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Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies

a technology of soluble polypeptide fractions and lag-3 proteins, which is applied in the field of soluble forms derived from the lag3 membrane protein, can solve the problems of not enabling the detection of significant decreases in viral titres

Inactive Publication Date: 2003-11-11
MERCK SERONO SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The soluble polypeptide fraction comprises, in particular, a peptide sequence derived from these domains by replacement, addition and/or deletion of one or more amino acids, and which possess a specificity equal to or greater than that of LAG-3 for its ligand, for example the whole of the first two immunoglobulin type domains of LAG-3, or the 4 immunoglobulin type domains of the extracytoplasmic domain of LAG-3.
Advantageously, the soluble polypeptide fraction is comprised of all or part of at least one of the four immunoglobulin type extracellular domains

Problems solved by technology

Nevertheless, clinical trials with soluble CD4 molecules, in particular of CD4-Ig, have not enabled a significant decrease in viral titres to be demonstrated.

Method used

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  • Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies
  • Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies
  • Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies

Examples

Experimental program
Comparison scheme
Effect test

example 1

Proliferation of active T lymphocyte lines in the presence of anti-LAG-3 monoclonal antibodies

The anti-LAG-3 monoclonal antibodies used were 17B4, described in BAIXERAS et al. (2) and deposited at the CNCM under No. I-1240 on Jul. 10, 1992, and 11E3, described in HUARD et al. (8).

These antibodies belong to the isotype IgG1. These antibodies were tested for their biological effects on activated T lymphocytes, stimulated by specific antigenic peptides or processed antigens presented by MHC Class II molecules expressed by autologous antigen presenting cells, expressing LAG-3.

An anti-CD48 monoclonal antibody designated 10 H3 was used as irrelevant IgG1 antibody (negative control).

The saturating concentrations of anti-LAG-3 and anti-CD48 antibodies were determined by immunofluorescence on PHA (phytohaemagglutinin)-blasts and cell lines transformed by Epstein-Barr virus (EBV). In the proliferation tests, the monoclonal antibodies were added in the proportion of 5 times the saturating conc...

example 2

Transient expression of LAG-3 fusion proteins

Soluble proteins derived from LAG-3 were obtained by a recombinant DNA technique using suitable vectors comprising DNA coding for LAG-3 and DNA coding for an immunoglobulin fragment. The transient expression system consisted of transfected Cos cells. This system makes it possible to produce several mg of recombinant fusion proteins. Recombinant DNA techniques were carried out as described by MANIATIS et al. (22). The modifications were made as recommended by the manufacturer.

Construction of LAG-3 D1-D4 Ig and LAG-3 D1D2 Ig

Fragments coding for the D1D2 or D1-D4 regions were amplified (30 cycles) from a fragment of cDNA (FDC sequence) encompassing LAG-3 cDNA (TRIEBEL et al. (1)), using Taq polymerase free from 5'-endonuclease activity and relatively resistant to an exposure to very high temperature; the amplification was followed by a denaturation at 98.degree. C. (with a Perkin Elmer Cetus "DNA thermal cycle"). Specific primers were used a...

example 3

Production of soluble subfragments of LAG-3

In order to produce large amounts of recombinant proteins, a stable expression system consisting of transfected mammalian cells was developed. The host cells are anchorage-dependent hamster ovary (CHO) cells isolated from CHO cells deficient in dihydrofolate reductase (DHFR) and consequently necessitating glycine, a purine and thymidine for their growth. The pivotal role of DHFR in the synthesis of nucleic acid precursors, combined with the sensitivity of DHFR-deficient cells with respect to tetrahydrofolate analogues such as methotrexate (MTX), has two major advantages. Transfection of these cells with expression vectors containing the DHFR gene permits the secretion of recombinant DHFR-resistant clones, and the culturing of these cells on selective media containing increasing amounts of MTX results in amplification of the DHFR gene and the DNA associated therewith.

Construction of LAG-3 D1, LAG-3 D1D2, LAG-3 D1-D4

Fragments of DNA coding fo...

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Abstract

Soluble polypeptide fraction consisting of all or part .[.one.]. .Iadd.of .Iaddend.at least .Iadd.one .Iaddend.of the four immunoglobulin-type extracellular LAG-3 protein domains (amino acids 1-159, 160-.[.230.]. .Iadd.239.Iaddend., 240-330 and 331-412 of the SEQ ID NO:1 sequence) or consisting of one peptide sequence derived from these domains by replacement, addition or deletion of one or more amino acids. The fraction of the invention has a specificity at least equal to that of LAG-3 in relation to its ligand.

Description

BACKGROUND OF THE INVENTION1. Field of the InventionThe invention relates to soluble forms derived from the LAG-3 membrane protein which are useful as immunosuppressants, as well as antibodies capable of preventing the specific binding of the LAG-3 protein to MHC (major histocompatibility complex) Class II molecules as immunostimulants.2. Description of the Related ArtIn WO-A 91 / 10682, a protein designated LAG-3 has been described.The LAG-3 protein is a protein selectively expressed by NK cells and activated T lymphocytes. Similarity of the amino acid sequence, the comparative exon / intron organization and the chromosomal localization show that LAG-3 is related to CD4. The initial characterization of the LAG-3 gene has been described by TRIEBEL et al. (1).The corresponding DNA codes for a type I transmembrane protein of 498 amino acids containing 4 extra-cellular sequences of the immunoglobulin type. LAG-3 is a member of the immunoglobulin superfamily.The mature protein comprises 476...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K14/705C07K19/00A61K38/00C12N15/09A61K38/17A61K39/395A61P37/06C07K16/28C07K16/42C12N15/12C12P21/02C12P21/08C12R1/91
CPCA61K38/00C07K14/70503C07K16/2803C07K19/00C07K2319/00A61P37/06A61K38/17C07K14/705
Inventor FAURE, FLORENCEHERCEND, THIERRYHUARD, BERTRANDTRIEBEL, FREDERIC
Owner MERCK SERONO SA
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