Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-03-15
2002-07-16
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C424S208100, C530S300000, C530S350000
Reexamination Certificate
active
06420141
ABSTRACT:
This application is a 371 of PCT/FR98/02727 filed Dec. 14, 1998.
Aids is induced by HIV-1 virus infection. Such virus consists in various proteins. Amongst such proteins, Tat is together with Nef protein produced the earliest in the viral cycle.
To fight against the effects of Tat protein various approaches have been proposed including genetic approaches to block Tat transcriptional action. Thus, clinical assays have taken place with antisense (Hybridon company), in vitro experiments have shown feasibility of ribozymes or mimics using the RNA of the TAR region (Tat binding site to mRNA).
Pharmacological molecules as inhibitors of Tat transcriptional effect have also been disclosed.
All these methods aim at blocking virus replication by blocking Tat transcriptional action.
Immunological methods have also been proposed for a long time.
For a specific immunization against native Tat protein, WO-A-91/18454 reports the use of polypeptides from retroviral proteins including native Tat protein, being obtained by proteolytic cleavages, chemical synthesis or genetic recombination, as immunogens to prevent the retroviral replication and the cytotoxic activity against in particular lymphocytes and nervous cells effected by retroviral proteins, in particular Tat. The toxicity of native proteins or the fragments thereof prevents however their use for an immunization.
WO-A-95/31999 reports the use of native Tat protein of HIV-1, fragments of said protein or polypeptides with deletions/substitutions as immunogens so as to block HIV-1 replication by blocking Tat extracellular protein capture by uninfected cells. In spite of the described immunogenicity, given the toxic effects of the native Tat protein in particular, the immune troubles it induces, and of the absence of neutralizing antibodies through immunization with the immunogens described therein, there is indeed a need to develop new inactivated Tat immunogens (which have lost all the nocuous effects from the native Tat protein) being adapted to induce a humoral and cellular immune response.
WO-A-96/27389 reports the use of new products, Tat toxoids and retroviral regulating proteins as immunogens adapted to induce an immune response against native Tat protein and prevent or amend the immunosuppressive effects thereof. Such toxoids, being inactive but immunogenic products, have been prepared by formaldehyde chemical treatment of the native protein or segments derived from such protein. However inactivation with aldehyde is uneasy. And for an industrial production regular results are necessary, mainly for inactivation. Moreover, Health agencies prefer products with a constant and well defined structure to authorize the sales thereof. An ideal product should further have a good stability over the time.
That is the reason why the present application aims at providing new toxoids and a simple and efficient new method to manufacture such toxoids, for example from HIV-1 regulating proteins which participate in the HIV-1 immunosuppressive effect or from alpha interferon the production of which is deranged by HIV-1 infection.
The toxoid strategy has great advantages with respect to aminoacid substitutions/deletions: the regions of native Tat protein which are responsible for the Tat pleomorphic effects are not well identified and it is difficult to predict a priori the substitutions/deletions which provide a total innocuity for the preparation thereof. For example mutation of C25 residue in G cancels Tat transactivating effect without suppressing the immunosuppressive effect thereof. On the other side, substitutions/deletions may alter strongly the structure of the modified molecule with respect to the native molecule (linear and conformational antigens) and thus prevent the development of an efficient immune reaction against the native molecule. This last point is particularly important for generating neutralizing antibodies against native protein.
An object of the present application is thus to provide a viral regulation protein or a fragment of a viral regulation protein or alpha interferon or a fragment of alpha interferon, characterized in that it is carboxymethylated.
In preferred implementation conditions for the invention, the above-mentioned protein or fragment comes from HIV-1, HIV-2, HTLV-1 or HTLV-2 virus, particularly HIV-1 or HIV-2 virus.
In other preferred implementation conditions for the invention, the above-mentioned protein or fragment is a viral regulation protein or a fragment of a viral regulation protein.
In further preferred implementation conditions for the invention, the above-mentioned protein or fragment comes from Tat, particularly from HIV-1.
Another object of the present application is also to provide a process for producing a protein or a fragment such as defined here-above, characterized in that said viral regulation protein or said fragment is submitted to a carboxymethylation step to obtain the expected compound which is isolated.
According to the invention, the proteins or fragments which have the same behaviour as toxins on the immune system, either the overproduced IFN&agr;, Tat or Nef, for example are modified with the carboxymethylation. Such chemical modification leads to new proteins or fragments which are biologically inactive, but hold their immunogenicity. In other words, such proteins or fragments which have the same behaviour as toxins on the immune system are recognized by antibodies produced against the carboxymethylated proteins or fragments according to the invention. Such carboxymethylated proteins or fragments hold enough immunogenic properties to create antibodies neutralizing or blocking said native protein and simultaneously have lost at least 90%, preferably at least 95%, more preferably at least 99% of the toxic biological properties of said native protein, namely its well known usual biological properties.
The carboxymethylated immunogenic compound according to the invention may consist in the totality or a fragment of the protein, including regulation protein, and may comprise, as it is well known by the skilled man in the art, one or more modifications in the aminoacids of such protein or fragment such as deletions, substitutions, additions or finctionalizations such as acylating aminoacids, inasmuch such modifications stay in the above-mentioned limits (no toxicity, immunological characters). For example, generally substituting an isoleucine residue for a leucine residue does not modify such properties. The modifications should generally concern less than 30% aminoacids, preferably less than 20%, more preferably less than 10%.
A fragment may comprise from 8 to 110 aminoacids for example, preferably from 12 to 60 aminoacids, more preferably from 12 to 40 aminoacids. Such a fragment may also comprise on the C- or N-terminal side(s) from 1 to 5 supplementary aminoacids, i.e. different from the origin segment. A fragment should also comprise one cystein at least to allow for carboxymethylation.
Generally, as to the modifications, the homology or similarity between the modified immunogene and the protein or part of native protein as well as the dimensions of the immunogenic compound and the use procedures or coupling of the immunogenic compound according to the invention with an immunogenic protein such as the tetanus toxoid, it may be referred in particular to equivalent WO-A-86/06414 or EP-A-0,220,273 or further PCT/US.86/0083 1 the teaching of which is incorporated therein by reference.
The immunogenic compounds according to the invention derived from regulation proteins will be called sometimes below in the experimental part “viral toxoids”.
In fact, as for the conventional bacterial toxoids, they are free from own toxicity, but are adapted to provoke an immunization per administration to a subject.
To verify that the native regulation protein is well recognized by antibodies raised against said modified regulation protein, it is possible for example to check immunologically through Elisa in the presence of specific antibodies, the building of antigen-antibody complexes, as it will b
Carcagno Miguel
Rappaport Jay
Zagury Jean-Francois
Bierman, Muserlian and Lucas
Neovacs
Park Hankyel T.
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