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Multivalent antigen-binding proteins

A technology for binding proteins and multivalent antigens, applied in the direction of hybrid immunoglobulin, anti-bacterial immunoglobulin, anti-hormonal immunoglobulin, etc., can solve problems such as obstacles and affecting binding activity, and achieve small size, economy and effectiveness The effect of mass production

Inactive Publication Date: 2000-12-27
贝克知产公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In fact, in the absence of conformational constraints inherent in the presence of the corresponding light chain at the binding site, it is generally believed that the binding regions in this type of structure hinder each other, adversely affecting binding activity unfavorably

Method used

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  • Multivalent antigen-binding proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1. Induced humoral immune response in llamas

[0075] Male llamas were immunized subcutaneously and intramuscularly with water-in-oil emulsion (1:9 V / V, antigen in water: Specol (Bokout et al.)). 0.75-1.5 ml water-in-oil emulsion per immunization site, vaccination contains: 100 g antigen. Antigens used were: hCG (Sigma), azo-stained RR6 (ICI) linked to BSA via a reactive triazine group, and Streptococcus mutant HG982 cells. Immunizations were given according to the following schedule: the second immunization three weeks after the first immunization. The third time was performed two weeks after the second time. Antigen-specific ELISAs track the immune response.

[0076] Anti-RR6 responses were measured using Nunc Covalink plates, which were stained with azo dyes. After incubation with (diluted) serum samples, they were incubated with polyclonal rabbit-anti-llama antiserum (obtained by immunizing rabbits with llama antibodies conjugated to llama immunoglobulin...

Embodiment 2

[0077] Cloning, expression and screening of embodiment 2 llama HC-V fragments

[0078] 2.1 Isolation of gene fragments encoding llama HC-V region

[0079] 200 ml of blood samples from immunized llamas were centrifuged to obtain enriched lymphocyte populations by step centrifugation in Ficoll (Pharmacia), and total RNA was isolated by acid guanidinium thiocyanate extraction (for example, by the method of Chomczynnski and Sacchi, 1987). After synthesizing first-strand cDNA (for example, with the Amersham First-Strand cDNA Kit), PCR amplify the DNA fragment encoding the HC-V segment and part of the long or short hinge region using special primers:

[0080] PstIVH-2B 5'-AGGTSMAR CTGCAG SAGTCWGG-3' (see SEQ.ID.NO:2) S=C and G, M=A and C, R=A and G, W=A and T,

[0081] Hind IIILam-075'-AACAGTT AAGCTT CCGCTTGCGGCCGCGGAGCTGGGGTCTTCGCTGTGGTGCG-3'

[0082] (short hinge) (see SEQ.ID.NO: 3)

[0083] Hind IIILam-0...

Embodiment 3

[0129] Example 3 Preparation of llama HC-V double heads by Saccharomyces cerevisiae

[0130] 3.1 Construction of episomal expression plasmid encoding anti-hCG / anti-RR6 bispecific double head

[0131] In the anti-hCG HC-V fragments H14 and HI15 (anti-α-subunit), the PstI site was removed and the XhoI site was introduced by PCR using the following primers: °C

[0132] MPG158WB

[0133] XhoI5'-GAATTAAGCGGCCGCCCAGGTGAAACTG CTCGAG TCWGGGGGA-3' (see SEQ.ID.NO: 27) and MPG159WB

[0134] BstEII 3'-CCCTGGGT CCAGTGG CAGAGGAGTGGCAGAGGAGTCTTGTTT-5' (see SEQ.ID.NO: 28) sequence in this way:

[0135] PstICAG GTC CAG CTG CAG GAG TCT GGG Q V Q L Q E S G (see SEQ.ID.NO:29 and NO:30) becomes

[0136] XhoICAG GTG AAA CTG CTC GAG TCW GGG Q V K L L E S G (see SEQ.ID.NO:31 and NO:32)

[0137] The PCR fragment was digested with XhoI and BstEII, and the 330bp fragment was purified by agarose gel electrophoresis and separated ...

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Abstract

A multivalent antigen binding protein comprises a single polypeptide chain comprising, in series, two or more single domain binding units which are preferably heavy chain variable domains derived from an immunoglobulin naturally devoid of light chains. Methods for their production and uses thereof, in particular for diagnosis, immunoassay and purification methods are disclosed.

Description

field of invention [0001] The present invention relates to multivalent and multispecific antigen binding proteins, methods for their preparation and their use. In particular, the invention relates to polypeptides comprising two or more single domain binding units, wherein the single domain binding units are preferably derived from the heavy chain variable domains of immunoglobulins naturally devoid of light chains. Background technique [0002] Antibodies are protein molecules belonging to the group of immunoglobulins produced by the immune system in response to antigens. The structure of most antibody molecules is based on a unit comprising four peptides, two identical heavy chains and two identical light chains, covalently linked by disulfide bonds. Each strand folds in a discontinuity. The C-terminal regions of the heavy and light chains are conserved in sequence and are called constant regions. The N-termini of the heavy and light chains, also known as the V regions, ...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61K39/395C07K16/00C07K16/12C07K16/26C07K16/44C07K16/46C07K19/00C12N1/15C12N15/13C12N15/62G01N33/531G01N33/577
CPCC07K16/468C07K16/26G01N33/531A61K38/00C07K2317/569C07K2317/34C07K16/44C07K2317/22C07K16/1275Y10S977/927C07K2319/00C07K16/00
Inventor L·G·J·弗伦肯S·豪厄尔A·M·莱德伯尔C·P·E·范德洛格特
Owner 贝克知产公司
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