei, S. perfingens, Clostridium tetani, Enterococcus faecalis, Hae 30 nepalensis, S. saprophyticus, S. Succinus, S, xylosus), S. mophilius influenzae, Haemophilius ducreyi, Coccidioides Sciuri group (e.g., S. fleurettii, S. lentus, S. Sciuri, S. Ste immitis, Bordetella pertussis, Coxiella burnetii, Ureaplasma panovicii, S. vitulinus), S. simulans group (e.g., S. simulans), urealyticum, Mycoplasma genitalium, Trichomatis vagina and S. warneri group (e.g., S. pasteuri, S. warneri). lis, Helicobacter pylori, Helicobacter hepaticus, Legionella In some embodiments, S. aureus can be differentiated pneumophila, Mycobacterium tuberculosis, Mycobacterium 35 from a protein A- and protein G-negative microbe (e.g., E. bovis, Mycobacterium africanum, Mycobacterium leprae, coli) using the assays and/or methods described herein. Mycobacterium asiaticum, Mycobacterium avium, Myco In some embodiments, S. aureus can be differentiated bacterium cellatum, Mycobacterium celonae, Mycobacte from S. epidermidis using the assays and/or methods rium fortuitum, Mycobacterium genavense, Mycobacterium described herein. haemophilum, Mycobacterium intracellulare, Mycobacte 40 In some embodiments, S. epidermidis cannot be differen rium kansasii, Mycobacterium malmoense, Mycobacterium tiated from a protein A- and protein G-negative microbe marinum, Mycobacterium scrofiliaceum, Mycobacterium (e.g., E. coli) using the assays and/or methods described simiae, Mycobacterium Szulgai, Mycobacterium ulcerans, herein. Mycobacterium xenopi, Corynebacterium diptheriae, Rho In some embodiments, a protein G-expressing microbe dococcus equi, Rickettsia aeschlimannii, Rickettsia africae, 45 includes Streptococcus species. Examples of Streptococcus Rickettsia Conorii, Arcanobacterium haemolyticum, Bacillus species can include, but are not limited to, alpha-hemolytic anthracis, Bacillus cereus, Lysteria monocytogenes, Yers including Pneumococci (e.g., S. pneumonia), and the Viri inia pestis, Yersinia enterocolitica, Shigella dysenteriae, dans group (e.g., S. mutans, S. mitis, S. sanguinis, S. Sali Neisseria meningitides, Neisseria gonorrhoeae, Streptococ varius, S. salivarius ssp. thermophilus, S. constellatus); and cus bovis, Streptococcus hemolyticus, Streptococcus mutans, 50 beta-hemolytic including Group A (e.g., S. pyogenes), Group Streptococcus pyogenes, Streptococcus pneumoniae, B (e.g., S. agalactiae), Group C (e.g., S. equi, and S. Staphylococcus aureus, Staphylococcus epidermidis, ZOOepidemicus), Group D (e.g., enterococci, Streptococcus Staphylococcus pneumoniae, Staphylococcus saprophyticus, bovis and Streptococcus equinus), Group F Streptococci, and Vibrio cholerae, Vibrio parahaemolyticus, Salmonella typhi, Group G Streptococci. Salmonella paratyphi, Salmonella enteritidis, Treponema 55 In some embodiments, a protein G-expressing microbe pallidum, Human rhinovirus, Human coronavirus, Dengue includes Group C and Group G streptococci. virus, Filoviruses (e.g., Marburg and Ebola viruses), Han One skilled in the art can understand that the engineered tavirus, Rift Valley virus, Hepatitis B, C, and E. Human microbe-targeting molecules or Substrates, products and kits Immunodeficiency Virus (e.g., HIV-1, HIV-2), HHV-8, described herein can be used to target any microorganism Human papillomavirus, Herpes virus (e.g., HV-I and HV-II), 60 with a microbe surface-binding domain described herein Human T-cell lymphotrophic viruses (e.g., HTLV-I and modified for each microorganism of interest. A skilled HTLV-II), Bovine leukemia virus, Influenza virus, Gua artisan can determine the cell-surface proteins or carbohy narito virus, Lassa virus, Measles virus, Rubella virus, drates for each microorganism of interest using any micro Mumps virus, Chickenpox (Varicella virus), Monkey pox, biology techniques known in the art. Epstein Bahr virus, Norwalk (and Norwalk-like) viruses, 65 Biofilm: Rotavirus, Parvovirus B19, Hantaan virus, Sin Nombre Accordingly, in some embodiments, the microbe-target virus, Venezuelan equine encephalitis, Sabia virus, West ing molecules or Substrates, products and kits herein can be US 9,593,160 B2 93 94 used to detect microbes and/or associated microbial matter yellow dwarf virus, wheat spindle streak virus, soil born present in a biofilm or to treat equipment Surfaces to prevent mosaic virus, wheat streak virus in maize, maize dwarf or inhibit formation of a biofilm. For example, Listeria mosaic virus, maize chlorotic dwarf virus, cucumber mosaic monocytogenes can form biofilms on a variety of materials virus, tobacco mosaic virus, alfalfa mosaic virus, potato used in food processing equipment and other food and virus X, potato virus Y. potato leaf roll virus and tomato non-food contact surfaces (Blackman, J Food Prot 1996; golden mosaic virus. 59:827-31; Frank, J Food Prot 1990; 53:550-4: Krysinski, J Military and Bioterrorism Applications: Food Prot 1992: 55:246-51; Ronner, J Food Prot 1993; In yet other embodiments, the engineered microbe-target 56:750-8). Biofilms can be broadly defined as microbial ing molecules and product comprising thereof can be used to cells attached to a surface, and which are embedded in a 10 detect or combat bioterror agents (e.g., B. Anthracis, and matrix of extracellular polymeric Substances produced by Smallpox). the microorganisms. Biofilms are known to occur in many In accordance with some embodiments described herein, environments and frequently lead to a wide diversity of an engineered microbe-binding molecule or microbe-bind undesirable effects. For example, biofilms cause fouling of ing substrate can be modified to bind to any of the microbes, industrial equipment Such as heat exchangers, pipelines, and 15 e.g., the ones described herein, including the associated ship hulls, resulting in reduced heat transfer, energy loss, microbial matter (e.g., but not limited to, fragments of cell increased fluid frictional resistance, and accelerated corro wall, microbial nucleic acid and endotoxin). Sion. Biofilm accumulation on teeth and gums, urinary and Embodiments of the various aspects described herein can intestinal tracts, and implanted medical devices such as be illustrated by the following numbered paragraphs. catheters and prostheses frequently lead to infections (Char 1. An engineered microbe-targeting molecule comprising: acklis W. G. Biofilm processes. In: Characklis W G and a. at least one microbe Surface-binding domain; Marshall K C eds. New York: John Wiley & Sons, 1990: b. a Substrate-binding domain adapted for orienting the 195-231: Costerton et al., Annu Rev Microbiol 1995; microbe Surface-binding domain away from a Sub 49:711-45). In some embodiments, the engineered microbe strate; and targeting microparticles, e.g., encapsulating a drug or a 25 c. at least one linker between the microbe surface-binding chemical for treatment of a biofilm, can be sprayed on domain and the Substrate-binding domain. contaminated equipment Surfaces. The bacteria present in 2. The engineered molecule of paragraph 1, wherein the the biofilm bind to the microbe-targeting microparticles, microbe-Surface binding domain comprises a carbohy which release the drug to treat the bacteria for targeted drug drate recognition domain (CRD) or a fragment thereof. delivery. 30 3. The engineered molecule of paragraph 1 or 2, wherein the In addition, L. monocytogenes attached to Surfaces such as CRD or a fragment thereof further comprises at least a stainless steel and rubber, materials commonly used in food portion of a carbohydrate-binding protein. processing environments, can Survive for prolonged periods 4. The engineered molecule of paragraph 3, wherein the (Helke and Wong, J Food Prot 1994:57:963-8). This would portion of the carbohydrate-binding protein excludes at partially explain their ability to persist in the processing 35 least one of complement and coagulation activation plant. Common Sources of L. monocytogenes in processing region. facilities include equipment, conveyors, product contact 5. The engineered molecule of any of paragraphs 2-4, Surfaces, hand tools, cleaning utensils, floors, drains, walls, wherein the CRD or the carbohydrate-binding protein is and condensate (Tomkin et al., Dairy, Food Environ Sanit derived from a lectin, a ficolin, or a fragment thereof. 1999; 19:551-62; Welbourn and Williams, Dairy, Food 40 6. The engineered molecule of paragraph 5 wherein the Environ Sanit 1999; 19:399-401). In some embodiments, the lectin is C-type lectin, or a fragment thereof. engineered microbe-targeting molecules can be configured 7. The engineered molecule of paragraph 6, wherein the to include a “smart label, which is undetectable when C-type lectin is collectin, or a fragment thereof. conjugated to the engineered microbe-targeting molecules, 8. The engineered molecule of paragraph 7, wherein the but produces a color change when released from the engi 45 collectin is mannose-binding lectin (MBL) or a fragment neered molecules in the presence of a microbe enzyme. thereof. Thus, when a microbe binds to the engineered microbe 9. The engineered molecule of any of paragraphs 2-8. targeting molecules, the microbe releases enzymes that wherein the CRD is of SEQ ID NO. 4 or a fragment release the detectable label from the engineered molecules. thereof. An observation of a color change indicates a risk for bacteria 50 10. The engineered molecule of any of paragraphs 2-9, contamination on a particular Surface, and thus some wherein the CRD or a fragment thereof further comprises embodiments of the engineered microbe-targeting mol a neck region of the carbohydrate-binding protein or a ecules and products can be used for early detection of fragment thereof. biofilm formation. 11. The engineered molecule of any of paragraphs 1-10, Plant Microbes: 55 wherein the Substrate-binding domain comprises at least In still further embodiments, the engineered microbe one amine. targeting molecules or Substrates and products described 12. The engineered molecule of any of paragraphs 1-11, herein can be used to target plant microbes and/or associated wherein the Substrate-binding domain comprises at least microbial matter. Plant fungi have caused major epidemics one oligopeptide comprising an amino acid sequence of with huge Societal impacts. Examples of plant fungi include, 60 AKT. but are not limited to, Phytophthora infestans, Crinipellis 13. The engineered molecule of any of paragraphs 1-12, perniciosa, frosty pod (Moniliophthora roreri). oomycete wherein the linker is adapted to provide flexibility and Phytophthora capsici, Mycosphaerella fijiensis, Fusarium orientation of the carbohydrate recognition domain to Ganoderma spp fungi and Phytophthora. An exemplary bind to the microbe surface. plant bacterium includes Burkholderia cepacia. Exemplary 65 14. The engineered molecule of any of paragraphs 1-13. plant viruses include, but are not limited to, soybean mosaic wherein the linker is adapted to facilitate expression and virus, bean pod mottle virus, tobacco ring spot virus, barley purification. US 9,593,160 B2 95 96 15. The engineered molecule of any of paragraphs 1-14. 34. The engineered lectin of paragraph 33, wherein the CRD wherein the linker comprises a portion of a Fc region of is of SEQ ID NO. 4. an immunoglobulin. 35. The engineered lectin of paragraph 33 or 34, wherein the 16. The engineered molecule of paragraph 15, wherein the CRD or a fragment thereof further comprises at least a immunoglobulin is selected from the group consisting of 5 portion of mannose-binding lectin (MBL). IgA, Ig|D, IgE, IgG, and IgM. 36. The engineered lectin of any of paragraphs 33-35, 17. The engineered molecule of paragraph 15 or 16, wherein wherein the portion of the MBL excludes at least one of the immunoglobulin is IgG1. complement and coagulation activation region. 18. The engineered molecule of any of paragraphs 15-17, 37. The engineered lectin of any of paragraphs 33-36, 10 wherein the CRD further comprises a neck region of the wherein the portion of the Fc region comprises at least one MBL. region selected from the group consisting of a hinge 38. The engineered lectin of any of paragraphs 33-37, region, a CH2 region, a CH3 region, and any combina wherein the Substrate-binding domain comprises at least tions thereof. one amine. 19. The engineered molecule of any of paragraphs 15-18, 15 39. The engineered lectin of any of paragraphs 33-38, wherein the portion of the Fc region comprises at least one wherein the Substrate-binding domain comprises at least hinge region, at least one CH2 region and at least one CH3 one oligopeptide comprising an amino acid sequence of region. AKT. 20. The engineered molecule of any of paragraphs 15-19. 40. The engineered lectin of any of paragraphs 33-39, wherein the portion of the Fc region comprises at least one wherein the linker is adapted to provide flexibility and mutation. orientation of the carbohydrate recognition domain to 21. The engineered molecule of paragraph 20, wherein the bind to the microbe surface. at least one mutation is selected to increase half-life of the 41. The engineered lectin of any of paragraphs 33-40, engineered molecule. wherein the linker is adapted to facilitate expression and 22. The engineered molecule of any of paragraphs 20-21, 25 purification. wherein the mutation is selected to modulate antibody 42. The engineered lectin of any of paragraphs 33-41, dependent cell-mediated cytotoxicity. wherein the linker comprises a portion of a Fc region of 23. The engineered molecule of any of paragraphs 20-22, an immunoglobulin. wherein the mutation is selected to modulate comple 43. The engineered lectin of paragraph 42, wherein the ment-dependent cytotoxicity. 30 immunoglobulin is selected from the group consisting of 24. The engineered molecule of any of paragraphs 20-23, IgA, Ig|D, IgE. IgG, and IgM. wherein the mutation occurs at amino acid residue 82 of 44. The engineered lectin of paragraph 42 or 43, wherein the SEQ ID NO. 9 from asparagine to aspartic acid. immunoglobulin is IgG1. 25. The engineered molecule of any of paragraphs 15-24. 45. The engineered lectin of any of paragraphs 42-44. wherein N-terminus of the Fc region is adapted for linking 35 wherein the portion of the Fc region comprises at least one to the Substrate-binding domain. region selected from the group consisting of a hinge 26. The engineered molecule of any of paragraphs 1-25, region, a CH2 region, a CH3 region, and any combina wherein the linker is part of the carbohydrate-binding tions thereof. protein, the neck region, the Fc region, or any combina 46. The engineered lectin of any of paragraphs 42-45. tions thereof. 40 wherein the portion of the Fc region comprises at least one 27. The engineered molecule of any of paragraphs 1-26, hinge region, at least one CH2 region and at least one CH3 wherein the engineered molecule is a dimer. region. 28. The engineered molecule of paragraph 27, wherein the 47. The engineered lectin of any of paragraphs 42-46, dimer is formed by dimerizing the Fc region of two wherein the portion of the Fc region comprises at least one engineered molecules. 45 mutation. 29. The engineered molecule of any of paragraphs 1-28, 48. The engineered lectin of paragraph 47, wherein the further comprising a detectable label. mutation is selected to increase half-life of the engineered 30. The engineered molecule of paragraph 29, wherein the molecule. detectable label is selected from the group consisting of 49. The engineered lectin of paragraph 48, wherein the biotin, a fluorescent dye or particle, a luminescent or 50 mutation occurs at an amino acid residue 232 of SEQ ID bioluminescent marker, a radiolabel, an enzyme, a micro NO. 9 from lysine to alanine. bial enzyme substrate, a quantum dot, an imaging agent, 50. The engineered lectin of any of paragraphs 47-49, and any combinations thereof. wherein the mutation is selected to modulate antibody 31. The engineered molecule of paragraph 30, wherein the dependent cell-mediated cytotoxicity. enzyme causes a color change in the presence of an 55 51. The engineered lectin of any of paragraphs 47-50, enzyme Substrate. wherein the mutation is selected to modulate comple 32. The engineered molecule of paragraph 31, wherein the ment-dependent cytotoxicity. enzyme is a horseradish peroxidase or alkaline phos 52. The engineered lectin of any of paragraphs 47-51, phatase. wherein the mutation occurs at amino acid site 82 of SEQ 33. An engineered mannose-binding lectin molecule com 60 ID NO. 9 from asparagine to aspartic acid. prising: 53. The engineered lectin of any of paragraphs 47-52, a. at least one carbohydrate recognition domain (CRD) or wherein N-terminus of the Fc region is adapted for linking a fragment thereof; to the Substrate-binding domain. b. a Substrate-binding domain adapted for orienting the 54. The engineered lectin of any of paragraphs 47-53, CRD away from a substrate; and 65 wherein the linker is part of the mannose-binding lectin, c. at least one linker between the CRD and the substrate the neck region, the Fc region, or any combinations binding domain. thereof. US 9,593,160 B2 97 98 55. The engineered lectin of any of paragraphs 33-54, 77. The engineered molecule of any of paragraphs 61-76, wherein the engineered molecule is a dimer. wherein the portion of the Fc region comprises at least one 56. The engineered lectin of paragraph 55, wherein the region selected from the group consisting of a hinge dimer is formed by dimerizing the Fc region of two region, a CH2 region, a CH3 region, and any combina engineered lectin molecules. tions thereof. 57. The engineered lectin of any of paragraphs 33-56, further 78. The engineered molecule of any of paragraphs 61-77, comprising a detectable label. wherein the portion of the Fc region comprises at least one 58. The engineered lectin of paragraph 57, wherein the hinge region, at least one CH2 region and at least one CH3 detectable label or imaging agent is selected from the region. group consisting of biotin, a fluorescent dye or particle, a 10 79. The engineered molecule of any of paragraphs 61-78, luminescent or bioluminescent marker, a radiolabel, an wherein the portion of the Fc region comprises at least one enzyme, a microbial enzyme Substrate, a quantum dot, an mutation. imaging agent, and any combinations thereof. 80. The engineered molecule of paragraph 79, wherein the 59. The engineered lectin of paragraph 58, wherein the at least one mutation is selected to increase half-life of the enzyme causes a color change in the presence of an 15 engineered microbe-binding molecule. enzyme Substrate. 81. The engineered molecule of any of paragraphs 61-80, 60. The engineered lectin of paragraph 59, wherein the wherein the mutation is selected to modulate antibody enzyme is a horseradish peroxidase or alkaline phos dependent cell-mediated cytotoxicity. phatase. 82. The engineered molecule of any of paragraphs 61-81, 61. An engineered microbe-targeting molecule comprising: wherein the mutation is selected to modulate comple a. at least one microbe Surface-binding domain; and ment-dependent cytotoxicity. b. at least a portion of a Fc region of an immunoglobulin. 83. The engineered molecule of any of paragraphs 61-82, 62. The engineered molecule of paragraph 61, wherein the wherein the mutation occurs at amino acid residue 82 of portion of the Fc region is linked to N-terminal of the 25 SEQ ID NO. 9 from asparagine to aspartic acid. microbe Surface-binding domain. 84. The engineered molecule of any of paragraphs 61-83, 63. The engineered molecule of paragraph 61 or 62, wherein wherein the engineered molecule is a dimer. the microbe Surface-binding domain comprises a carbo 85. The engineered molecule of paragraph 84, wherein the hydrate recognition domain (CRD) or a fragment thereof. dimer is formed by dimerizing the Fc region of two 64. The engineered molecule of paragraph 63, wherein the 30 engineered molecules. CRD or a fragment thereof further comprises at least a 86. The engineered molecule of any of paragraphs 61-85, portion of a carbohydrate-binding protein. further comprising a detectable label. 65. The engineered molecule of paragraph 64, wherein the 87. The engineered molecule of paragraph 86, wherein the portion of the carbohydrate-binding protein excludes at detectable label is selected from the group consisting of least one of complement and coagulation activation 35 biotin, a fluorescent dye or particle, a luminescent or region. bioluminescent marker, a radiolabel, an enzyme, a micro 66. The engineered molecule of any of paragraphs 63-65, bial enzyme substrate, a quantum dot, an imaging agent, wherein the CRD or the carbohydrate-binding protein is and any combinations thereof. derived from a lectin, a ficolin, or a fragment thereof. 88. The engineered molecule of paragraph 87, wherein the 67. The engineered molecule of paragraph 66, wherein the 40 enzyme causes a color change in the presence of an lectin is C-type lectin, or a fragment thereof. enzyme Substrate. 68. The engineered molecule of paragraph 67, wherein the 89. The engineered molecule of paragraph 88, wherein the C-type lectin is collectin, or a fragment thereof. enzyme is a horseradish peroxidase or alkaline phos 69. The engineered molecule of paragraph 68, wherein the phatase. collectin is mannose-binding lectin (MBL) or a fragment 45 90. A microbe-targeting Substrate or a product comprising a thereof. Substrate, and at least one engineered microbe-targeting 70. The engineered molecule of any of paragraphs 63-69, molecule of any of paragraphs 1-32 and 61-89 or at least wherein the CRD is of SEQ ID NO. 4 or a fragment one engineered mannose-binding lectin molecule of any thereof. of paragraphs 33-60, wherein the substrate comprises on 71. The engineered molecule of any of paragraphs 63-70, 50 its Surface said at least one engineered microbe-targeting wherein the CRD or a fragment thereof further comprises molecule or at least one engineered mannose-binding a neck region of a carbohydrate-binding protein. lectin molecule. 72. The engineered molecule of any of paragraphs 61-71, 91. The microbe-targeting substrate or the product of para wherein said at least a portion of the Fc region of the graph 90, wherein the substrate-binding domain of the immunoglobulin further comprises a Substrate-binding 55 engineered microbe-targeting molecule or mannose-bind domain. ing lectin molecule is adapted for binding to the Substrate. 73. The engineered molecule of paragraph 72, wherein the 92. The microbe-targeting substrate or the product of para Substrate-binding domain comprises at least one amine. graph 90 or 91, wherein the substrate is selected from the 74. The engineered molecule of any of paragraphs 61-73, group consisting of a nucleic acid scaffold, a protein wherein the Substrate-binding domain comprises at least 60 scaffold, a lipid scaffold, a dendrimer, microparticle or a one oligopeptide comprising an amino acid sequence of microbead, a nanotube, a microtiter plate, a medical AKT. apparatus or implant, a microchip, a filtration device, a 75. The engineered molecule of any of paragraphs 61-74, membrane, a diagnostic strip, a dipstick, an extracorpo wherein the immunoglobulin is selected from the group real device, a spiral mixer, and a hollow-fiber reactor. consisting of IgA, Ig), IgE. IgG, and IgM. 65 93. The microbe-targeting substrate or the product of any of 76. The engineered molecule of any of paragraphs 61-75, paragraphs 90-92, wherein the substrate is a micropar wherein the immunoglobulin is IgG1. ticle. US 9,593,160 B2 99 100 94. The microbe-targeting Substrate or the product of para 111. The kit of any of paragraphs 105-108, wherein the one graph 93, wherein the microparticle is a magnetic or more microbe-targeting Substrates include dipsticks. microparticle. 112. The kit of any of paragraphs 105-109, wherein the one 95. The microbe-targeting substrate or the product of para or more microbe-targeting Substrates include a population graph 93, wherein the microparticle is a fluorescent 5 of microbe-targeting microbeads. microparticle or a quantum dot. 113. The kit of paragraph 108, 109 or 112, wherein the 96. The microbe-targeting substrate or the product of para population of microbes or microbe-targeting microbeads graph 93, wherein the microparticle is a drug delivery is provided in one or more separate containers. vehicle. 114. The kit of any of paragraphs 108, 109, and 112-113, 97. The microbe-targeting substrate or the product of any of 10 wherein the population of the microbeads or microbe paragraphs 90-96, wherein the substrate is a dipstick. targeting microbeads comprises at least one distinct Sub 98. The microbe-targeting substrate or the product of any of set, the distinct Subset comprising microbeads or microbe paragraphs 90-96, wherein the substrate is a membrane. targeting microbeads having a dimension different from 99. The microbe-targeting substrate or the product of para 15 the rest of the population. graph 97 or 98, wherein the dipstick or the membrane 115. The kit of any of paragraphs 110-114, wherein the comprises on its Surface at least an area adapted for use as microbe-targeting microbeads each further comprises a a reference area. detection label. 100. The microbe-targeting substrate or the product of any 116. The kit of any of paragraphs 105-115, further compris of paragraphs 90-99, wherein the substrate is a living cell, ing one or more containers each containing a population or a biological tissue or organ. of detectable labels, wherein each of the detectable label 101. The microbe-targeting substrate or the product of any is conjugated to a molecule. of paragraphs 90-100, wherein the substrate is function 117. The kit of paragraph 116, wherein at least one of the alized. containers contains a distinct population of detectable 102. The microbe-targeting substrate or the product of any 25 labels. of paragraphs 90-101, wherein the substrate is treated to 118. The kit of any of paragraphs 116-117, wherein the become less adhesive to a biological molecule. molecule is an engineered microbe-targeting molecule of 103. The microbe-targeting substrate or the product of any of paragraphs 1-32 and 61-89 or an engineered paragraph 102, wherein the biological molecule is mannose-binding lectin molecules of any of paragraphs Selected from the group consisting of blood cells and 30 33-6O. components, proteins, nucleic acids, peptides, Small mol 119. The kit of paragraph 118, wherein the molecule com ecules, therapeutic agents, cells or fragments thereof, and prises at least a carbohydrate recognition domain (CRD) any combinations thereof. or a fragment thereof. 104. A pharmaceutical composition comprising at least one 120. The kit of paragraph 119, wherein at least one popu engineered microbe-targeting molecule of any of para 35 lation of the molecule comprises SEQ ID NO. 4 or a graphs 1-32 and 61-89 or at least one engineered man fragment thereof. nose-binding lectin molecule of any of paragraphs 33-60 121. The kit of any of paragraphs 116-120, wherein the or at least one microbe-targeting Substrate of any of molecule further comprises a Fc region of an immuno paragraphs 90-103, and a pharmaceutically acceptable globulin. carrier. 40 122. The kit of any of paragraphs 116-120, wherein the 105. A kit comprising: molecule includes an antibody specific to the microbe. a. one or more containers containing a population of 123. The kit of any of paragraphs 116-122, wherein the engineered microbe-targeting molecules of any of para detectable label comprises an enzyme that produces a graphs 1-32 and 61-89 or a population of engineered color change in the presence of an enzyme substrate. mannose-binding lectin molecules of any of paragraphs 45 124. The kit of paragraph 123, wherein the enzyme is a 33-60; and horseradish peroxidase, an alkaline phosphatase, or any b. at least one reagent. combinations thereof. 106. The kit of paragraph 105, further comprising one or 125. The kit of any of paragraphs 105-124, further compris more Substrates to which the engineered microbe-target ing one or more containers containing an enzyme Sub ing molecules or engineered mannose-binding lectin mol 50 strate that changes color in the presence of the enzyme. ecules are conjugated. 126. The kit of any of paragraphs 116-125, wherein the 107. The kit of paragraph 105 or 106, wherein the substrates detectable label comprises a fluorescent molecule. are selected from the group consisting of a nucleic acid 127. The kit of any of paragraphs 105-126, wherein the at scaffold, a protein scaffold, a lipid scaffold, a dendrimer, least one reagent is a wash buffer, a dilution buffer, a stop microparticle or a microbead, a nanotube, a microtiter 55 buffer, a buffered solution containing a chelating agent, a plate, a medical apparatus or implant, a microchip, a coupling agent used for conjugation of the engineered filtration device, a membrane, a diagnostic strip, a dip molecule to the Substrate, or any combinations thereof. Stick, an extracorporeal device, a spiral mixer, and a 128. The kit of any of paragraphs 105-127, further compris hollow-fiber reactor. ing at least one microtiter plate. 108. The kit of any of paragraphs 105-107, wherein the 60 129. The kit of any of paragraphs 108-128, wherein the Substrates include a population of the microbeads. population of microbeads or microbe-targeting micro 109. The kit of paragraph 108, wherein the microbeads are beads is lyophilized. magnetic microbeads. 130. The kit of any of paragraphs 105-129, further compris 110. A kit comprising: ing at least one blood collection container. a. one or more microbe-targeting Substrates of any of 65 131. The kit of paragraph 130, wherein the population of the paragraphs 90-104; and microbe-targeting microbeads is pre-loaded into said at b. at least one reagent. least one blood collection container. US 9,593,160 B2 101 102 132. The kit of paragraph 130 or 131, wherein the blood 153. The method of any of paragraphs 137-152, further collection container further comprises an anti-coagulant comprising washing the Substrate with after detachment agent. of the microbe. 133. The kit of any of paragraphs 112-132, wherein the 154. The method of any of paragraphs 137-153, wherein the engineered microbe-targeting microbeads are microbe microbe-targeting molecule binds to a substrate. targeting magnetic microbeads. 155. A composition for treating and/or preventing a micro 134. The kit of paragraph 133, further comprising a magnet bial infection or a microbial contamination comprising at adapted for collecting the microbe-targeting magnetic least one engineered microbe-targeting molecule of any of microbeads in the blood collection container. paragraphs 1-32 or 61-89 or at least one engineered 135. The kit of any of paragraphs 105-134, further compris 10 mannose-binding lectin molecule of any of paragraphs ing a reference for comparison with a readout determined 33-60 or at least one microbe-targeting substrate of any of from a test sample. paragraphs 90-103. 136. The kit of any of paragraphs 110-135, wherein one or 156. The composition of paragraph 155, wherein the com more microbe-targeting Substrates are individually pack position is formulated for treating and/or preventing a aged. 15 microbial infection or a microbial contamination present 137. A method of detaching a microbe and/or microbial in an environment Surface. matter from a microbe-targeting molecule, the method 157. The composition of paragraph 156, wherein the envi comprising incubating the Substrate with buffer having an ronmental Surface includes a medical device, an implant acidic pH. able device, a surface in a hospital or clinic (e.g., an 138. The method of paragraph 137, wherein the buffer has operating room or an intensive-care unit), a machine or a pH about 6.5 or lower. working Surface for manufacturing or processing food or 139. The method of paragraph 137 or 138, wherein the pharmaceutical products, a cell culture, a water treatment buffer comprises 0.2M glycine and has a pH of about 2.8. plant, a water reservoir or a botanical plant. 140. A method of detaching a microbe and/or microbial 158. The composition of any of paragraphs 155-157, matter from a microbe-targeting molecule, the method 25 wherein the composition is formulated for treating and/or comprising incubating the Substrate with a buffer com preventing a microbial infection in a body fluid of a prising an ion which forms a salt with Ca2+ ion and Subject. wherein the said salt is insoluble in the buffer. 159. The composition of any of paragraphs of 155-158, 141. The method of paragraph 140, wherein said ion is wherein the composition is formulated for treating and/or Selected from the group consisting of phosphate, oxalate, 30 preventing a microbial infection in a tissue of a Subject. carbonate, Sulfate, fluoride, gluconic acid, oxido-trioxo 160. The composition of paragraph 158 or 159, wherein the manganese, Stearic acid, and any combinations thereof. subject is a mammalian subject. 142. The method of paragraph 140 or 141, wherein said ion 161. The composition of any of paragraphs 155-160, is present at a concentration of about 0.05M to about 5M. wherein said at least one engineered microbe-targeting 143. The method of any of paragraphs 140-142, wherein the 35 molecule is present in an amount effective to reduce the buffer comprises about 0.1M sodium phosphate and has growth and/or spread of the microbe. pH of about 6.8. 162. The composition of any of paragraphs 155-161, further 144. The method of any of paragraphs 140-143, wherein the comprising at least one of an antimicrobial agent and a interaction between the microbe and the microbe-target drug delivery vehicle. ing molecule is mediated by a Ca2+ ion. 40 163. The composition of paragraph 162, wherein at least one 145. The method of any of paragraphs 140-144, wherein the of the engineered microbe-targeting molecule and the aqueous solution further comprises a chelating agent. antimicrobial agent is coated on a Surface of the drug 146. The method of paragraph 145, wherein the chelating delivery vehicle. agent is selected from the group consisting of 1.2-bis(2- 164. The composition of paragraph 162 or 163, wherein the Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; ethyl 45 drug delivery vehicle is selected from the group consist enediaminetetraacetic acid (EDTA); ethylene glycol-bis ing of a peptide particle, a polymeric particle, a den (2-aminoethylether)-N.N.N',N'-tetraacetic acid (EGTA); drimer, a vesicle, a liposome, a hydrogel, a nucleic acid ethylene glycol-bis(B-aminoethyl ether)-N.N.N',N'-tet scaffold, an aptamer, and any combinations thereof, raacetic acid, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'- 165. The composition of any of paragraphs 162-164, tetraacetic acid (BAPTA), nitrile-2.2".2"-triacetic acid 50 wherein the antimicrobial agent is fused with said at least (NTA), and any combinations thereof. one engineered microbe-targeting molecule. 147. The method of any of paragraphs 137-146, wherein the 166. The composition of any of paragraphs 162-165, Substrate is microparticle. wherein the antimicrobial agent is selected from the group 148. The method of any of paragraphs 137-147, wherein the consisting of silver nanoparticle, an antimicrobial metal Substrate is a magnetic microparticle. 55 loendopeptidase, an antimicrobial peptide, an antibiotic, 149. The method of any of paragraphs 137-148, wherein the and any combinations thereof. microbe-targeting molecule is an engineered microbe 167. The composition of any of paragraphs 155-166, targeting molecule of any of paragraphs 1-32 or 61-89, or wherein a microbe causing the microbial infection or an engineered mannose-binding ligand of any of para microbial contamination is a protein A-expressing graphs 33-60. 60 microbe, a protein G-expressing microbe or any combi 150. The method of any of paragraphs 137-149, further nations thereof. comprising heating or cooling the buffer during said 168. The composition of paragraph 167, wherein the protein contacting. A-expressing microbe includes Staphylococcus or the 151. The method of any of paragraphs 137-150, further protein G-expressing microbe includes Streptococcus. comprising shaking the Substrate in the buffer. 65 169. The composition of paragraph 167 or 168, wherein the 152. The method of any of paragraphs 137-151, wherein said protein A-expressing microbe includes Staphylococcus incubation is for at least 5 minutes. CaS US 9,593,160 B2 103 104 170. The composition of any of paragraphs 167-169, comprises a first Subset of microbe-targeting Substrates wherein the microbe is resistant to at least one antimi and a second Subset of microbe-targeting Substrates; crobial agent. and 171. The composition of paragraph 170, wherein the anti wherein the first subset of microbe-targeting substrates microbial agent is an antibiotic. 5 each has a first pre-determined dimension; and 172. The composition of paragraph 171, wherein the anti wherein the second Subset of microbe-targeting Substrates biotic is selected from the group consisting of aminogly each has a second pre-determined dimension. cosides, ansamycins, carbacephem, carbapenems, cepha 188. The assay of paragraph 187, wherein the first subset and losporins, glycopeptides, lincosamides, lipopeptide, the second subset are added to the test sample to form a 10 single mixture. macrollides, monobactams, nitrofurans, penicillins, poly 189. The assay of paragraph 187, wherein the second subset peptides, quinolones, Sulfonamides, tetracyclines, methi is added to the test sample after isolation of the first subset cillin, Vancomycin, and any combinations thereof. previously added to the test sample. 173. The composition of any of paragraphs 167-172, 190. The assay of any of paragraph 186-189, wherein the wherein the protein A-expressing microbe includes methi 15 microbe-targeting Substrate is in a form of a microbead. cillin-resistant Staphylococcus aureus. 191. The assay of paragraph 190, wherein the first pre 174. The composition of any of paragraphs 167-173, determined dimension and the second pre-determined wherein the protein A-expressing microbe includes van dimension of the microbead range from about 10 nm to comycin-resistant Staphylococcus aureus. about 10 um. 175. The composition of any of paragraphs 155-174. 192. The assay of paragraph 190, wherein the first pre wherein the composition is adapted for use as a wound determined dimension and the second pre-determined dressing. dimension of the microbead range from about 50 nm to 176. The composition of any of paragraphs 155-175, about 200 nm. wherein the immunoglobulin is a human immunoglobu 193. The assay of any of paragraphs 190-192, wherein the lin. 25 microbead is a magnetic microbead. 177. A method for removing a microbe and/or microbial 194. The assay of any of paragraphs 186-193, further matter from a target area comprising contacting the target comprising analyzing the microbe-targeting Substrate for area with a composition of any of paragraphs 155-176. the presence or absence of a bound microbe and/or 178. A method for treating and/or preventing a microbial microbial matter, wherein the presence of a microbe infection or microbial contamination in a target area 30 targeting Substrate-bound microbe and/or microbial mat comprising contacting the target area with a first compo ter indicates that the test sample is infected with a sition of any of paragraphs 155-176. microbe; and the absence of a microbe-targeting sub 179. The method of paragraph 177 or 178, wherein the target strate-bound microbe and/or microbial matter indicates area includes an environmental Surface. the test sample contains no detectable microbes or micro 35 bial matter. 180. The method of paragraph 179, wherein the environ 195. The assay of any of paragraphs 186-194, wherein the mental Surface includes a medical device, an implantable microbial matter includes endotoxin. device, a Surface in a hospital or clinic (e.g., an operating 196. An assay for determining the presence or absence of a room or an intensive-care unit), a machine or working protein-A expressing microbe, a protein-G expressing Surface for manufacturing or processing food or pharma 40 microbe, or microbial matter thereof, in a test sample, the ceutical products, a cell culture, a water treatment plant, assay comprising: a water reservoir or a botanical plant. contacting a test sample with a microbe-targeting Sub 181. The method of paragraph 177 or 178, wherein the target strate of any of paragraphs 90 to 103 in the presence of area is present in a body fluid of a Subject. a chelating agent. 182. The method of paragraph 177 or 178, wherein the target 45 197. The assay of paragraph 196, further comprising ana area is present in a tissue of a subject. lyzing the microbe-targeting Substrate for the presence or 183. The method of paragraph 182, further comprising absence of a bound microbe, wherein the presence of a replacing the first composition in contact with the tissue microbe-targeting Substrate-bound microbe indicates the with a second composition of any of paragraphs 157-178 presence of a protein-A expressing microbe or a protein after a period of time. 50 G-expressing microbe in the test sample; and the absence 184. The method of paragraph 182 or 183, further compris of a microbe-targeting Substrate-bound microbe indicates ing administering an additional treatment to the tissue. the absence of a protein-A expressing or a protein G-ex 185. The method of paragraph 184, wherein the additional pressing microbe in the test sample. treatment includes a negative-pressure treatment, a 198. The assay of paragraph 197, wherein in the absence of vacuum-assisted debridement, administration of an anti 55 a microbe-targeting Substrate-bound microbe, the test microbial agent, or any combinations thereof. sample is further contacted with the microbe-targeting 186. An assay for determining the presence or absence of a Substrate in the presence of free calcium ions. microbe and/or microbial matter in a test sample, the 199. An assay for detecting a protein-A expressing microbe, assay comprising: a protein-G expressing microbe, or microbial matter contacting a test sample with a microbe-targeting Sub 60 thereof, in a test sample, the assay comprising: strate of any of paragraphs 90-103. i. contacting a test sample with a microbe-targeting Sub 187. An assay of determining the presence or absence of a strate of any of paragraphs 90 to 103; microbe and/or microbial matter in a test sample, the ii. contacting the microbe-binding molecule with a solu method comprising: tion comprising a chelating agent; and contacting a test sample with a plurality of microbe 65 iii. analyzing the microbe-targeting Substrate for the pres targeting Substrates of any of paragraphs 90-103. ence or absence of a bound microbe, wherein the wherein the plurality of microbe-targeting Substrates presence of a microbe-targeting Substrate-bound US 9,593,160 B2 105 106 microbe indicates the presence of a protein A-express 215. The assay of any of paragraphs 196-214, wherein the ing microbe or a protein G-expressing microbe in the protein A-expressing microbe includes Staphylococcus, test sample; and the absence of a microbe-targeting or the protein G-expressing microbe includes Streptococ Substrate-bound microbe indicates the absence of a C.S. protein A-expressing microbe or a protein G-express 5 216. The assay of paragraph 215, wherein the protein ing microbe in the test sample. A-expressing microbe includes Staphylococcus aureus. 200. The assay of paragraph 199, further comprising isolat 217. The assay of paragraph 215, wherein the Staphylococ ing the microbe-targeting Substrate from the test sample cus species excludes Staphylococcus epidermidis. before contacting with the Solution comprising the chelat 218. The assay of any of paragraphs 186-217, further ing agent. 10 comprising analyzing at least one microbe-targeting Sub 201. The assay of any of paragraphs 186-200, further strate upon contact with the test sample before contacting comprising isolating the microbe-targeting Substrate from the microbe-binding molecule with the solution compris the test sample or the Solution comprising the chelating ing the chelating agent. agent before the analyzing step. 219. The assay of paragraph 186-218, wherein the microbe 202. The assay of paragraph 201, wherein the analyzing 15 targeting Substrate is in a form of a microbead. comprises an immunoassay, ELISA, Gram staining, 220. The assay of paragraph 219, wherein the microbead is immunostaining, microscopy, spectroscopy, immunofluo a magnetic microbead. rescence, western blot, PCR, RT-PCR, fluorescence in situ 221. A method of determining the presence or absence of hybridization, sequencing, mass spectroscopy, and any Staphylococcus aureus infection in a Subject, comprising combinations thereof. performing the assay of any of paragraphs 190-214. 203. The assay of any of paragraphs 186-202, further wherein the binding of a microbe to said at least one comprising culturing the microbe bound on the microbe engineered microbe-targeting Substrate in the presence of targeting Substrate. a chelating agent is indicative of Staphylococcus aureus 204. The assay of any of paragraphs 186-203, further infection in the subject. comprising Subjecting the microbe bound on the microbe 25 222. The method of paragraph 221, further comprising targeting Substrate to an antibiotic. administering or prescribing to the Subject a first antimi 205. The assay of any of paragraphs 186-204, wherein the crobial agent when the subject is detected with Staphy microbe-targeting Substrate is preformed from at least a lococcus aureus. Substrate and said at least one engineered microbe-bind 223. The method of paragraph 221 or 222, further compris ing molecule before the contacting. 30 ing analyzing the test sample or the Solution comprising 206. The assay of any of paragraphs 186-204, wherein the the chelating agent after isolating the engineered microbe microbe-targeting substrate is formed from at least said targeting substrate therefrom to determine the presence or Substrate and said at least one engineered microbe-bind absence of a protein A-negative or a protein G-negative ing molecule during the contacting. microbe. 207. The assay of any of paragraphs 196-206, wherein the 35 224. The method of paragraph 223, further comprising presence of the chelating agent reduces the likelihood of administering or prescribing to the Subject a second a protein A- and protein G-negative microbe, if present, in antimicrobial agent when the subject is detected with a the test sample, to bind with said at least one engineered protein A-negative or a protein G-negative microbe. microbe-binding molecule. 225. The method of paragraph 224, wherein the protein 208. The assay of any of paragraphs 186-207, further 40 A-negative or the protein G-negative microbe include E. comprising detaching the bound microbe from the coli. microbe-targeting Substrate. 226. The method of any of paragraphs 221-225, further 209. The assay of paragraph 208, further comprising con comprising administering or prescribing to the Subject a tacting the isolated microbe-targeting Substrate with a low composition comprising at least one engineered microbe pH buffer. 45 targeting molecule of any of paragraphs 1-32 or 61-89, or 210. The assay of any of paragraphs 196-209, wherein the at least one engineered mannose-binding lectin molecule chelating agent is a metal-ion chelating agent. of any of paragraphs 33-60. 211. The assay of any of paragraphs 196-210, wherein the chelating agent chelates a calcium ion. SOME SELECTED DEFINITIONS 212. The assay of paragraph 211, wherein the calcium 50 chelating agent is selected from the group consisting of Unless stated otherwise, or implicit from context, the 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic following terms and phrases include the meanings provided acid, ethylenediaminetetraacetic acid (EDTA); ethylene below. Unless explicitly stated otherwise, or apparent from glycol-bis(2-aminoethylether)-N.N.N',N'-tetraacetic acid; context, the terms and phrases below do not exclude the ethylene glycol-bis(D-aminoethyl ether)-N.N.N2.N2-tet 55 meaning that the term or phrase has acquired in the art to raacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N, which it pertains. The definitions are provided to aid in N,N',N'-tetraacetic acid (BAPTA), a buffer containing describing particular embodiments of the aspects described citrate, N,N-Bis(2-(bis-(carboxymethyl)amino)ethyl)- herein, and are not intended to limit the claimed invention, glycine (DTPA), nitrilo-2,2'2"-triacetic acid (NTA), a because the scope of the invention is limited only by the buffer that precipitates a calcium ion from the test sample, 60 claims. Further, unless otherwise required by context, sin a low pH buffer, any derivatives thereof, and any com gular terms shall include pluralities and plural terms shall binations thereof. include the singular. 213. The assay of any of paragraphs 209-212, wherein the As used herein the term “comprising or “comprises” is low pH buffer has a pH less than 7. used in reference to compositions, methods, and respective 214. The assay of any of paragraphs 209-213, wherein the 65 component(s) thereof, that are essential to the invention, yet low pH buffer is selected from the group consisting of open to the inclusion of unspecified elements, whether arginine and pyrophosphate. essential or not. US 9,593,160 B2 107 108 As used herein the term “consisting essentially of refers feline species, e.g., domestic cat, canine species, e.g., dog. to those elements required for a given embodiment. The fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, term permits the presence of additional elements that do not e.g., trout, catfish and salmon. Patient or subject includes materially affect the basic and novel or functional charac any Subset of the foregoing, e.g., all of the above, but teristic(s) of that embodiment of the invention. excluding one or more groups or species such as humans, The term “consisting of refers to compositions, methods, primates or rodents. In certain embodiments of the aspects and respective components thereof as described herein, described herein, the Subject is a mammal, e.g., a primate, which are exclusive of any element not recited in that e.g., a human. The terms, “patient' and “subject” are used description of the embodiment. interchangeably herein. Other than in the operating examples, or where otherwise 10 In some embodiments, the Subject is a mammal. The indicated, all numbers expressing quantities of ingredients mammal can be a human, non-human primate, mouse, rat, or reaction conditions used herein should be understood as dog, cat, horse, or cow, but are not limited to these examples. modified in all instances by the term “about.” The term Mammals other than humans can be advantageously used as "about when used in connection with percentages may Subjects that represent animal models of disorders. meant 1%. 15 A Subject can be one who has been previously diagnosed The singular terms “a,” “an,” and “the include plural with or identified as Suffering from or having a disease or referents unless context clearly indicates otherwise. Simi disorder caused by any microbes or pathogens described larly, the word 'or' is intended to include “and” unless the herein. By way of example only, a subject can be diagnosed context clearly indicates otherwise. Thus for example, ref with sepsis, inflammatory diseases, or infections. erences to “the method’ includes one or more methods, The term “therapeutic agents' is art-recognized and refers and/or steps of the type described herein and/or which will to any chemical moiety that is a biologically, physiologi become apparent to those persons skilled in the art upon cally, or pharmacologically active Substance that acts locally reading this disclosure and so forth. or systemically in a subject. Examples of therapeutic agents, Although methods and materials similar or equivalent to also referred to as “drugs’, are described in well-known those described herein can be used in the practice or testing 25 literature references such as the Merck Index, the Physicians of this disclosure, Suitable methods and materials are Desk Reference, and The Pharmacological Basis of Thera described below. The term “comprises' means “includes.” peutics, and they include, without limitation, medicaments; The abbreviation, "e.g. is derived from the Latin exempli Vitamins; mineral Supplements; Substances used for the gratia, and is used herein to indicate a non-limiting example. treatment, prevention, diagnosis, cure or mitigation of a Thus, the abbreviation “e.g. is synonymous with the term 30 disease or illness; substances which affect the structure or “for example.” function of the body; or pro-drugs, which become biologi The terms “microbe-binding” and “microbe-targeting as cally active or more active after they have been placed in a used interchangeably herein refers to an ability of a mol physiological environment. Various forms of a therapeutic ecule or composition to bind and/or capture a microbe agent may be used which are capable of being released from and/or microbial matter. 35 the Subject composition into adjacent tissues or fluids upon The term "FcMBL microbead' as used herein refers to a administration to a Subject. Examples include steroids and microbead comprising on its surface at least one FcMBL esters of steroids (e.g., estrogen, progesterone, testosterone, molecule. In some embodiments, the microbead comprises androsterone, cholesterol, norethindrone, digoxigenin, on its surface a saturating amount of the FcMBL molecules. cholic acid, deoxycholic acid, and chenodeoxycholic acid), A microbead can be magnetic or non-magnetic. 40 boron-containing compounds (e.g., carborane), chemothera The term “FcMBL magnetic microbead' as used herein peutic nucleotides, drugs (e.g., antibiotics, antivirals, anti refers to a magnetic microbead comprising on its Surface at fungals), enediynes (e.g., calicheamicins, esperamicins, least one FcMBL molecule. In some embodiments, the dynemicin, neocarzinostatin chromophore, and kedarcidin magnetic microbead comprises on its Surface a Saturating chromophore), heavy metal complexes (e.g., cisplatin), hor amount of the FcMBL molecules. 45 mone antagonists (e.g., tamoxifen), non-specific (non-anti The term “antibody” as used herein refers to immuno body) proteins (e.g., Sugar oligomers), oligonucleotides globulin molecules and immunologically active portions of (e.g., antisense oligonucleotides that bind to a target nucleic immunoglobulin molecules (molecules that contain an anti acid sequence (e.g., mRNA sequence)), peptides, proteins, gen binding site which specifically binds an antigen), includ antibodies, photodynamic agents (e.g., rhodamine 123), ing monoclonal antibodies (including full length monoclo 50 radionuclides (e.g., I-131, Re-186, Re-188, Y-90, Bi-212, nal antibodies), polyclonal antibodies, multispecific At-211, Sr-89, Ho-166, Sm-153, Cu-67 and Cu-64), toxins antibodies (for example, bispecific antibodies), chimeric (e.g., ricin), and transcription-based pharmaceuticals. antibodies, humanized antibodies, human antibodies, and As used here in, the term "peptidomimetic' means a single chain antibodies (scFVs). peptide-like molecule that has the activity of the peptide on The term "peptide' refers to a polymer of amino acids, or 55 which it is structurally based. Such peptidomimetics include amino acid analogs, regardless of its size or function. In chemically modified peptides, peptide-like molecules con some embodiments, the term "peptide' refers to small taining non-naturally occurring amino acids, and peptoids, polypeptides, e.g., a polymer of about 15-25 amino acids. and have an activity Such as the cardiac specificity of the The term "oligonucleotide' as used herein refers to a short peptide upon which the peptidomimetic is derived (see, for nucleic acid polymer, typically with twenty or fewer bases. 60 example, Goodman and Ro, Peptidomimetics for Drug As used herein, a “subject’ means a human or animal. Design, in “Burger's Medicinal Chemistry and Drug Dis Usually the animal is a vertebrate Such as a primate, rodent, covery”, Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), domestic animal or game animal. Primates include chim pages 803-861). panzees, cynomologous monkeys, spider monkeys, and A variety of peptidomimetics are known in the art and can macaques, e.g., Rhesus. Rodents include mice, rats, wood 65 be encompassed within embodiments described herein chucks, ferrets, rabbits and hamsters. Domestic and game including, for example, peptide-like molecules which con animals include cows, horses, pigs, deer, bison, buffalo, tain a constrained amino acid, a non-peptide component that US 9,593,160 B2 109 110 mimics peptide secondary structure, or an amide bond different amino acid residue that has similar chemical prop isostere. A peptidomimetic that contains a constrained, non erties. Conservative amino acid Substitutions include naturally occurring amino acid can include, for example, an replacement of a leucine with an isoleucine or valine, an C.-methylated amino acid; O.C.-dialkylglycine or CL-amino aspartate with a glutamate, or a threonine with a serine. cycloalkane carboxylic acid; an NC-COcyclized amino acid; “Conservative amino acid substitutions' result from replac an NC.-methylated amino acid; C.B- or Y-amino cycloalkane ing one amino acid with another having similar structural carboxylic acid; an O.f3-unsaturated amino acid; a B.f3 and/or chemical properties, such as the replacement of a dimethyl or B-methyl amino acid; CfB-substituted-2,3- leucine with an isoleucine or valine, an aspartate with a methano amino acid; an N Cö or CC-Cöcyclized amino glutamate, or a threonine with a serine. Thus, a “conserva acid; a Substituted proline or another amino acid mimetic. A 10 tive Substitution of a particular amino acid sequence refers peptidomimetic which mimics peptide secondary structure to substitution of those amino acids that are not critical for can contain, for example, a nonpeptidic B-turn mimic; Y-turn polypeptide activity or Substitution of amino acids with mimic; mimic of B-sheet structure; or mimic of helical other amino acids having similar properties (e.g., acidic, structure, each of which is well known in the art. A pep basic, positively or negatively charged, polar or non-polar, tidomimetic also can be a peptide-like molecule which 15 etc.) Such that the Substitution of even critical amino acids contains, for example, an amide bond isostere such as a does not substantially alter activity. Conservative substitu retro-inverso modification; reduced amide bond; methyl tion tables providing functionally similar amino acids are enethioether or methylene-sulfoxide bond; methylene ether well known in the art. For example, the following six groups bond; ethylene bond; thioamide bond; transolefin or fluo each contain amino acids that are conservative Substitutions roolefin bond; 1,5-disubstituted tetrazole ring; ketomethyl for one another: 1) Alanine (A), Serine (S), Threonine (T): ene or fluoroketomethylene bond or another amide isostere. 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), One skilled in the art understands that these and other Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine peptidomimetics are encompassed within the meaning of the (I), Leucine (L), Methionine (M), Valine (V); and 6) Phe term "peptidomimetic' as used herein. nylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Methods for identifying a peptidomimetic are well known 25 Creighton, Proteins, W. H. Freeman and Company (1984).) in the art and include, for example, the screening of data In addition, individual substitutions, deletions or additions bases that contain libraries of potential peptidomimetics. For that alter, add or delete a single amino acid or a small example, the Cambridge Structural Database contains a percentage of amino acids in an encoded sequence are also collection of greater than 300,000 compounds that have “conservative substitutions.” Insertions or deletions are typi known crystal structures (Allen et al., Acta Crystallogr. 30 cally in the range of about 1 to 5 amino acids. Section B, 35:2331 (1979)). This structural depository is Although preferred embodiments have been depicted and continually updated as new crystal structures are determined described in detail herein, it will be apparent to those skilled and can be screened for compounds having Suitable shapes, in the relevant art that various modifications, additions, for example, the same shape as a peptide described herein, Substitutions, and the like can be made without departing as well as potential geometrical and chemical complemen 35 from the spirit of the invention and these are therefore tarity to a cognate receptor. Where no crystal structure of a considered to be within the scope of the invention as defined peptide described herein is available, a structure can be in the claims which follow. Further, to the extent not already generated using, for example, the program CONCORD indicated, it will be understood by those of ordinary skill in (Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). the art that any one of the various embodiments herein Another database, the Available Chemicals Directory (Mo 40 described and illustrated may be further modified to incor lecular Design Limited, Informations Systems; San Leandro porate features shown in any of the other embodiments Calif.), contains about 100,000 compounds that are com disclosed herein. mercially available and also can be searched to identify potential peptidomimetics of a peptide described herein, for EXAMPLES example, having specificity for the microbes. 45 The terms “homology” as used herein refers to sequence The following examples illustrate Some embodiments and similarity between two peptides or between two nucleic acid aspects of the invention. It will be apparent to those skilled molecules. Homology can be determined by comparing a in the relevant art that various modifications, additions, position in each sequence which may be aligned for pur substitutions, and the like can be performed without altering poses of comparison. When an equivalent position in the 50 the spirit or scope of the invention, and Such modifications compared sequences is occupied by the same base or amino and variations are encompassed within the scope of the acid, then the molecules are identical at that position; when invention as defined in the claims which follow. The fol the equivalent site occupied by the same or a similar amino lowing examples do not in any way limit the invention. acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (simi 55 Example 1 lar) at that position. Expression as a percentage of homology refers to a function of the number of identical or similar Expression and Purification of Exemplary amino acids at positions shared by the compared sequences. Engineered MBL Molecules A sequence which is “unrelated' or “non-homologous shares less than 40% identity. Determination of homologs of 60 Engineered MBL for optimized binding to pathogens the genes or peptides described herein may be easily ascer without the complement activation and coagulation side tained by the skilled artisan. effects which are present in WTMBL were constructed. The The term “conservative substitution,” when describing a MBL carbohydrate recognition domain & various lengths of polypeptide, refers to a change in the amino acid composi the neck domain were cloned and fused to the Fc fragment tion of the polypeptide that does not substantially alter the 65 of human IgG1 comprising the hinge, CH2 and CH3 regions polypeptide’s activity, fore examples, a conservative Substi to form the fusion proteins. In one embodiment, the MBL tution refers to Substituting an amino acid residue for a carbohydrate recognition domain and at least a portion of the US 9,593,160 B2 111 112 neck domain was cloned and fused to the Fc fragment of 0.22 micron nylon filter and stored at 4°C. This one-step human IgG1 to form the fusion protein FcMBL.81 (SEQ ID purification gave a recovery of at least about 90% (data not NO. 6). In one embodiment, the MBL carbohydrate recog shown). nition domain without a neck region was cloned and fused To analyze the purified proteins, a reduced SDS-PAGE to the Fc fragment of human IgG1 to form the fusion protein was performed using the Invitrogen System. Western blot FcMBL.111 (SEQ ID NO. 8). The complement and coagul ting was then performed onto PVDF membranes using an lation activation regions of the MBL (e.g., the collagen triple iBlot system (Invitrogen) and the PVDF membranes were helix and hinge MASP binding regions) was removed from then probed with biotinylated anti-human MBL antibodies the fusion proteins. (R&D Systems). The results of the purified proteins In some embodiments, the AKT tripeptide was inserted 10 FcMBL.81 and MBL wild-type (WT) are shown in FIG. 4. into the N terminus of Fc (at the hinge region: H of the Fc-X vector shown in FIG. 3) for single-site biotinylation of the Example 2 FcMBL.81 (The amino acid sequence for such embodiment Testing the Potency/Biological Activity of the MBL with the AKT tripeptide fused to the N terminal portion of 15 the Fc, designated as AKTFcMBL.81, is shown in SEQ ID Constructs NO. 7). The mono-biotin engineered MBL molecules AKT FcMBL.81 were then conjugated to streptavidin-coated To determine calcium-dependent binding of the Fc MBL beads and the carbohydrate binding MBL heads were ori proteins to a mannan-coated ELISA plate, 96-well ELISA ented away from the substrate for optimized binding to plate was first coated with 0.5 mg/ml mannan (M3640, pathogens. Sigma). The purified Fc MBL.81 and Fc MBL.111 fusion In some embodiments, the asparagine N82 (N297 in proteins (Supernatant from 293 cell expression purified using Kabat numbering) of SEQID NO. 6 was mutated to aspartic recombinant protein A using the AKTA system & acid (D) to remove the glycosylation of Fc to remove confirmed >90% pure by SDS-PAGE) were diluted and antibody dependent cellular cytotoxicity (ADCC) and 25 added to the mannan-coated ELISA plate. In some sample Complement Dependent Cytotoxicity (CDC) functionality. wells, EDTA was also added to chelate calcium. A secondary Four different engineered MBL fusion protein construct antibody anti-human Fc HRP (109-036-098 Jackson Lab) were produced: was then added to all sample wells. The O.D. values of each (1) Fc MBL. 111 (SEQ ID NO. 8) consists of the Fc portion sample were measured at 450 nm. of IgG (Kabat numbering 216-447) fused to the MBL 30 Presented herein indicated that the Fc MBL.81 fusion CRD head (amino acids 111 to 228). protein binds to mannan in the presence of calcium, but Such (2) Fc MBL.81 (SEQID NO. 6) consists of the Fc portion binding is reduced by ~100 fold in the presence of EDTA of IgG (Kabat numbering 216-447) fused to the MBL (FIG. 5). These assays can be repeated and compared with CRD head AND neck region (amino acids 81-228). the WTMBL from SinoBiologicals. As shown in FIG. 5, the (3) AKT-Fc MBL.81 (SEQ ID NO. 7) consists of the Fc 35 FcMBL.111 fusion protein is inactive in the mannan bind portion of IgG (Kabat numbering 216-447) fused to the ing, regardless of the presence of calcium. Without wishing MBL CRD head AND neck region (amino acids 81-228). to be bound by theory, the neck regions are needed in some The 3 amino acid fragment AKT is fused to the N terminal embodiments provided herein to provide flexibility and portion of the Fc. orientation of the engineered microbe-targeting molecules (4) Fc MBL.81 D consists of the Fc portion of IgG (Kabat 40 (e.g., engineered MBL molecules) for binding to the carbo numbering 216-447) fused to the MBL CRD head AND hydrates on pathogens. The findings presented herein indi neck region (amino acids 81-228), in addition, the Fc cate that both the Fc MBL.81 and WT MBL binding to glycosylation site has been removed by Substituting aspar mannan is calcium-dependent and can be reversed by EDTA tic acid (D) for asparagine (N) at position 82 (297 in chelation. Further, the findings indicated that Fc MBL. 111 Kabat numbering) 45 (the MBL CRD head fused to Fc) appears to be a relatively A major advantage of the Fc fusion technology is the ease poor binder to mannan, as compared to Fc MBL.81. of expression and purification of fusion proteins (Lo et al. The AKT-FcMBL.81 fusion protein appears to show a (1998) Protein Engineering. 11: 495-500). The N terminal higher background binding to mannan in the presence of Fc has been shown to improve expression levels, protein EDTA than the Fc MBL.81 fusion protein. This can be, not folding and secretion of the fusion partner. In addition, the 50 being bound by theory, due to the AKT binding site on the Fc has a staphylococcal protein A binding site, which is N terminus of the fusion protein, which is designed for extremely useful for one-step purification on protein A aminoxy biotinylation for oriented binding on Streptavidin affinity chromatography. Thus, in some embodiments, dif beads. Thus, the AKT-FcMBL.81 fusion protein can be a bit ferent engineered MBL nucleic acid sequences encoding the sticky. In some embodiments, the AKT-FcMBL.81 may not amino acid sequences discussed above can be inserted in the 55 be ideal for the mannan binding assay. Fc-X vector disclosed in the Lo et al. Id. Human U 293 cells were then transfected with Fc MBL DNA using the lipo Example 3 fectamine reagent (Invitrogen). The engineered MBL fusion proteins can be purified on a 5 ml HiTrap Protein A column Activity in Complement and Coagulation Assays using the GE Akta Avant 25 system. 60 with MBL Null Serum For protein purification, an exemplary loading buffer is 100 mM Phosphate 150 mM. NaCl pH 7, and an exemplary WTMBL activates complement and coagulation through elution buffer is 100 mM Phosphate 150 mM. NaCl pH 3. the MASP proteins. In this example provided herein, MBL Following elution, the protein was immediately neutralized null serum was used as a source of complement and coagul with 1 N. NaOH and Tween 80 (Pierce SurfactAmp) was 65 lation proteins, while the WTMBL and the FcMBL.81 were added to a final concentration of 0.01%. The engineered used as the sources of MBL to activate complement activa MBL fusion proteins were then sterile-filtered through a tion of clotting function. US 9,593,160 B2 113 114 Assays to measure complement activation has been dis N-terminal amine in a PLP-mediated transamination reac cussed in Michelow et al. (2010) JBC 285: 24729. Briefly, tion, followed by the addition of aminooxy-biotin to the triplicate samples of diluted chimeric proteins were added to aldehyde. mannan-coated microtiter plates with 1% MBL-null human For the Tosylactivated microbeads, the MBL can be serum as a source of MASP Normal human serum comple directly and covalently coupled to the surface of the micro ment standard (Quidel, San Diego, Calif.) containing native beads by replacing the tosyl groups with its primary amines MBL was used to generate a standard curve. After incuba (lysine residues). Alternatively, aminooxy chemistries can tion at 37° C. and rinsing, deposited human C4 fragments be used to allow for oriented binding to the Tosylactivated (Sigma-Aldrich) were detected with anti-human C4c anti DYNABEADSR) microbeads without using biotin-strepta bodies (Dako Denmark A/S), followed by addition of bioti 10 vidin. This should lead to a more stable system and reduce nylated secondary antibodies (Jackson ImmunoResearch non-specific binding to sticky Streptavidin. Laboratories, West Grove, Pa.), avidin-containing Vectastain ABC-alkaline phosphatase reagent (VectorLaboratories, Example 5 Burlingame, Calif.), and p-nitrophenyl phosphate, and mea surement at A405 nm. 15 Comparison of C. albicans Capture by Fc Methods to determine coagulation and search for throm MBL.81-Coated Magnetic Microbeads with WT bin-like activity have been previously established, e.g., MBL-Coated Magnetic Microbeads discussed in Takahashi et al. (2011) Immunobiology 216: 96). Briefly, the assay was designed to detect MBL-MASP It was sought to determine whether the small -90 kDa complex-mediated activities by using plates that were coated AKT-FcMBL dimers assembled on the surface of the with Mannan in carbonate binding buffer, pH 9.5. After DYNABEADS(R) microbeads with the CRD heads oriented rinsing the prepared mannan-coated plates with TBS, pH away from the microbead substrate, and whether the AKT 7.4, supplemented with 10 mMCaCl (TBS-CaCl), the FcMBL.81 conjugated to the magnetic microbeads (via wells were incubated with diluted MBL proteins with or biotin-streptavidin coupling) has the same avidity of binding without 1% MBL null serum as a MASP source. The wells 25 as the large (~650 kDa) multimeric wild-type biotinylated were incubated at room temperature for 1 h and then rinsed MBL, which is randomly attached to the streptavidin micro thoroughly to wash off endogenous prothrombin and throm bead. bin. Thrombin-like activity of MBL/MASP complex was Briefly, the 650 kDa MBL from Sino Biological was measured by incubating the wells with a rhodamine 110 biotinylated and then coupled to MYONETM Streptavidin based thrombin substrate (tosyl Gly-Phe-Arg-amide, 30 microbeads described in Example 4. The wide-type MBL R22124. Invitrogen). coupled to such microbeads was designated as “Sino MBL The findings presented herein indicate that no C4 depo microbeads' below. AKT-FcMBL.81 was also coupled to sition on mannan-coated plates from Fc MBL.81 activation MYONETM Streptavidin microbeads. Equivalent masses of of MBL null serum, but C4 was deposited from the WT the two proteins were coupled to equal numbers of micro MBL control (Data not shown). Further, no coagulation 35 beads. activation (thrombin-like activity) by the Fc MBL.81 fusion To perform the MBL microbead capture experiments, protein was determined (data not shown). Thus, unlike the 1500 Candida were incubated for 10 minutes with either 1 WTMBL, the Fc MBL.81 does not activate complement or ul of the AKT FcMBL, microbeads or 1 ul of the Sino MBL coagulation. microbeads in TBS-Tween buffer supplemented with 5 mM 40 Calcium. Microbeads with bound Candida were removed by Example 4 capturing them on a magnet for 2 minutes. Then, about /10 of the captured material was plated on YEPD Agar plates Exemplary Methods for Production of MBL and incubated at 30° C. for 1-2 days, followed by counting Magnetic Microbeads and comparing with the total counts from an equivalent 45 dilution of the 1500 Candida starting culture. Different microbe-targeting molecules (e.g., engineered FIG. 6A shows that greater than 95% of the Candida was MBL molecules) can be coupled to magnetic microbeads bound to the AKT Fc MBL.81 microbeads, while greater using different sizes or types of microbeads and Surface than 92% was bound to the Sino MBL microbeads. There chemistries. Examples of magnetic microbeads that can be were no significant differences in the results using these two used for the microbe-targeting magnetic microbeads 50 types of microbeads, indicating that the engineered MBL include, but are not limited to, 1 micron MYONETM T1 magnetic microbeads described herein showed at least com streptavidin microbeads (streptavidin coupled via tosyl parable, or indeed better, binding than the WT MBL mag groups) from Invitrogen (DYNABEADSR), 1 micron Tosy netic microbeads at the indicated pathogen density. lactivated microbeads from Invitrogen (DYNABEADS(R), Next, it was sought to evaluate the performance of the and 100 nm (128 nm average diameter) Streptavidin Plus 55 engineered MBL magnetic microbeads at higher pathogen microbeads (streptavidin coupled via carboxyl groups) from densities, e.g., above 10 yeast cells. First, C. albicans was Ademtech. cultured overnight for 2 days, and then washed 2 times with For binding to the MYONETM and Ademtech streptavidin PBS. The final pellet of C. albicans was resuspended in microbeads, the MBL can be biotinylated (~4 biotins per TTBS w/ Calcium TBS, 0.1% Tween-20, 5 mM CaCl, molecule) using Thermo EZ-Link Sulfo-NHS-LC-Biotin, 60 with a reading of OD600 around 0.5-0.7. The unlabeled which reacts with primary amines (lysine residues). For MYONETM T1 streptavidin microbeads were washed 2 single-site biotinylation, for example, using the method times in PBS 0.1% BSA and diluted in original volume of described in Witus et al. (2010) JACS. 132: 16812, the PBS 0.1% BSA. To a 1.5 mL tube, about 1 mL of above C. AKT-FcMBL.81 fusion protein is biotinylated only at the albicans mixture was first added, followed by either 2 ul of N-terminal amine for oriented binding to Streptavidin 65 unlabeled microbeads, 2 ul of FcMBL labeled microbeads, microbeads. Briefly, the PLP-mediated bioconjugation is a or 2 ul of WT MBL labeled microbeads. A C. albicans two-step process, in which an aldehyde is first added to the mixture was used as a no-bead control. All the mixtures were