# US 11,440,946 B2 · Universal antibody-mediated biosensor

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- Number: US 11,440,946 B2
- Document ID: US11440946B2
- Status: Granted
- Kind: B2
- Application number: 15/557,603
- Filed: 2016-03-11
- Published: 2018-03-01
- Granted: 2022-09-13
- Earliest claimed: 2015-03-13
- Assignees: University of Maryland, Baltimore
- Applicants: University of Maryland, Baltimore
- Inventors: Dan Schulze; Scott E. Strome
- Web page: https://patentgrep.com/patent/US11440946B2

## CPC

- C07K 14/70535: {Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/12: against material from bacteria
- C12Q 1/02: involving viable microorganisms
- G01N 33/5038: {involving detection of metabolites per se}
- G01N 33/5041: {involving analysis of members of signalling pathways}
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 33/6854: {Immunoglobulins}
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/03: containing a transmembrane segment
- G01N 2333/70535: Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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)

## Abstract

A universal antibody-mediated biosensor is provided that comprise a biosensor cell line stably expressing a novel chimeric fusion protein that can be used to detect target agents in a sample. The fusion protein has an extracellular antibody-binding domain that binds antibodies without regard to their binding specificity and a signaling domain that induces cellular activation upon antigen binding. Because the fusion protein binds to the Fc region of any antibody, it can serve as a universal pathway between extracellular signaling and intracellular activation. The biosensor can be used to detect the presence of selected antigens in a sample.

## Claims

### Claim 1

A biosensor cell stably expressing a chimeric fusion protein, wherein the chimeric fusion protein comprises an Fcγ receptor (FcγR) antibody-binding domain and a signaling domain, wherein the FcγR antibody-binding domain is an FcγRI antibody-binding domain, wherein the signaling domain is an immunoglobulin alpha (Igα) signaling domain, and wherein the fusion protein is the FcγRI/Igα fusion protein set forth in SEQ ID NO:8.

### Claim 2 (depends on 1)

The biosensor cell of claim 1, wherein the FcγRI antibody-binding domain is set forth in SEQ ID NO:1 or 3.

### Claim 3 (depends on 1)

The biosensor cell of claim 1, wherein the Igα signaling domain is set forth in SEQ ID NO:5.

### Claim 4 (depends on 1)

A method of detecting a target agent in a sample, comprising
(a) contacting the sample with (i) an antibody having binding specificity for the target agent and (ii) the biosensor cell of claim 1 stably expressing the chimeric fusion protein that binds the antibody, under conditions promoting (a′) binding of the target agent by the antibody and (b′) binding of the antibody to the FcγRI antibody-binding domain of the chimeric fusion protein expressed by the biosensor cell, and
(b) assaying the biosensor cell for cellular activation resulting from binding of the target agent by the antibody, and binding of the antibody to the FcγRI antibody-binding domain of the chimeric fusion protein expressed by the biosensor cell.

### Claim 5 (depends on 4)

The method of claim 4, wherein the sample is an air sample, a liquid sample, a vegetable sample, or a dry sample.

### Claim 6 (depends on 4)

The method of claim 4, wherein the sample is a biological sample selected from the group consisting of blood, serum, sweat, urine, cerebrospinal fluid, mucus, semen, stool, bronchoalveolar lavage fluid, and tissue.

### Claim 7 (depends on 4)

The method of claim 4, wherein the target agent is an environmental toxin, pollutant, or drug.

### Claim 8 (depends on 4)

The method of claim 4, wherein the target agent is a biologic agent selected from the group consisting of a bio-warfare agent, an allergen, a parasitic antigen, a fungal antigen, a viral antigen, a bacterial antigen, a cellular antigen, and an antibody.

### Claim 9 (depends on 4)

The method of claim 4, wherein cellular activation is an increase in intracellular Ca2+ levels.

### Claim 10 (depends on 4)

The method of claim 4, wherein the FcγRI antibody-binding domain is set forth in SEQ ID NO:1 or 3.

### Claim 11 (depends on 10)

The method of claim 10, wherein the Iga signaling domain is set forth in SEQ ID NO:5.

### Claim 12

A chimeric fusion protein comprising an Fcγ receptor (FcγR) antibody-binding domain and a signaling domain, wherein the FcγR antibody-binding domain is an FcγRI antibody-binding domain, wherein the signaling domain is an Igα signaling domain, and wherein the fusion protein is the FcγRI/Igα fusion protein set forth in SEQ ID NO:8.

### Claim 13 (depends on 12)

The chimeric fusion protein of claim 12, wherein the FcγRI antibody-binding domain is set forth in SEQ ID NO:1 or 3.

### Claim 14 (depends on 12)

The chimeric fusion protein of claim 12, wherein the Igα signaling domain is set forth in SEQ ID NO:5.