Engineered opsonin for pathogen detection and treatment

• US 9,150,631 B2
• Filed: 01/19/2011
• Issued: 10/06/2015
• Est. Priority Date: 01/19/2010
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Super, Michael, Way, Jeffrey Charles, Ingber, Donald E.
• Primary Examiner(s): Alstrum Acevedo, James H
• Assistant Examiner(s): DABKOWSKI, ERINNE R
• Application Number:
  US13/574,191
• Publication Number:
  US 20130035283A1
• Time in Patent Office: 1,721 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C07K 14/4726 Lectins
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: C07K 17/00 Carrier-bound or immobilised peptides carrier-bound or immobilised enzymes C12N11/00; Preparation thereof
• CPC Class Codes: C07K 2317/53 Hinge
• CPC Class Codes: C07K 2317/66 comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: G01N 2333/40 from Candida
• CPC Class Codes: G01N 27/745 for detecting magnetic beads used in biochemical assays concerning the assays G01N33/54326; sensors therefor G01R33/1269; automatic analysers therefor G01N35/0098
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: G01N 33/56961 Plant cells or fungi
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

1. A recombinant opsonin comprising:

a carbohydrate recognition domain of mannose binding lectin (MBL) consisting of (i) SEQ ID NO;

2 or (ii) amino acid residues 31 (glycine) to 148 (isoleucine) of SEQ ID NO;

2; and

a peptide comprising a portion of an immunoglobulin Fc that links to the carbohydrate recognition domain.

2. The recombinant opsonin of claim 1, 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.

3. The recombinant opsonin of claim 1, wherein the peptide comprises a Glycine+Serine segment, or a Proline+Alanine+Serine segment.

4. The recombinant opsonin of claim 1, wherein the portion of the immunoglobulin Fc comprises a hinge or a CH2-CH3 interface.

5. The recombinant opsonin of claim 1, wherein the recombinant opsonin comprises a lectin consisting of amino acid residues 81 (proline) to 228 (isoleucine) of MBL, fused to a Fc portion of human IgG (Fcγ

) (SEQ ID NO;

     3).

6. The recombinant opsonin of claim 1, wherein the peptide comprising the portion of the immunoglobulin Fc comprises an amino acid sequence of SEQ ID NO:

1.

7. The recombinant opsonin of claim 6, wherein the amino acid sequence of the peptide is modified to exclude N-linked glycosylation.

8. The recombinant opsonin of claim 7, wherein the amino acid residue 82 in the SEQ ID NO:

1 is changed from asparagine (N) to aspartic acid (D).

9. The recombinant opsonin of claim 1, wherein N-terminus of the recombinant opsonin consists essentially of an amino acid sequence of alanine-lysine-threonine (AKT).

10. The recombinant opsonin of claim 1, wherein the recombinant opsonin is attached to a solid substrate.

11. The recombinant opsonin of claim 10, wherein the solid substrate is a magnetic microbead.

12. The recombinant opsonin of claim 10, 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.

13. The recombinant opsonin of claim 1, wherein the carbohydrate recognition domain is linked to C-terminal of the peptide.

14. The recombinant opsonin of claim 1, wherein the carbohydrate recognition domain binds to at least one carbohydrate that is present on a microbe.

15. The recombinant opsonin of claim 10, wherein N-terminus of the recombinant opsonin is attached to the solid substrate.