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  Pharmaceutical Patents  

 

Title:  Mutated immunogenic peptides derived from R9M, polynucleotides coding for same and therapeutic uses thereof
United States Patent: 
7,476,719
Issued: 
January 13, 2009

Inventors: 
Firat; Huseyin (Paris, FR), Langlade-Demoyan; Pierre (Paris, FR), Vilmer; Etienne (Paris, FR), Lemonnier; Franc is (Bourg la Reine, FR), Rohrlich; Pierre (Saint Mande, FR), Yotnda; Patricia (Houston, TX)
Assignee: 
Institut Pasteur, Institut National De La Sante Et De La Recherche Medicale, Assistance Publique Hopitaux De Paris
Appl. No.:  10/448,521
Filed:
 May 30, 2003


 

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Abstract

The invention concerns the optimisation of the wild R9M peptide and the use of the resulting peptides for therapeutic vaccination and/or preventive vaccination against leukemia in humans. More particularly, the invention concerns mutated immunogenic peptides derived from the human TEL/AML1 fusion protein comprising the wild R9M peptide sequence Arg-Ile-Ala-Glu-Czs-Ile-Leu-Gly-Met. The invention also concerns polynucleotides coding for the mutated R9M immunogenic peptides, cellular expression vectors comprising nucleic acid sequences expressing the mutated R9M immunogenic peptides and polyclonal or monoclonal antibodies capable of being fixed on at least one of said peptides/polynucleotides. The invention further concerns the use of said peptides, polynucleotides and/or antibodies for preparing vaccines, anti-tumoral medicines and compositions and for in vitro and in vivo stimulation of the immune response in humans.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to the optimization of the wildtype R9M peptide and to the use of the peptides obtained for purposes of therapeutic vaccination and/or preventive vaccination against leukemia in humans.

More particularly, the invention relates to mutated immunogenic peptides derived from the human fusion protein TEL/AML1 comprising the wildtype R9M peptide sequence (Arg-Ile-Ala-Glu-Cys-Ile-Leu-Gly-Met) (SEQ ID NO: 1), the said peptides being mutated in the R9M peptide sequence. Preferably, the immunogenic peptides of the invention are characterized in that they slow down the development of lymphoblastic tumor cells and induce an immune response to the development of lymphoblastic tumor cells after their administration to a leukemic patient. Among the preferred peptides of the present invention are peptides having as peptide sequence: RIAESILGM (SEQ ID NO: 2), RIAEAILGM (SEQ ID NO: 3), RIAE.alpha.-butILGM (SEQ ID NO: 6), YIAESILGM (SEQ ID NO: 4), YIAEAILGM (SEQ ID NO: 5), and YIAE.alpha.-butILGM (SEQ ID NO: 7).

The invention also concerns the polynucleotides coding for the mutated immunogenic R9M peptides, the cellular expression vectors comprising the nucleic acid sequences expressing the mutated immunogenic R9M peptides and the polyclonal or monoclonal antibodies capable of binding to at least one of the peptides/polynucleotides previously mentioned.

The invention also relates to medicines and pharmaceutical compositions containing the peptides, the polynucleotides and/or the antibodies that are the objects of the invention.

The present invention also relates to the use of peptides, polynucleotides and/or antibodies such as defined above as anti-tumor agents for the preparation of an anti-tumor vaccine and for the stimulation in vitro and in vivo of the immune response in humans.

One of the major advantages of the present invention is that the mutated R9M peptides obtained possess a high affinity for the HLA-A2.01 molecule and form with this molecule a stable complex making them capable of inducing in vivo or in vitro a cytotoxic immune response.

DETAILED DESCRIPTION OF THE INVENTION

The wildtype R9M peptide has the peptide sequence Arg-Ile-Ala-Glu-Cys-Ile-Leu-Gly-Met (RIAECILGM; SEQ ID No. 1). This peptide sequence corresponds to a region of the human fusion protein TEL/AML1 (GENBANK.TM. No. S78496).

The present invention relates to the optimization of the R9M peptide and to the use of the peptides obtained for therapeutic vaccination and/or preventive vaccination against leukemia in humans. The nomenclature used to describe the sequence of the peptides of the present invention is the international nomenclature using the three letter code or the one letter code, and in which the amino-terminus is presented on the left and the carboxyl terminus on the right.

It is advisable to point out that throughout the whole of the description "amino acid" is intended to designate both the natural amino acids and the non-natural amino acids. "Natural amino acid" is intended to designate the amino acids in the L form which can be found in the natural proteins, i.e. alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

However, the present invention also relates to the peptides having non-natural amino acids, i.e. the preceding amino acids in their D form as well as to the homo forms of certain amino acids like arginine, lysine, phenylalanine and serine or the nor forms of leucine or valine. In certain cases, it is advantageous to use alpha-aminobutyric acid and it is also possible to envisage the use of other amino acids like for example agmatine (Agm), alpha-aminoisobutyric acid (Aib), N-formyl-Trp (F-Trp), sarcosine, statin, ornithine, deaminotyrosine and other modified amino acids. All the amino acids can be used provided they are not toxic to humans and that they do not modify the capacity of the peptides obtained to induce an immune response in vivo or in vitro.

By "peptide derivative" of a wildtype protein is meant all of the peptides which possess a peptide sequence substantially identical, at least in part, to the peptide sequence of the wildtype protein. They may be for example chemically modified peptides having a peptide sequence 100% identical with a portion of the wildtype protein. They may also be hybrid peptides having a first portion 100% identical with a first portion of the wildtype protein and a second portion in no way/partially identical with a second portion of the wildtype protein. They may also be peptides having a total/partial homology with a portion of the wildtype protein. According to a partial embodiment of the invention, the peptides comprise a peptide sequence derived substantially from the R9M peptide sequence of the human fusion protein TEL/AML1, this sequence having at least nine (9) consecutive amino acids.

By "mutated" peptides derived from a wildtype protein are meant all of the peptides which have been obtained following a modification of said wildtype protein, whether it is a modification by addition, deletion or substitution of one or more amino acids of the wildtype protein. It may also be a modification provided by the addition of carbon chains bound to at least one of the amino acids of the wildtype protein or to at least one of the amino acids of the peptides which corresponds to a substitution or a modification of one of the amino acids of the wildtype protein. More particularly, the present invention covers the peptides which derive from the human fusion protein TEL/AML1 which comprises the wildtype epitope R9M and which have been mutated in the R9M sequence. However, the present invention covers any polypeptide comprising a peptide sequence mutated or derived from the R9M wildtype sequence and capable of producing biological effects at least equivalent to those whose specific sequence is provided hereafter.

A person well-acquainted with the field of the invention will know how to obtain different mutated and derived peptides and will also know how to determine which of all of the peptides obtained are the peptides having adequate biological activity. Preferably, the sequence of the peptide (including its chemical modifications if necessary) will be such that the peptide obtained will possess an enhanced affinity for the HLA-A2.01 molecule such that it can form with this molecule a stable complex having a longer half-life (DC.sub.50) than the half-life of the wildtype R9M peptide/HLA-A2.01 complex. The term DC.sub.50 characterizing the half-life of the peptide HLA-A2.01 complex is described in the publication "H-2 class 1 knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies" (European Journal of Immunology: 29:3112-21, 1999). This half-life ought normally to make it capable of inducing in vivo or in vitro a cytotoxic immune response. Preferably, the peptide obtained will slow down the growth of lymphoblastic tumor cells when administered to a leukemic receiver and will trigger a cytotoxic T response to the leukemic cells of common type B-ALL with t(12;21) of the leukemic receiver.

Advantageously, the inventors have discovered that the substitution of the amino acid cysteine at position 5 of the wildtype R9M peptide sequence by a neutral amino acid selected from serine, alanine and alpha-aminobutyric acid and/or the substitution of the amino acid arginine at position 1 of the wildtype R9M peptide sequence by an aromatic amino acid such as tyrosine conferred an enhanced biological activity on the mutated peptides. In this connection, the inventors have discovered that the peptides having as peptide sequence RIAESILGM (SEQ ID No. 2), RIAEAILGM (SEQ ID No. 3), YIAESILGM (SEQ ID No. 4), YIAEAILGM (SQ ID No. 5), RIAE.alpha.-butILGM (SEQ ID No. 6) and YIAE .alpha.-butILGM (SEQ ID No. 7), were particularly useful. By ".alpha.-but" is meant alpha-aminobutyric acid which is linked to glutamic acid.

It is also possible to make provision for other modifications (chemical or peptidic) making it possible for the peptides to cross certain biological barriers, to show a better solubilization, to facilitate their incorporation in special galenical forms such as for example liposomes or microparticles. Moreover, it is advisable to observe in this respect that the peptides according to the present invention may be available in a deglycosylated or glycosylated form if necessary.

The peptides according to the present invention can be prepared by any suitable procedure. In particular, they can be obtained by chemical synthesis but it is also possible to obtain them by a biological route by using, in particular, different vectors in suitable cell cultures such as will be described hereafter. It is also advisable to note that, in certain cases and depending on the method of preparation, it may be necessary to renature certain tertiary structures of the peptides obtained.

The DNA sequences coding for the peptides of the invention can be easily determined from the amino acid sequences. Table 1 (see Original Patent) gives the nucleotide sequence for the wildtype R9M peptide and the nucleotide sequences deduced for several mutated R9M peptides -- see Original Patent.

Thus, the object of the invention is also a procedure for the preparation of a peptide of the invention by transformation of a cell host with the aid of an expression vector (plasmid, cosmid, virus, etc) comprising the DNA sequences coding for the peptides of the invention, followed by the placing in culture of the thus transformed cell host and the recovery of the peptide in the culture medium. Hence, the invention also relates to any cell host transformed by an expression vector such as defined above and comprising the regulatory elements permitting the expression of the nucleotide sequence coding for a peptide according to the invention. The use of vectors for the expression of proteins and peptides in the cells of a host, in particular the human host, is known and will not be described in detail. The specific constructions obviously depend on the host, the epitope and the vector selected.

The mutated peptides of the present invention and the polynucleotides encoding them can be used in many ways as antitumor agents or for the preparation of an antitumor vaccine. For example, they can be used in an in vitro stimulation procedure of the cytotoxic CD8 response, comprising the separation or not of the lymphoid cells of a patient and the incubation in vitro of the said cells in the presence of at least one mutated immunogenic peptide and/or in the presence of at least one polynucleotide, objects of the invention. Those cells which express the epitopes derived from TEL/AML1 can stimulate in vitro a specific anti-leukemic cytotoxic response, the CD8 T cells stimulated in vitro being subsequently injected into the leukemic patient. It is also possible to use the peptides and polynucleotides, objects of the invention, in an in vivo induction procedure of the anti-leukemic CD8 T response via the injection of cells expressing the epitopes derived from TEL/AML1 described previously into a leukemic patient, the injected cells permitting the induction of an anti-leukemic CD8 T response in vivo.

The mutated peptides of the present invention and the polynucleotides encoding them can also be used to prepare polyclonal or monoclonal antibodies binding to at least one peptide/polynucleotide object of the invention. The present invention thus also relates to such purified antibodies which can be obtained by very well known procedures.

The mutated peptides of the present invention and the polynucleotides encoding them can be used for the production of a medicine with the objective of being administered in vivo for the purposes of therapeutic and/or preventive vaccination against leukemia in humans, especially the common acute lymphoblastic leukemia B-ALL with t(12;21). These medicines may comprise at least one of the elements selected in the group constituted by the immunogenic peptides and the polynucleotides described above and lytic T-cells sensitized in vitro by the placing in contact of an immunogenic peptide according to the invention. The polyclonal or monoclonal antibodies previously mentioned may themselves also be used for the preparation of a medicine intended for the treatment of leukemia such as described hereafter.

In a privileged embodiment of the invention at least one portion of the immunogenic peptides/polynucleotides according to the invention is conjugated to a support to which it is absorbed or bound covalently or non-covalently at its C-terminus and/or N-terminus. The support may be constituted of carrier molecules (natural or synthetic), physiological and non-toxic. The carrier molecules can make it possible in particular to increase the immunogenicity of the peptides of the invention through the intermediary of complementary reactive groups borne respectively by the carrier molecule and the peptide. As an example of carrier molecules, mention should be made of natural proteins such as tetanus anatoxin, ovalbumin, serum albumins, hemocyamines, the PPD (purified protein derivative) of tuberculin, etc. As examples of synthetic macromolecular supports, mention should be made for example of the polylysines or the poly(D,L-alanine)-poly (L-lysine). As examples of hydrocarbon or lipid supports, mention should be made of the saturated or unsaturated fatty acids. The support may also take the form of liposomes, particles, vesicles, microspheres or latex or polystyrene beads.

The invention also relates to therapeutic compositions comprising a medicine or several polyclonal or monoclonal antibodies such as described previously and a pharmaceutically acceptable vehicle. These compositions may be advantageous for the treatment or the prevention of the common acute lymphoblastic leukemia B with t(12;21) in humans. Naturally, the use of antibody-based compositions usually requires that these latter are compatible with administration to humans. They may be, in particular, antibodies humanized by known procedures or directly expressed in situ from the DNA sequence.

The therapeutic compositions according to the present invention may be available in any solid or liquid form usual for pharmaceutical administration, i.e. for example forms of administration as liquid, as gel or any other support permitting for example controlled release. Of the usable compositions, mention may be made in particular of the injectable compositions more particularly designed for injection into the blood circulation in humans. The compositions of the invention may also contain constituents which increase the immunogenicity of the peptides, in particular other immunogenic peptides, specific or unspecific adjuvants of immunity such as Freund adjuvant, polysaccharides or equivalent compounds.

The present invention relates in addition to compositions designed to be administered in order to express in situ the peptides previously described. For example, by injecting the "naked DNA" coding for the immunogenic peptides of the invention, this injection leads in a certain number of cases to the expression of the encoded peptide and to an immune response to the said peptide. It will also be possible to use systems of "naked DNA" but which contain their proper expression system or expression vectors such as previously described. The expression vectors are likely, in certain cases, to improve the activity of the peptides expressed. The vaccination systems making use of the DNA sequences are known and have already been extensively described in the literature.

The invention also relates to tumor cells obtained by double transfection of EL4S3-Rob (murine .beta.2-microglobulin negative) mice with:

1) the human gene encoding the HHD molecule; and

2) the human gene encoding for the fusion protein TEL/AML1 comprising: a) the wildtype R9M peptide sequence (Arg-Ile-Ala-Glu-Cys-Ile-Leu-Gly-Met) (SEQ ID NO: 1); or b) a mutated R9M peptide sequence coding for mutated immunogenic peptide such as defined above.

These cells make it possible to obtain an animal model of anti-leukemic vaccination, which validates the vaccination experiments. The methods for obtaining these kinds of cells are well known in the field and will not be described in detail. The method for obtaining the EL4 S3-Rob has been described in detail by Pascolo et al. in J. Exp. Med., 185:2043-51 (1997).

The present invention covers very particularly the cell line doubly transfected by the genes coding for the HLA-A2.01 and the TEL/AML1 translocation. This cell line is called "EL4 Rob.HHD.TEL.AML1" and was deposited as a biological sample with the National Culture Collection of Micro-organisms (CNCM, Pasteur Institute, Paris) on 1, Dec. 2000, and was assigned the registration No. I-2587.

The invention also relates to a selection procedure for therapeutic molecules capable of inducing a protective immune response in vivo to fungal, bacterial, viral or tumoral wildtype peptides. This procedure is characterized in that: a) a tumor cell line, EL4S3-Rob, doubly transfected by nucleotide sequences coding for the HHD molecule and nucleotide sequences coding for the said fungal, bacterial, viral or tumor wildtype peptides is administered to an compatible animal model, the said animal model having a genotype compatible with that of said transfected tumor cell and having been previously immunized with the said molecules which it is desired to select; b) the capacity of the said transfected cells to induce a response in vivo to the epitopes of the said fungal, bacterial, viral or tumor peptides is compared with the capacity of the said fungal, bacterial, viral or tumor wildtype peptides to induce a response in vivo to the epitopes of the said wildtype peptides in an animal model subjected to prior immunization with the wildtype peptide sequences of the said therapeutic molecules. In a preferential embodiment of the invention, the compatible animal model is obtained by using, on the one hand, a mouse having an inactive and unexpressed MHC replaced by the gene coding for HLA-A2.01 and, on the other hand, a tumor cell line transfected by at least the gene coding for the HLA-A2.01 molecule. It is, however, understood that the invention applies to any compatible animal model construction, for example by replacing the gene coding for HLA-A2.01 by a gene coding for HLA-B7 etc.

Although throughout the descriptive statement of the present invention the term "peptide" is used, it is understood that the invention is not limited to the compounds formed by the union of a limited number of amino acids. In fact, the flexibility of the recombinant technologies makes it possible to produce proteins comprising a multiplicity of identical or different epitopes and likely to improve the immunogenic activity of the final product. Thus the present invention also covers the immunogenic polymers comprising between two and ten peptides selected from the mutated peptides previously defined. Similarly, the present invention covers the polypeptides coding for a peptide sequence other than a wildtype R9M sequence, these polypeptides incorporating one or more peptide sequences coding for a peptide selected from the mutated peptides previously defined. Finally, the present invention includes the oligonucleotides having a nucleotide sequence coding for a peptide sequence other than a wildtype R9M sequence, these oligonucleotides incorporating one or more polynucleotides such as previously defined.
 

Claim 1 of 1 Claim

1. An isolated mutated immunogenic peptide consisting of the R9M wildtype peptide sequence (SEQ ID NO:1) mutated by substitution at P1 and/or P5 of the wildtype R9M peptide sequence, which consists of a peptide sequence selected from SEQ ID NOS:2 to 7.

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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