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Human anti-epidermal growth factor receptor single-chain antibodies

a growth factor receptor and anti-epidermal technology, applied in the field of molecular biology and monoclonal antibody technology, can solve the problems of poor prognosis of patients with tumors that overexpress the epidermal growth factor receptor, poor immunological or allergic response, and uneven distribution of monoclonal antibodies around the tumor

Inactive Publication Date: 2006-05-25
UAB RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many patients with tumors that overexpress the epidermal growth factor receptor have a poorer prognosis than those who do not.
However, the problem with rat and mouse monoclonal antibodies or even the human-mouse chimeric antibody is the possibility of an immune or allergic response with prolonged treatment (10-13).
However due to the heterologous vascular structure around the tumor and the molecular size of the antibodies, monoclonal antibodies penetrate the tumor poorly and are unevenly distributed around the tumor.
The prior art is deficient in the lack of a 100% human single-chain antibody that binds to the epidermal growth factor receptor.

Method used

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  • Human anti-epidermal growth factor receptor single-chain antibodies
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  • Human anti-epidermal growth factor receptor single-chain antibodies

Examples

Experimental program
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Effect test

example 1

Isolating EGFR-Specific Human Single-Chain Antibody (scFv)

[0042] In the screening of the phage library for anti-epidermal growth factor receptor scFv, an IgM scFv display library with a calculated complexity of 2×107 independent clones was constructed in pSEX81 (FIG. 1) as described (15) using peripheral leukocyte cDNA prepared from healthy donors. The IgM phage display library was screened for expression of scFvs which specifically bind the EGF receptor (EGFR). The phage library was suspended in 500 ml 2× YT-GA medium (17 g Tryptone, 10 g yeast extract, 100 mM glucose, 100 μg / ml ampicillin and H2O to 1 liter) to an initial OD600 of 0.025. The cells were grown with shaking (240 rpm) at 37° C. until an OD600 of 0.1 at which point the cells were superinfected with helper phage, M13K07 (Amersham Pharmacia Biotech), at an MOI of 20. After the addition of helper phage, the cells were mixed gently and left undisturbed for 15 min at 37° C. followed by shaking (240 rpm) for 45 min. The me...

example 2

Sequencing and Analysis of scFv Clones

[0051] After 3 rounds of phage panning, individual clones were identified by ELISA as described above. Plasmid DNA was isolated and sequenced according to standard manufacturer's protocol for the ABI DNA sequencer (UAB Automated DNA Sequencing Core Facility). Plasmid DNA was sequenced both directions initially using pelB and gene III primers. Internal sequencing primers were determined from the initial sequence data and synthesized by Operon (Alameda, Calif.). After completion of the scFv sequence, the data was analyzed using SeqWeb software (Genetics Computer Group, Madison, Wis.) for alignment of complementary-determining regions (CDRs) with known variable-chain sequence data.

[0052] Two clones, pSEX81-6 and pSEX81-63, have been sequenced and their putative amino acid sequences are shown in FIG. 2. The clones are in the order, variable heavy chain (VH)-linker-variable light chain (VL), with both clones containing a lambda VL chain.

[0053] Wh...

example3

Targeting the scFv to a Cellular Compartment and Expression of Secretory scFv

[0054] In eukaryotic cells, scFvs can be targeted to specific subcellular compartments by engineering the nucleotide sequence to express a protein with the appropriate signal sequences. In this way the scFvs can be modified to be directed to a subcellular compartment where the antibody might prove to be most effective. Recently, Lotti et al. showed that the C-terminal sequence KKXX from the adenovirus E19 protein would enhance the localization of the protein to the cis-golgi complex with some retention in the ER (18). To direct the scFv to the cytoplasm, the hydrophobic amino acid core of the immunoglobulin secretory signal sequence was removed (19). The addition of a nuclear localization signal from the large T-antigen of SV40 virus, PKKKRKV (SEQ ID No. 3), to the N-terminal end can target the scFv to the nucleus (20).

[0055] To target mitochondria, the N-terminal presequence of the subunit VIII of human...

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Abstract

Human anti-epidermal growth factor receptor (EGFR) single-chain antibodies (scFvs) were isolated from a human IgM phage display library using purified epidermal growth factor receptor as antigen. Two isolates with different amino acid sequences were identified by ELISA as epidermal growth factor receptor-specific. The scFvs bind to the full length epidermal growth factor receptor and the truncated and / or mutated epidermal growth factor receptor on human cells. These anti-EGFR-scFvs are useful as therapeutic and / or diagnostic agents.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This is a divisional application of U.S. Ser. No. 10 / 703,277, filed Nov. 6, 2003, which is a divisional of U.S. Ser. No. 09 / 976,118, filed Oct. 12, 2001, now issued as U.S. Pat. No. 6,699,473, which claims benefit of provisional U.S. Ser. No. 60 / 240,353, filed Oct. 13, 2000, now abandoned.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates generally to the fields of molecular biology and monoclonal antibody technology. More specifically, the present invention relates to human single-chain antibodies that bind specifically to the epidermal growth factor receptor. [0004] 2. Description of the Related Art [0005] The epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein consisting of an extracellular ligand binding domain, a transmembrane region and an intracellular domain with tyrosine kinase activity. The binding of growth factors, EGF or TGFa, to the epidermal growth ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K51/00A61K39/395C07K16/28C07K16/46A61K45/00C12N15/09A61K51/10
CPCA61K51/103A61K2039/505C07K16/2863C07K2317/21C07K2317/622
Inventor RAISCH, KEVINCURIEL, DAVIDBONNER, JAMES
Owner UAB RES FOUND
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