# US 8,772,451 B2 · Soluble TCR molecules and methods of use

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- Number: US 8,772,451 B2
- Document ID: US8772451B2
- Status: Granted
- Kind: B2
- Application number: 10/985,271
- Filed: 2004-11-10
- Published: 2005-09-29
- Granted: 2014-07-08
- Earliest claimed: 2003-11-10
- Assignees: Altor BioScience Corporation
- Applicants: Shari A. Price-Schiavi; Heather J. Belmont; Kimberlyn F. Card; Xiaoyun Zhu; Hing C. Wong
- Inventors: Shari A. Price-Schiavi; Heather J. Belmont; Kimberlyn F. Card; Xiaoyun Zhu; Hing C. Wong
- Web page: https://patentgrep.com/patent/US8772451B2

## CPC

- A61K 51/1027: {against receptors, cell-surface antigens or cell-surface determinants}
- A61K 47/6415: {Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins}
- A61K 47/642: {the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate}
- A61K 47/6425: {the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell}
- A61K 47/665: {the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems}
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- C07K 14/55: IL-2
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- G01N 33/5758: {involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites}
- G01N 33/5759: {involving compounds localised on the membrane of tumour or cancer cells}
- G01N 33/6875: {Nucleoproteins}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/32: fusions with soluble part of a cell surface receptor, "decoy receptors"
- G01N 2333/7051: T-cell receptor (TcR)-CD3 complex
- G01N 2333/70539: MHC-molecules, e.g. HLA-molecules
- 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)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- 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)
- 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

Disclosed are compositions and methods for detecting cells or tissue comprising a peptide antigen presented in the context of an MHC or HLA complex. The invention has a wide range of applications including providing a highly sensitive method for detecting cancer cells.

## Claims

### Claim 1

A method for detecting cells or tissue comprising a peptide antigen presented on cells or tissue in the context of an MHC complex, wherein the peptide antigen is an endogenous human p53 antigen, the method comprising:
a) contacting the cells or tissue with at least one denaturing agent, wherein the denaturing agent is sufficient to fix the cells or tissue, thereby fixing the cells or tissue;
b) contacting the fixed cells or tissue that have not been manipulated to express artificial levels of the peptide antigen with at least one soluble single chain TCR(scTCR) molecule under conditions that form a specific binding complex between the presented peptide antigen and the soluble scTCR, wherein the soluble scTCR comprises the Vα and Vβ domains of SEQ ID NO: 16, and wherein the soluble single chain TCR molecule is a monomer;
c) washing the cells or tissue under conditions appropriate to remove any soluble scTCR molecule not bound to the presented peptide antigen; and
d) detecting the specific binding complex as being indicative of cells or tissue comprising the presented peptide antigen.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cells or tissue are further contacted with at least one blocking agent.

### Claim 3 (depends on 2)

The method of claim 2, wherein the method further comprises contacting the cells or tissue with the blocking agent before step b) to reduce non-specific binding between the soluble scTCR and the cells.

### Claim 4 (depends on 2)

The method of claim 2, wherein the blocking agent is a peroxide, serum protein, antibody, or fragment thereof.

### Claim 5 (depends on 1)

The method of claim 1, wherein the amount of the peptide antigen present on the cells is less than about 100,000 copies.

### Claim 6 (depends on 5)

The method of claim 5, wherein the amount of the peptide antigen present on the cells is less than about 400 copies.

### Claim 7 (depends on 1)

The method of claim 1, wherein the peptide antigen is a tumor-associated peptide antigen.

### Claim 8 (depends on 1)

The method of claim 1, wherein the cells or tissue are suspended.

### Claim 9 (depends on 1)

The method of claim 1, wherein the soluble scTCR molecule is detectably-labeled.

### Claim 10 (depends on 9)

The method of claim 9, wherein the detectable label is biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule.

### Claim 11 (depends on 1)

The method of claim 1, wherein the MHC complex is HLA-A2.

### Claim 12 (depends on 1)

The method of claim 1, wherein the method further comprises performing a control to detect any binding between the soluble scTCR to cells that do not comprise the peptide antigen.

### Claim 13 (depends on 1)

The method of claim 1, wherein the cell or tissue is a tumor cell or tissue.

### Claim 14 (depends on 1)

The method of claim 1, wherein the cells are tumor cells.