# US 9,518,114 B2 · Treating bladder tumor cells using fibronectin attachment protein as a target

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- Number: US 9,518,114 B2
- Document ID: US9518114B2
- Status: Granted
- Kind: B2
- Application number: 13/884,427
- Filed: 2011-11-11
- Published: 2013-09-05
- Granted: 2016-12-13
- Earliest claimed: 2010-11-12
- Assignees: Purdue Research Foundation
- Applicants: Ruben Claudio Aguilar; Timothy Ratliff; David Thompson; Scott Crist
- Inventors: Ruben Claudio Aguilar; Timothy Ratliff; David Thompson; Scott Crist
- Web page: https://patentgrep.com/patent/US9518114B2

## CPC

- A61K 38/164: {from bacteria}
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61K 47/6835: {the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site}
- A61K 47/6881: {Cluster-antibody conjugates, i.e. the modifying agent consists of a plurality of antibodies covalently linked to each other or of different antigen-binding fragments covalently linked to each other}
- A61K 47/6913: {the liposome being modified on its surface by an antibody}
- A61K 47/6927: {the form being a solid microparticle having no hollow or gas-filled cores}
- C07K 16/12: against material from bacteria
- C07K 16/1278: Bacillus (G)
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 16/3038: {Kidney, bladder}
- A61K 2039/505: {comprising antibodies}
- 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)
- 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)

## Abstract

Composition and methods are disclosed for utilizing micro-aggregation of FAP-containing complexes to promote their fast internalization. This approach allows the uptake of cytotoxic cargo coupled to either FAP-Antibodies or FAP-liposome complexes by tumor bladder cells. Importantly, this approach is efficient even under serum-free conditions such as the ones found in the lumen of the bladder.

## Claims

### Claim 1

A method of enhancing targeted uptake of therapeutic or diagnostic agents by a patient's cells, said method comprising the steps of:
providing a multivalent complex comprising at least one carrier and a plurality of fibronectin attachment proteins or functional equivalents thereof attached to the carrier such that each fibronectin attachment protein or functional equivalent thereof is positioned within submicrons of at least one other fibronectin attachment protein or functional equivalent to form a cluster and wherein said complex is linked to a therapeutic or diagnostic agent; and
administering said complex to said patient;
wherein upon contact between receptors of a patient cell and the fibronectin attachment proteins or functional equivalents thereof of the complex, the cluster further comprises the receptors of the patient cell.

### Claim 2 (depends on 1)

The method of claim 1 wherein:
the multivalent complex further comprises multiple ligands, each ligand bound to a fibronectin attachment protein or the function equivalent thereof and held less than 150 nm apart from each other; and
the fibronectin attachment proteins or functional equivalents within the cluster are attached to the carrier through a linker.

### Claim 3 (depends on 2)

The method of claim 2 wherein the therapeutic of diagnostic agent is targeted to bladder cancer cells and said administering step comprises introducing said complex into the lumen of the patient's bladder.

### Claim 4 (depends on 2)

The method of claim 2 wherein each said fibronectin attachment protein or functional equivalent is a recombinant fusion peptide comprising a non-native peptide tag, and said ligands bind to said non-native peptide tag.

### Claim 5 (depends on 2)

The method of claim 2 wherein the ligands are antibodies that specifically bind to fibronectin attachment protein or the functional equivalent thereof, and the cluster induces uptake of the carrier by the patient cell.

### Claim 6 (depends on 4)

The method of claim 4 wherein the ligands are antibodies that specifically bind to said non-native peptide tag.

### Claim 7 (depends on 4)

The method of claim 4 wherein the carrier comprises a liposome, wherein at least a portion of said plurality of ligands are presented on the external surface of the liposome.

### Claim 8 (depends on 7)

The method of claim 7 wherein the diagnostic agent or therapeutic agent is encapsulated within the liposome and the liposome comprises a diameter of 50 nm.

### Claim 9 (depends on 7)

The method of claim 7 wherein the fibronectin attachment protein or functional equivalent thereof comprises a histidine peptide tag and the ligand is a Ni2+NTA-bearing liposome.

### Claim 10 (depends on 3)

The method of claim 3 wherein said fibronectin attachment protein or functional equivalent thereof comprises the RWFV sequence (residues 213-216) of the native peptide.

### Claim 11

A method of enhancing the targeted uptake of therapeutic or diagnostic agents by a patient's bladder cells, said method comprising the steps of
providing a complex that comprises:
a plurality of recombinant fusion peptides, each peptide comprising a fibronectin attachment protein and a non-native peptide tag;
a plurality of ligands bound to said recombinant fusion peptides;
a nanoparticle or nano-carrier wherein said ligands are linked to said nanoparticle or nano-carrier surface and spaced less than 150 nm apart from each other such that each fibronectin attachment protein is positioned within submicrons of at least one other fibronectin attachment protein of the complex to form a cluster; and
a therapeutic or diagnostic agent linked to said ligand or peptide; and
contacting said cells with said complex.

### Claim 12 (depends on 11)

The method of claim 11 wherein said ligands are antibodies specific for said non-native peptide tag and said therapeutic or diagnostic agent is linked to the constant domain of the antibodies.

### Claim 13 (depends on 11)

The method of claim 11 wherein the therapeutic or diagnostic agent is targeted to bladder cancer cells and said contacting step comprises introducing said complex into the lumen of the patient's bladder.

### Claim 14

A method for enhancing selective uptake of diagnostic or therapeutic agents by a patient's bladder tumor cells comprising the steps of:
providing a stable multivalent complex of fibronectin attachment proteins, wherein the fibronectin attachment protein complex comprises at least one carrier, a plurality of fibronectin attachment proteins attached to the carrier, and a plurality of ligands that bind to fibronectin attachment protein and position at least two fibronectin attachment proteins within submicrons of each other to form a cluster, and wherein said complex is linked to a therapeutic or diagnostic agent; and
contacting said bladder tumor cells with said complex;
wherein upon contact between receptors of a bladder tumor cell and the fibronectin attachment proteins of the complex, the cluster further comprises the receptors of the bladder tumor cell.

### Claim 15 (depends on 14)

The method of claim 14 wherein said fibronectin attachment protein is a recombinant fusion peptide comprising a non-native peptide tag, and said ligands bind to said non-native peptide tag.

### Claim 16 (depends on 14)

The method of claim 14 wherein the ligands are antibodies that specifically bind to fibronectin attachment protein and the cluster induces uptake of the carrier by the patient cell.

### Claim 17 (depends on 15)

The method of claim 15 wherein the ligands are antibodies that specifically bind to said non-native peptide tag.

### Claim 18 (depends on 14)

The method of claim 14 wherein said ligands are held less than 150 nm apart from each other and the fibronectin attachment proteins within the cluster are covalently attached to one another through a linker.

### Claim 19 (depends on 14)

The method of claim 14 wherein said contacting step comprises introducing said complex into the lumen of the patient's bladder.

### Claim 20 (depends on 16)

The method of claim 16 wherein the multivalent complex is formed by providing the antibodies at a fibronectin attachment proteins or functional equivalents thereof to antibodies dilution ratio of between 1:600 and 1:1800 to facilitate formation of the cluster.