Engineered opsonin for pathogen detection and treatment

Abstract

The present invention provides for engineered molecular opsonins that may be used to bind biological pathogens or identify subclasses or specific pathogen species for use in devices and systems for treatment and diagnosis of patients with infectious diseases, blood-borne infections or sepsis. An aspect of the invention provides for mannose-binding lectin (MBL), which is an abundant natural serum protein that is part of the innate immune system. The ability of this protein lectin to bind to surface molecules on virtually all classes of biopathogens (viruses, bacteria, fungi, protozoans) make engineered forms of MBL extremely useful in diagnosing and treating infectious diseases and sepsis.

1 .- 30 . (canceled) 
     
     
         31 . A recombinant opsonin comprising:
 a carbohydrate recognition domain of mannose-binding lectin (MBL), wherein the carbohydrate recognition domain binds to at least one carbohydrate that is present on a surface of a microbe; and   a peptide comprising a portion of an immunoglobulin Fc that links to the carbohydrate recognition domain, and wherein the recombinant opsonin excludes a functional domain of MBL that binds to a mannan-binding lectin associated serine protease (MASP), the functional domain being present in cysteine-rich N terminal domain of MBL or collagen segment of MBL.   
     
     
         32 . The recombinant opsonin of  claim 31 , wherein said carbohydrate recognition domain comprises the neck and lectin domains of MBL. 
     
     
         33 . The recombinant opsonin of  claim 31 , wherein N-terminus of the recombinant opsonin comprises at least one cysteine residue that allows chemical cross-linking to a surface of a solid substrate. 
     
     
         34 . The recombinant opsonin of  claim 31 , wherein the peptide comprises a Glycine+Serine segment, or a Proline+Alanine+Serine segment. 
     
     
         35 . The recombinant opsonin of  claim 31 , wherein the portion of the immunoglobulin Fc comprises a hinge or a CH2-CH3 interface. 
     
     
         36 . The recombinant opsonin of  claim 31 , wherein the carbohydrate recognition domain consists of (i) SEQ ID NO: 2, or (ii) amino acid residues 31 (glycine) to 148 (isoleucine) of SEQ ID NO: 2. 
     
     
         37 . The recombinant opsonin of  claim 31 , wherein the recombinant opsonin comprises a lectin having amino acid residues 81 (proline) to 228 (isoleucine) of MBL, fused to an Fc portion of human IgG (Fcγ) (SEQ ID NO: 3). 
     
     
         38 . The recombinant opsonin of  claim 31 , wherein the peptide comprising the portion of the immunoglobulin Fc comprises an amino acid sequence of SEQ ID NO: 1. 
     
     
         39 . The recombinant opsonin of  claim 38 , wherein the amino acid sequence of the peptide is modified to exclude N-linked glycosylation. 
     
     
         40 . The recombinant opsonin of  claim 39 , wherein the amino acid residue 82 in the SEQ ID NO: 1 is changed from asparagine (N) to aspartic acid (D). 
     
     
         41 . The recombinant opsonin of  claim 31 , wherein N-terminus of the recombinant opsonin consists essentially of an amino acid sequence of alanine-lysine-threonine (AKT). 
     
     
         42 . The recombinant opsonin of  claim 31 , wherein the recombinant opsonin is attached to a solid substrate. 
     
     
         43 . The recombinant opsonin of  claim 42 , wherein N-terminus of the recombinant opsonin is attached to the solid substrate. 
     
     
         44 . The recombinant opsonin of  claim 42 , wherein the solid substrate is a magnetic microbead. 
     
     
         45 . The recombinant opsonin of  claim 42 , wherein the solid substrate is selected from a group consisting of a magnetic microbead, a paramagnetic microbead, a microporous membrane, a hollow fiber, any other fluid filtration membrane, flow device, a living cell, an extracellular matrix of a biological tissue or organ, and a phagocyte. 
     
     
         46 . The recombinant opsonin of  claim 31 , wherein the carbohydrate recognition domain is linked to C-terminal of the peptide. 
     
     
         47 . A method comprising:
 contacting a fluid with a recombinant opsonin, the recombinant opsonin comprising a carbohydrate recognition domain of mannose-binding lectin (MBL), wherein the carbohydrate recognition domain binds to at least one carbohydrate that is present on a surface of a microbe; and   a peptide comprising a portion of an immunoglobulin Fc that links to the carbohydrate recognition domain, and wherein the recombinant opsonin excludes a functional domain of MBL that binds to a mannan-binding lectin associated serine protease (MASP), the functional domain being present in cysteine-rich N terminal domain of MBL or collagen segment of MBL.   
     
     
         48 . The method of  claim 47 , wherein the recombinant opsonin is conjugated to a magnetic particle prior to the contacting, and the separating is achieved by applying magnetic force to the fluid after the opsonin-binding microorganism has bound to the recombinant opsonin. 
     
     
         49 . The method of  claim 47  wherein the fluid is a biological fluid derived from a subject selected from the group consisting of blood, cerebrospinal fluid, joint fluid, urine, semen, saliva, tears, and fluids collected by needle, biopsy, or aspiration procedures; or is a fluid derived from a water or a food sample. 
     
     
         50 . The method of  claim 49 , wherein the subject is suffering from infection or sepsis.