# US 10,865,235 B2 · Engineered microbe-targeting molecules and uses thereof

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- Number: US 10,865,235 B2
- Document ID: US10865235B2
- Status: Granted
- Kind: B2
- Application number: 16/683,630
- Filed: 2019-11-14
- Published: 2020-07-30
- Granted: 2020-12-15
- Earliest claimed: 2011-07-18
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Donald E. Ingber; Michael Super; Jeffrey Charles Way; Mark J. Cartwright; Julia B. Berthet; Dinah R. Super; Martin Rottman; Alexander L. Watters
- Web page: https://patentgrep.com/patent/US10865235B2

## CPC

- C07K 16/12: against material from bacteria
- A61K 47/68: the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K 47/6815: {Enzymes}
- A61P 31/04: Antibacterial agents
- C07K 14/42: Lectins, e.g. concanavalin, phytohaemagglutinin
- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- G01N 33/56911: {Bacteria}
- G01N 33/56916: {Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia}
- G01N 33/56938: {Staphylococcus}
- G01N 33/56961: {Plant cells or fungi}
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- G01N 2333/42: Lectins, e.g. concanavalin, phytohaemagglutinin
- G01N 2333/4724: Lectins
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Described herein are engineered microbe-targeting or microbe-binding molecules, kits comprising the same and uses thereof. Some particular embodiments of the microbe-targeting or microbe-binding molecules comprise a carbohydrate recognition domain of mannose-binding lectin, or a fragment thereof, linked to a portion of a Fc region. In some embodiments, the microbe-targeting molecules or microbe-binding molecules can be conjugated to a substrate, e.g., a magnetic microbead, forming a microbe-targeting substrate (e.g., a microbe-targeting magnetic microbead). Such microbe-targeting molecules and/or substrates and the kits comprising the same can bind and/or capture of a microbe and/or microbial matter thereof, and can thus be used in various applications, e.g., diagnosis and/or treatment of an infection caused by microbes such as sepsis in a subject or any environmental surface. Microbe-targeting molecules and/or substrates can be regenerated after use by washing with a low pH buffer or buffer in which calcium is insoluble.

## Claims

### Claim 1

A composition comprising:
a plurality of magnetic beads, wherein the magnetic beads are conjugated to a plurality of molecules each comprising a first molecule comprising a carbohydrate recognition domain of a mannose-binding lectin (MBL) linked to an Fc region of an antibody (FcMBL); and
a second molecule comprising a detectable label conjugated to a targeting agent specific for a microbe.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the FcMBL molecules are dimerized.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the FcMBL molecules are dimerized via interactions of their respective Fc regions.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the MBL comprises an amino acid sequence of SEQ ID NO: 2.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the targeting agent comprises a C-type lectin.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the targeting agent comprises a collectin.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the targeting agent comprises mannose-binding lectin.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the targeting agent comprises a carbohydrate recognition domain of mannose-binding lectin, or a fragment thereof, linked to a portion of a Fc region.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the first molecule further comprises the carbohydrate recognition domain and a neck region of the MBL and wherein the Fc region comprises an Fc fragment of human IgG1.

### Claim 10 (depends on 9)

The composition of claim 9, wherein the Fc region is linked N-terminal of the carbohydrate recognition domain.

### Claim 11 (depends on 1)

The composition of claim 1, wherein the MBL comprises a carbohydrate recognition domain having the sequence of SEQ ID NO: 4 or a fragment thereof.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the MBL comprises a carbohydrate recognition domain having a sequence having at least 90% identity to the sequence of SEQ ID NO: 4.

### Claim 13 (depends on 1)

The composition of claim 1, wherein the detectable label comprises an enzyme.

### Claim 14 (depends on 13)

The composition of claim 13, wherein the enzyme is horseradish peroxidase (HRP).

### Claim 15 (depends on 13)

The composition of claim 13, which further comprises a substrate for the enzyme.

### Claim 16 (depends on 15)

The composition of claim 15, wherein the substrate is 3,3′,5,5′-tetramethylbenzidine.

### Claim 17 (depends on 1)

The composition of claim 1, wherein the targeting agent specific for the microbe comprises an engineered microbe-targeting molecule or an antibody.

### Claim 18 (depends on 1)

The composition of claim 1, wherein the targeting agent specific for the microbe comprises MBL.