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Humanised antibodies to epidermal growth factor receptor

A humanized and human antibody technology, applied in the direction of antibodies, anti-receptors/cell surface antigens/cell surface determinant immunoglobulins, antibody mimics/scaffolds, etc., can solve the problem of enhancing the ability of cell growth

Inactive Publication Date: 2003-07-23
SCANCELL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They unexpectedly found that these humanized antibodies bound EGFR-expressing cells similarly to the original murine antibody, but had an enhanced ability to inhibit the growth of these cells

Method used

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  • Humanised antibodies to epidermal growth factor receptor
  • Humanised antibodies to epidermal growth factor receptor
  • Humanised antibodies to epidermal growth factor receptor

Examples

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Embodiment Embodiment 1

[0076] Examples Example 1-Construction of chimeric antibodies from α340

[0077] Use Qiagen RNeasy kit according to the instruction manual from 5×10 6 Total RNA was isolated from hybridoma α340 cells (Durrant et al., Prenatal Diagnostics, 14, 131, 1994). The RNA was converted to cDNA with Promega (Southampton, UK) reverse transcriptase, buffer and dNTP. Variable region heavy chain (V H ) And light chain (V L ) cDNAs were amplified using the primer set of Jones and Bendig (Bio / Technology, 9, 188, 1991). The amplified DNA is gel purified and cloned into the vector pGem  T Easy (Promega). The PCR products were bidirectionally sequenced with 373A Applied Biosystems automatic sequencer (Applied Biosystems, Warrington, UK). V obtained H And V L The DNA sequence is shown in Figure 1, and the protein sequence is shown in Figure 2 (when used here, V L With V K the same).

[0078] The location of the complementarity determining region (CDR) is determined with reference to other antibody se...

Embodiment 2

[0081] Cut out mouse V from pUC19 as a HindIII-BamHI fragment H And V L Expression cassette, which contains mouse heavy chain immunoglobulin promoter, leader signal peptide, leader intron, V H Or V L Sequence and splice site. It was transferred to the expression vectors pSVgpt and pSVhyg (Figure 4 and Figure 5), which contained human IgG1 or K constant regions and markers for selection in mammalian cells, respectively. Confirmed that V in the expression vector H And V L The DNA sequence is correct. Example 2-Design of α340 Deimmunized Sequence

[0082] The following examples describe methods for reducing the human immune response triggered by existing therapeutic antibodies. The realization method consists of two steps: the first step is to compare the mouse heavy chain and light chain sequences with the human germline sequence database. The most similar germline sequence is selected as the human template for the deimmunized sequence, and the changes necessary to transform the mur...

Embodiment 3

[0087] The introduced mutations included two of the α340 antibody CDRs. V K Region CDR1 contains V K I-L mutation of b. V H Region CDR3 similarly contains V H V-A mutation of e. These substitutions have a considerable impact on the antigen binding ability of the α340 antibody, indicating the importance of producing other variants with different mutations. Example 3-Construction of deimmunized antibody sequence

[0088] The deimmunized variable region was constructed by overlapping PCR recombination method. Cloned mouse V H And V K The gene serves as a template for mutagenesis of the framework region into the desired deimmunization sequence. Synthesize several sets of mutagenic primer pairs containing the region to be changed.

[0089] The application of mutagenic primer pairs requires an annealing temperature of 48℃-50℃. All amplifications used pfu turbo proofreading polymerase (Stratagene, La Jolla, California). α340 Chimeric V H And V K The construct is used as a template to int...

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Abstract

The present invention provides a humanised form of the antibody 340 obtainable from the cell line deposited with the ECACC under accession number 97021428. Such antibodies have been found to have an increased ability to kill cells compared to the murine antibody 340. Also provided are nucleic acids encoding such antibodies, as well as the use of the antibodies in medicine, in particular in the treatment of cancer.

Description

Invention field [0001] The present invention relates to humanized antibodies and fragments thereof, in particular, humanized antibodies specific for epidermal growth factor receptor (EGFR). Background of the invention [0002] EGFR is a tumor-associated cell surface antigen and a well-known antibody target. Durrant et al. (Prenatal Diagnosis, 14, 131-140, 1994) describe a mouse monoclonal antibody, called "340", which binds to EGFR with high specificity. The cell line expressing this antibody is deposited in ECACC with the deposit number of 97021428. Monoclonal antibody 340 is directed against the osteosarcoma cell line 791T. Immunoprecipitation studies have shown that 340 recognizes a membrane glycoprotein with a molecular weight of 170kDa from osteosarcoma tumors and placental tissues. The terminal amino acid sequencing of the purified antigen showed sequence identity with the epidermal growth factor receptor. To confirm that the 340 antigen is an EGF receptor, radiolabeled EGF...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K31/7088A61K39/395A61P35/00C07K16/28C07K16/46C07K19/00C12N1/15C12N1/19C12N1/21C12N5/10C12N15/13C12P21/02
CPCC07K2319/00C07K16/467C07K16/2863A61K2039/505C07K2317/56C07K2317/24A61P35/00C07K16/28
Inventor J·R·M·艾里斯L·G·杜兰特
Owner SCANCELL
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