Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70514—CD4
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • C07K14/245—Escherichia (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

• the invention relates to polypeptides resulting from the fusion between, on the one hand, at least a part of the CD4 molecule (also called T4), more particularly that comprising the region near its N-terminal end which contains a site. of fixation for HIV viruses and, on the other hand, a bacterial periplasmic protein affine maltose (MalE). It also relates more particularly to hybrid polypeptides of this type, or even the aforementioned part of CD4 molecule separated from such hybrid polypeptides, more particularly when these various polypeptides consist of products of expression in E. coli of DNA sequences.
• CD4 molecule also called T4
• MalE bacterial periplasmic protein affine maltose
• these expression products having been exported into the bacterial periplasm, extracted and purified, preferably in a single step on affinity column and, if necessary, treated with selected proteases to recover a polypeptide whose essential sequence consists of that of the CD4 protein or of the above-mentioned part of CD4 protein, from these hybrid polypeptides.
• HIV relates to any retrovirus capable of inducing AIDS in the host, in particular man.
• HIV refers to HIV1, HIV2 or any pathogenic variants thereof.
• the figures surrounded by signs in parentheses refer to the bibliography attached to this text.
• the CD4 protein is an integral glycoprotein of the T-cell membrane (helper / inducer).
• the part External located on the N-terminal side) consists of four domains similar to those of the light chains of immunoglobulins. It is followed by a transmembrane part and a cytoplastic part (1).
• This protein serves as a receptor for the HIV virus (responsible for AIDS) through an interaction with the viral protein gpl20 (2). It has been shown that the first two domains (which do not have glycosylation sites) contain the gpl20 binding site as well as those of the anti-CD4 monoclonal antibodies, named I0T4 and OKT4A and having a neutralizing power on viral infectivity ( 3), (4), (5).
• a truncated protein, containing these two domains, and secreted by eukaryotic cells has been shown to have the ability to inhibit viral infection in in vitro tests (3), (4), (6), (7 ), (8), (9) and in the rhesus monkey (10).
• the hybrid proteins can also be expressed in eukaryotic cells such as the C1102 line (embryonic hamster fibrioblasts) when they are transfected with constructs associating the MalE and CD4 genes fused to transcription signals specific to eukaryotic cells (for example origin of replication and polyadenylation signals of the SV40 virus). Techniques known to those skilled in the art are applied for this.
• hybrid proteins the purification of which is simple and rapid. Indeed, like the protein MalE, the hybrid protein is capable of being produced in abundance and of being purified in one step on an amylose column with elution by maltose at neutral pH.
• the invention takes advantage of the fact that this environment appears in gram negative bacteria, including in particular E. coli, to promote both the establishment of disulfide bridges which are important for the biological activity of CD4, more particularly of the N part. -terminal of the latter, and this particularly when the sequence originating from CD4 and present in the polypeptide is devoid of the C-terminal region of CD4.
• this technique allows an evaluation of its therapeutic power of the CD4 protein, in the hybrid state, and gives access to certain studies such as: selection of CD4 mutants having modified properties ( stability, interaction with the virus).
• selection of CD4 mutants having modified properties stability, interaction with the virus.
• the omission of the C-terminal part of the CD4 molecule is accompanied by very increased production yields of the hybrid protein in host cells, in particular E. coli.
• the invention relates more particularly to a hybrid polypeptide containing a peptide chain derived from the fused CD4 protein, upstream from its N-terminal end with a first sequence and, where appropriate, downstream from its C-terminal end, with a second sequence, this first sequence and, where appropriate the second sequence being respectively derived of the protein MalE, this hybrid polypeptide being more particularly characterized
• the peptide chain derived from the CD4 protein contains at least the N-terminal region portion of this protein which itself comprises a binding site for the HIV virus and
• said first sequence derived from MalE and, where appropriate, the second have, when recourse to production techniques by genetic engineering for the production of the hybrid protein, lengths sufficient for the hybrid polypeptide to retain both the properties of the protein MalE with regard to its transport in the transfected microorganism then used, in particular E. coli outside the cytoplasm and for export in the periplasm of this organism and 1 • specific affinity of MalE for maltose or ylose it.
• a preferred hybrid polypeptide of the invention lacks the above second sequence of MalE. It has indeed been found that these latter types of hybrid polypeptides were much better tolerated by the bacteria in culture which produced them than those in which the sequence derived from CD4 was inserted between the two previously indicated sequences of MalE. It is also remarkable that the activity specific to the CD4 protein is conserved, even though the latter is fused with the chain originating from MalE at its own N-terminal end, itself close to a binding site. on HIV type viruses.
• the hybrid polypeptide according to the invention contains at least region VI of the CD4 protein, or even also region V2. It is more particularly devoid of the V3 and V4 regions of CD4.
• the invention relates more particularly still to the hybrid polypeptides as defined above, when they have been produced in a gram negative bacterium, such as E. coli, under the conditions mentioned above, or a polypeptide derived from the above, after separation of the chains containing the sequences derived from MalE.
• polypeptides according to the invention can be produced in particular by the techniques described in European patent application No. 299,810, to which reference was made in the foregoing. These are the same techniques which have been used in the examples Other characteristics of the invention also result from the description of these examples considered in combination with:
• polypeptide according to the invention contains the region which extends between amino acids 1 and 177 of the N-terminal part of the CD4 protein:
• the invention relates to any hybrid polypeptide of the above-mentioned genus, in which only part of the amino acid sequence 1-177 is contained, but which, like this one, is capable of ensuring the export of the polypeptide hybrid outside the cytoplasm.
• the hybrid protein contains at least the amino acid sequence extending from the 15th to the 85th amino acid (region VI) of CD4, in the absence of protein VI. It can also include the sequence extending between the 15th amino acid and the 160th amino acid of the previous sequence (regions VI and V2).
• deletions or substitutions can be made locally within the sequence amino acids, as long as they do not affect the ability of the MalE region to transport the expressed hybrid protein from the corresponding nucleic acid sequence.
• the invention thus makes it possible to obtain hybrid polypeptides formed between the amino acid sequence chosen within the N-terminal region of CD4 and a MalE protein which have at least the same level of activity as the soluble CD4 molecule. produced in mammalian cells (CHO). This similarity of activity is assessed by:
• the overall structure of the CD4 protein is shown schematically in FIG. 1, in which: - the part represented by a thickened line (and to which the arrow starting from HIV refers) corresponds to the fixation site to which reference is more particularly made in the present text, the designations to the right of the schematic representation of protein refers to the different parts of the protein (VI, V2, V3 and V4), as well as to positions of the CD4 protein sites recognized by the monoclonal antibodies identified in the figure,
• G refers to the normally glycosylated sites of the CD4 protein
• E extracellular
• M membrane
• C cytoplasmic
• fig. 1 Two expression vectors carrying a chimeric gene (fig. 1) were constructed. The first determines a hybrid protein (CIMER) where the 177 amino acids of the N-terminal part of CD4 are fused to the C-terminal end of MalE (369 amino acids). The second code for a hybrid protein (CISREM) comprising the same CD4 fragment fused this time at the N-terminal end of the MalE protein. In the latter case, in order to facilitate export into the periplasm, the N-terminal part of CD4 was itself fused to the signal peptide of MalE: the soluble part of CD4 is therefore found between the signal peptide of MalE and the mature protein (see fig. 1).
• CIMER hybrid protein
• CISREM hybrid protein
• FIG. 2 The constructions in question are schematically represented in FIG. 2: the main characteristics are recalled below (appreciated in combination with fig. 2).
• a DNA fragment (Fnu4H-NdeI) containing the codons -4 to +177 of the CD4 gene was inserted into a plasmid (derived from pBR322) carrying the genes for resistance to ampicillin and to tetracycline, the gene lacl ⁇ and the MalE gene placed under the control of the tac promoter (IPTG inducible).
• An adapter was inserted at the distal end of MalE, so that the insertion of CD4 is in phase at the last codon of malE.
• the structure of the protein is indicated above the diagram representing that of the plasmid.
• CD4-MalE fusion CISREM
• the same CD4 fragment was inserted downstream of the malE signal sequence (after the 27th codon of the mature protein).
• all of malE (codons -2 to +370) was inserted in phase after the 177th codon of CD4.
• the rest of the plasmid (resistance to ampicillin and tetracycline, lad 0 gene - and tac promoter) are identical to the previous one.
• the structure of the protein is indicated above the diagram representing that of the plasmid.
• the transcription of the chimeric genes is placed under the control of the tac promoter (13). It is insensitive to catabolic repression and can be induced at will by the addition of IPTG (Isopropyl 3-D-thiogalactoside): the presence on the plasmid of the lacl 0 - gene (which determines a repressor which binds to the tac promoter) ensures a low level of expression in the absence of an inducer.
• IPTG Isopropyl 3-D-thiogalactoside
• strains of E. coli carrying the mutations Ion " and degP which inactivate cytoplasmic and periplasmic proteases respectively (14), (15).
• the cells carrying the expression vectors are induced for three or four generations at 30 ⁇ C, they produce a protein mainly exported (CIMER) or partially exported (CISREM) in the periplasm, identifiable by its size (60 KD), and by the specific antibodies directed against its antigenic determinants MalE and CD4.
• CIMER protein mainly exported
• CISREM partially exported
• the fusion proteins are capable of complementing a strain deleted for MalE (normal growth in synthetic medium containing maltose as the sole source of carbon).
• MalE normal growth in synthetic medium containing maltose as the sole source of carbon.
• CISREM overproduction of CISREM tends to become toxic to bacteria while that of CIMER is not.
• the latter protein has been purified and its properties have been studied.
• the MalE part maintains its recognition site for maltose and maltodextrins.
• periplasmic content osmotic shock
• amyloidosis column The passage of the periplasmic content (osmotic shock) on an amyloidosis column leads to the specific binding of a fraction of the periplasmic proteins to amyloidosis. This fraction can be eluted from the column with maltose and purified in a single step with a yield of the order of 500 ⁇ g of hybrid protein per g of bacteria.
• the molecular weight of 60 kD was assessed by comparison of the gel migration distances of the hybrid protein and of known high molecular weight reference proteins respectively, under the conditions described in connection with the implementation. of the kit high molecular weight Amersham RAINBOW.
• the above reference proteins consisted of the following proteins:
• the CD4 part of the hybrid protein is capable of binding the HIV viral particles via their envelope protein gp! 20.
• i Experiments using "neutralizing" monoclonal antibodies show that the latter (I0T4 and 0KT4A already mentioned) react with the hybrid protein in ELISA tests or by immunoprecipitation at the same concentrations as soluble CD4 purified from mammalian cells.
• ii A direct interaction between the hybrid protein and a purified protein gpl20 or gpl60 of HIV1 could be demonstrated by co-immunoprecipitation and by immunoblotting.
• iii Finally, bringing the hybrid protein into contact with viral particles "neutralize" the latter.
• hybrid proteins results of fusions between the bacterial gene malE and a fraction of the human CD4 gene, and expressed in E. coli therefore retain the properties of the two components, in particular the neutralizing power of CD4 towards the HIV virus, in particular HIV1.
• the invention therefore also relates to compositions intended for therapeutic use in vivo, containing, in association with a pharmaceutical vehicle suitable for the chosen mode of administration, in particular parenterally, a dose effective for neutralizing in retrovirus a retrovirus belonging to the class of HIV, of the hybrid polypeptide MalE-CD4, as defined above or of the soluble peptide CD4 derived from the above-mentioned hybrid polypeptide, after cleavage and separation of its MalE sequences.
• the above effective dose may vary from patient to patient and should be appreciated by the clinician each time.
• the daily doses should be between 100 ⁇ g and 10 g, for example being of the order of 1 gram.
• hybrid proteins according to the invention can be produced in large quantities.
• the use of. coli as a CD4 expression agent makes it possible to envisage the introduction of modifications at the level of the CD4 gene and the development of selection techniques making it possible to isolate even more efficient mutants.
• These hybrid proteins also allow studies of CD4 structure, for example by X-rays. They can also be applied, after fixation on an affine column, in particular based on polymerized maltose or amylose, to the purification of HIV virus contained in a culture medium, in particular by selective retention of virus when such a medium is brought into contact with such an affine column.
• the invention also relates to a method of in vivo detection of the possible presence of HIV virus or of antigens of this virus in a biological sample, this method comprising bringing this biological sample into contact with a polypeptide, hybrid or not, such as defined above, under conditions authorizing the binding of this polypeptide to HIV or the HIV antigen possibly present and the detection of the complex possibly produced.

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• 1989
  • 1989-03-17 FR FR8903557A patent/FR2644463A1/fr not_active Withdrawn
• 1990
  • 1990-03-16 EP EP19900905557 patent/EP0426787A1/fr not_active Withdrawn
  • 1990-03-16 JP JP50532590A patent/JPH03504729A/ja active Pending
  • 1990-03-16 WO PCT/FR1990/000182 patent/WO1990011360A1/fr not_active Application Discontinuation

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