Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• V chains of the heterodimeric TCRs include the V- ⁇ chain linked covalently to a C- ⁇ chain and the V- ⁇ chain linked to a C- ⁇ chain.
• the C- ⁇ chain and C- ⁇ chain are each independently linked to a suitable cell transmembrane domain which domain is typically further linked independently to a suitable cytosolic domain.
• the invention also features a nucleic acid segment (DNA or RNA) that encodes at least one, preferably between from about one to five, more preferably about one to two, of the sc-TCR molecules provided herein. Also included are DNA vectors that include the nucleic acid segment.
• Gap parameters can be modified to suit a user's needs. For example, when employing the GCG software package, a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 can be used.
• sc- TCR molecules may include at least one C ⁇ chain (as shown in Figure 5, for example) or a functional fragment thereof, preferably between from about one to five of such chains, with about one of such a C ⁇ chain being suitable.
• the linker sequence comprises glycine, alanine or serine residues, particularly glycine and serine residues.
• the linker sequence is suitably linked to the ⁇ chain of the TCR molecule, although the linker sequence also could be attached to the ⁇ chain of the TCR molecule.
• the linker sequence may be linked to both ⁇ and ⁇ chains of the TCR molecule.
• the invention features particular sc-TCR molecules in which at least one of the peptide sequences, preferably one of same has the following sequence: Gly Gly Gly Gly Ser repeated as many as four or more times (SEQ ID NO. 5).
• a non-cell based assay such as a TCR Enzyme Linked Immunosorbant Assay (ELISA) .
• the TCR can be bound directly to a support and its ability to bind to MHC:p53 peptide complexes can be measured, or the MHC:p53 complex can be bound to the support and the ability of the complex to bind to the TCR can be measured.
• Suitable supports include, but are not limited to, wells of a microtiter plate, cell culture plates, smembrane, glass or polymer substrates, and the like.
• soluble or similar term is meant that a TCR molecule of the invention, usually but not exclusively a single chain construct, is not readily sedimented under low G-force centrifugation (e.g. less than about 30,000 revolutions per minute in a standard centrifuge) from an aqueous buffer, e.g., cell media. Further, the molecule is soluble if it remains in aqueous solution at a temperature greater than about 5-37°C and at or near neutral pH in the presence of low or no concentration of an anionic or non- ionic detergent. Under these conditions, a soluble protein will often have a low sedimentation value, e.g., less than about 10 to 50 Svedberg units.
• Sc-Fv DNA was then restriction digested and cloned into the "shuttle vector" downstream of the sc-TCR.
• the "shuttle vector” was digested with the appropriate restriction enzymes to drop out the previous linker DNA fragment and allow for ligation of linker sequences between the sc-TCR and the sc-Fv.
• the cells were diluted into 10 ml of 10% IMDM medium (IMDM, 10% FBS, 2mM glutamine) and grown in a T-25cm 2 TC flask overnight at 37°C with 5% CO2 The next day, the cells were plated in 96 well plates with selective medium (10% IMDM plus 1.0 mg/ml G418). After 1 week, the concentration of G418 was increased to 2 mg/ml. The growing colonies were re-fed approximately two weeks after transfection and screened about one week later.
• 10% IMDM medium IMDM, 10% FBS, 2mM glutamine

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• Health & Medical Sciences (AREA)
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• General Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Zoology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Cell Biology (AREA)
• Toxicology (AREA)
• General Chemical & Material Sciences (AREA)
• Peptides Or Proteins (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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• 2002
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