Catalytic antibodies and a method of producing same

a catalytic antibody and antibody technology, applied in the field of growth factor precursors, can solve the problems of difficult to predict the structure of transition state analogs, ineffective recombinant techniques in generating all components of humoral responses, and inefficient immunisation of mice with transition state analogs

Inactive Publication Date: 2009-10-29
KOENTGEN FRANK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]Yet another aspect of the present invention provides a growth factor precursor comprising a recombinant polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least two B cell surface molecule binding portions, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both...

Problems solved by technology

However, recombinant techniques have not been fully effective in generating all components of the humoral response.
However, this approach has a severe limitation in that it is difficult to predict the structure of transition state analogs which effect proteolysis of specific proteins.
Immunising a mouse with a transition state analog is by definition ineffici...

Method used

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  • Catalytic antibodies and a method of producing same
  • Catalytic antibodies and a method of producing same
  • Catalytic antibodies and a method of producing same

Examples

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example 1

Generation of LHL from Synthetic Oligonucleotides

[0152]LHL was generated from three overlapping synthetic oligos, a 115mer, a 116mer and a 105mer, using the proofreading DNA polymerase Pfu in two 20 cycle PCR reactions. The two PCR products (290 bp and 200 bp) were purified and blunt end cloned into the expression vector pASK75. The sequence was verified by automated sequencing. All subsequent PCRs were done in a similar fashion as described in the literature. The nucleotide and corresponding amino acid sequence for LHL is shown in SEQ ID NO: 1 and SEQ ID NO:2 respectively.

example 2

Expression of LHL in E. coli and Purification Over a Human IgG (huIgG) Affinity Column

[0153]The expression vector pASK75 directs protein expression via the ompA signal peptide into the periplasm of E. coli. Protein expression was induced with 200 ng / ml anhydrotetracycline for 16 hrs in midlog E. coli DH10B cultures. Cells were lysed and soluble LHL purified (>95%) over a huIgG affinity column. Extensive washes with 0.5% v / v Triton X-100 were performed on the affinity column in order to eliminate endotoxins from the preparations. Expression levels were estimated at 200 mg per litre of culture.

example 3

Generation of an LHL Protein Carrying the N-Terminal Flag Epitope and the C-Terminal Strep-Tag

[0154]A form of LHL (referred to herein as “LHL.seq”) was generated by PCR containing the FLAG epitope at its N-terminus and the so called strep-tag at its C-terminus. The nucleotide and corresponding amino acid sequence for LHL.seq is shown in SEQ ID NO:7 and SEQ ID NO:8, respectively. The FLAG epitope comprises the amino acids DYKDDDDK (SEQ ID NO:9) and the strep-tag the amino acids AWRHPQFGG (SEQ ID NO: 14). The FLAG epitope is recognised by several anti-FLAG monoclonal antibodies and the strep-tag by streptavidin. The strep-tag was used for purification of LHL.seq over a streptavidin column. LHL.seq was washed with 0.5% v / v Triton X-100, Tween20 and PBS while bound to the column in order to minimise endotoxin levels. LHL.seq was eluted with either 100 mM glycine pH2.0 or with 1 mg / ml diaminobiotin in PBS. In this method LHL.seq was not purified on the basis of binding immunoglobulin, th...

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Abstract

The present invention relates generally to a growth factor precursor and its use to select production of antigen specific catalytic antibodies. Such catalytic antibodies are produced following B cell activation and proliferation induced by catalytic cleavage products of a target antigen portion of the growth factor precursor of the present invention. A particularly useful form of the growth factor precursor is as a nucleic acid vaccine. The nucleic acid vaccine of the present invention preferably further comprises a molecular adjuvant. Another aspect of the present invention comprises a growth factor precursor in multimeric form. The growth factor precursor of the present invention is useful for generating catalytic antibodies for both therapeutic, diagnostic and industrial purposes.

Description

FIELD OF THE INVENTION[0001]The present invention relates generally to a growth factor precursor and its use to select production of antigen specific catalytic antibodies. Such catalytic antibodies are produced following B cell activation and proliferation induced by catalytic cleavage products of a target antigen portion of the growth factor precursor of the present invention. A particularly useful form of the growth factor precursor is as a nucleic acid vaccine. The nucleic acid vaccine of the present invention preferably further comprises a molecular adjuvant. Another aspect of the present invention comprises a growth factor precursor in multimeric form. The growth factor precursor of the present invention is useful for generating catalytic antibodies for both therapeutic, diagnostic and industrial purposes.BACKGROUND OF THE INVENTION[0002]The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied...

Claims

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Application Information

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IPC IPC(8): A61K39/395C12N9/00C07H21/00A61K39/44C07K16/18C12N15/19
CPCC07K16/18C07K2317/622C12N9/0002C07K2319/00C07K2317/624
Inventor KOENTGEN, FRANKSUESS, GABRIELE MARIATARLINTON, DAVID MATHEWTREUTLEIN, HERBERT RUDOLF
Owner KOENTGEN FRANK
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