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High-affinity antibodies

A kind of affinity and antibody technology, applied in the direction of antibodies, antineoplastic drugs, chemical instruments and methods, etc., can solve the problems of increasing treatment-related costs and unacceptable clinical side effects

Inactive Publication Date: 2001-10-03
KS BIOMEDIX LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this may cause unacceptable clinical side effects and will also increase treatment-related costs

Method used

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  • High-affinity antibodies

Examples

Experimental program
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Embodiment 1

[0028] The sheep were immunized with carcinoembryonic antigen (CEA) in complete Freund's adjuvant, and then boosted three times with the antigen in incomplete Freund's adjuvant. Animals were sacrificed and lymph nodes removed after the final booster.

[0029] Lymph node cells were washed and fused with sheep heteromyeloma fusion partner SFP3.2. Fused cells were cultured in medium containing HAT (Life Technologies) at approximately 10 6 The total concentration of cells / m was plated. Then conventional EIA and pickling EIA were used to screen the hybridomas secreting high-affinity antibodies against specific antigens in the samples.

[0030] A standard EIA screening assay is performed as follows:

[0031] The Maxisorb assay plate (NUNC) was coated with CEA (0.4 μg / ml CEA in phosphate buffered saline, pH 7.2), 100 μl per well, overnight at 4°C. Plates were then washed 3 times with phosphate buffered saline pH 7.2 containing 0.01% Tween 20 detergent. Then, 200 μl of pH 7.2 PBS...

Embodiment 2

[0036] Single-chain Fv fragments derived from the hybridoma 6H9 described above were produced as follows:

[0037] mRNA was purified from cultured hybridoma cells using oligo-dT cellulose. Single-stranded DNA (cDNA) complementary to mRNA is synthesized by reverse transcription. Universal primers designed from the heavy and light chain constant regions of sheep antibody genes were used in separate reverse transcription reactions to synthesize antibody variable region cDNA.

[0038] The cDNA is then amplified by polymerase chain reaction and double-stranded DNA is generated using primers designed from the heavy and light chain variable region framework sequences. Separate polymerase chain reactions were used to amplify the heavy and light chain regions. The products were then analyzed by agarose gel electrophoresis, and DNA bands corresponding to the light and heavy chain genes were excised from the gel for purification.

[0039] Equimolar amounts of heavy and light chain var...

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Abstract

High-affinity monoclonal antibodies, wherein the affinity is characterised by: (i) incubating first and second samples of the antibody in antigen-coated microtitre plate wells at a concentration chosen to be within the linear part of a standard curve at pH 7.2 for 1 hour at 37 DEG C; (ii) removing unbound antibody from both samples; (iii) incubating the first sample with PBS at pH 7.2 for 1 hour at 37 DEG C, and reducing the pH of the second sample to pH 3 or below and incubating for 1 hour at 37 DEG C; (iv) removing unbound antibody from both samples; (v) incubating both samples with anti-antibody alkaline phosphatase-conjugate for 1 hour at 37 DEG C; (vi) removing unbound conjugate from both samples; and (vii) adding PNPP substrate to the samples, measuring the absorbance of the samples at 405 nm, and determining the amount of antibody bound to antigen, wherein the amount bound in the second sample is > 50 % of that of the first sample.

Description

field of invention [0001] The present invention relates to antibodies and their therapeutic use. Background of the invention [0002] Antibodies have long been recognized as potentially powerful tools for treating cancer and other diseases. However, despite some notable exceptions, this potential has not been universally recognized. [0003] This relative lack of success may be due at least in part to the use of rodent-derived monoclonal antibodies that rarely have -9 M affinity. Antibodies with this level of affinity have limited therapeutic utility because it has proven difficult to deliver enough antibody to the target to achieve useful biological activity. Antibody binding to antigen is reversible and tends to dissociate rather than bind at the antibody concentrations actually used in vivo. In principle, it is possible to counteract the dissociation of the antigen by increasing the antibody concentration. However, this may cause unacceptable clinical side effects an...

Claims

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Application Information

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IPC IPC(8): G01N33/577A61K39/395A61K47/48A61P35/00C07K1/22C07K16/30C07K16/32C12N15/09C12N15/13C12P21/08
CPCC07K2317/622C07K16/3007C07K2317/20A61K47/4843A61K47/6815A61P35/00C07K16/30
Inventor P·J·哈里森
Owner KS BIOMEDIX LTD
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