Patent Application
20080026995
The present invention is illustrated, without however being limited, by the following figures:
P-glycoprotein or “P-170 protein” is a 170 kDa membrane-bound phosphoglycoprotein which was identified by Juliano and Ling (1976. Biochim Biophys Acta 455(1): 152-162). The murine P-170 protein consists of 1276 amino acids forming two equivalent entities. The hydrophobic domains of the molecule are numbered from 1 to 12 and are involved in the efflux of chemotherapeutic drugs. Extracellular loops 1, 2 and 4 of the murine P-170 protein have been selected for their pronounced extracellular topology, suggesting that they may be antigenic in nature. The human P-170 protein (Chen et al. 1986 Cell. 47(3): 381-389) is a 1280 amino acid protein having two homologous domains each comprising six transmembrane helical domains and a nucleotide binding site. The hydrophobic regions of these transmembrane domains form extracellular loops considered to be the fragments for recognition of the P-170 protein from the outside of the cell. Extracellular loops 1, 4 and 6 of the human P-170 protein have also been selected as having a high antigenic capacity due to their particularly marked extracellular locations.
The immunotherapy of the present invention is directed at allowing the effects of the chemotherapeutic drugs used in cancer chemotherapy to manifest themselves.
In the context of the invention, “cancer” is defined, in accordance with its usual meaning, by two main characteristics: cell growth and proliferation that is not regulated by external signals, and the ability to invade tissues along with, where appropriate, the ability to form metastases by colonizing distant sites.
These characteristics are the result of the intrinsic properties of cancer cells, i.e., of their caryotypic and genomic instability, of their uncontrolled proliferation, of their metastatic capacity accompanied by the acquisition of new phenotypes and also of the activation and derepression of oncogenes in said cancer cells. The term “cancer” is therefore, in the context of the present invention, any phase of cell growth or proliferation having the above characteristics, evolving in particular toward the development of primary tumors and/or of metastatic tumors (secondary tumors).
In addition, for the purposes of the present invention, the expression “treatment for multidrug resistance” is understood to mean all medical treatments intended to combat multidrug resistance, to limit the consequences thereof, to avoid death and/or, preferably, to re-establish sensitivity to anticancer medicinal products. Towards this goal, the treatment is preferably directed toward curing multidrug resistance, by inducing complete reversion to a chemotherapy-sensitive cell phenotype. This reversion may, however, be partial: consequently, the treatment of multidrug resistance will prove to be palliative, allowing prolonged remission of the patient. The treatment for multidrug resistance is also characterized by virtue of its prophylactic capacity, making it possible to prevent the appearance of de novo or acquired multidrug resistances in a patient. That means, a treatment for multidrug resistance according to the present invention includes a vaccination for the same.
The invention relates, in certain embodiments, to an immunogenic composition comprising conjugates comprising all or, in certain embodiments, part of the amino acid sequences of an extracellular loop of the P-170 protein that, under suitable administration conditions, allows the induction of anti-P-170 antibodies.
In the context of the present invention, the immunogenic composition also allows the induction of anti-P-170 antibodies for the treatment including the partial or full reversion of multidrug resistance as well as the prophylaxis against multidrug resistance in a patient suffering from a cancer.
In preferred embodiments, the conjugates comprise part, or preferably, all of the amino acid sequences of at least one extracellular loop, two extracellular loops, preferably of at least three extracellular loops, of the P-170 protein.
A “conjugate,” according to the invention, is a reagent formed by covalent bonding between molecules of fatty acids containing a carbon chain of between C12 and C24, and peptide according to the present invention, to form a lipid-peptide hybrid molecule. The peptide, which is, in a preferred embodiment obtained, by solid-phase synthesis, is covalently coupled to the fatty acid molecules constituting the lipid region of the molecule (also referred to herein as “non-pegylated conjugate”). Alternatively, the peptide may be covalently coupled to at least two spacer molecules, including, but not limited to polyethyleneglycol (PEG), polylysine or polyglycine and other suitable polymers, which, in turn are covalently attached, to a phospholipid including, but not limited to, phosphatidylethanolamine. As the person skilled in the art will appreciate, any other chemically suitable phospholipid can be used that allows reconstitution in liposomes and virosomes including their functional equivalents.
For the purposes of the present invention, the term “derived peptide” or “peptide derived” from a P-glycoprotein or loops thereof denotes all or part of the amino acid sequence making up an extracellular loop of the P-glycoprotein, as long as said “derived peptide” has at least one epitope of the extracellular loop from which it derives. The inventions also includes peptides based on the P-glycoprotein or loops thereof that are peptides comprising all or part of the amino acid sequence making up, for example, an extracellular loop of the P-glycoprotein as long as these peptides, under appropriate administration conditions, alone or with other such peptides, induce an antibody response. Such peptides advantageously have between 5 and 50 amino acid residues, preferably between 5 and 40, or between 10 and 30, advantageously between 10 and 25 residues. The following falls within this definition: (1) peptides whose amino acid sequence is identical to the corresponding sequence of the extracellular loop from which they are derived, or (2) peptides whose amino acid sequence is modified with respect to the sequence of the extracellular loop from which they are derived, wherein said modification may, in certain embodiments, consist of a point mutation by insertion, deletion or substitution, in particular, conservative substitution, of one or more residues, provided that the peptide formed thereby still carries an epitope of the P-glycoprotein or induces an antibody response as outlined above, respectively. In particular, an acceptable mutation is a mutation which does not disturb the conformation of the modified peptide when it is included in a composition of the invention. This can be verified by the ability of the modified peptide to induce antibodies when it is formulated in a composition in accordance with the invention.
The present invention also includes peptides that have substantial homology or substantial identity with the respective portion of the naturally occurring P-glycoprotein. The terms “substantial homology” or “substantial identity”, when referring to a peptide of the present invention, indicate that the peptide in question exhibits at least about 30% identity with the respective portion of the naturally-occurring protein, usually at least about 70% identity, more usually at least about 80% identity, preferably at least about 90% identity, and more preferably at least about 95% identity.
The homology of a peptide or protein of the present invention such as a P-glycoprotein, a loop thereof, such as loops 1, 2 and/or 4 of the murine P-170 protein and/or loops 1, 4, and/or 6 of the human P-170 protein, or portion of any of those loops, is determined as the degree of identity between two protein/peptide sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, p. 443-453). Gap with the following settings for polypeptide sequence comparison may be used: Gap creation penalty of 3.0 and Gap extension penalty of 0.1, the mature part of a polypeptide encoded by an analogous DNA sequence exhibits a degree of identity preferably of at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, more preferably at least 95% or 96%, more preferably at least 97% or 98%, and most preferably at least 99% with the amino acid sequence of the peptides/proteins of the present invention, in particular with any of the amino acid sequences shown in SEQ ID NOs: 1 to 3 and 7, 11, 13 or any subsequences thereof having at least 10 amino acid residues.
Protein analysis software matches similar sequences using measures of homology assigned to various substitutions, deletions and other modifications.
Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. However, other substitutions were shown to not substantially interfere with the functionality of the respective conjugate, e.g., substitutions of histidine with methionine and cysteine with glutamic acid.
The peptides of the invention are preferably obtained by chemical synthesis, in particular using the methods described hereinafter. The peptides may, in certain embodiments, contain one or more modified amino acids.
In the context of the invention, the expression “extracellular loop” of P-glycoprotein (or P-170 protein) denotes each amino acid sequence of the P-170 protein having an extracellular topology or a significant connection with the extracellular environment so that it can be selected as a sequence carrying an epitope of this protein.
The amino acid sequences making up the synthetic peptides used in the context of the invention may be antigenic per se, or may be antigenic when they are presented in a conformation that conserves the conformation of the amino acid sequence of the extracellular loop from which the peptide is derived, or a conformation sufficiently similar to confer on the peptide the ability to induce the production of the same or functionally equivalent antibodies under suitable administration conditions. The ability to induce the production of antibodies may, for example, be verified in mice immunized with the peptides prepared in according to the invention or by any other known means. Thus, the peptides of the invention advantageously have, in the immunogenic composition, a three-dimensional conformation that reproduces the conformation of the extracellular portion of loop from which the peptides are derived, or are so similar to said conformation, as to confer on the composition formed their immunogenic capacity. The appropriate presentation of the peptides advantageously results from their association with the other constituents of the immunogenic composition.
This ability to induce the production of antibodies is in particular present when the peptides of the invention are peptides that have been synthesized and modified to be in the form of conjugates and are combined with liposomes or virosomes including their functional equivalents.
In the context of the invention, the expression “antigenic structure” denotes molecules capable of reacting with antibodies so as to form antigen/antibody complexes. An “antigenic structure” of the immunogenic composition may or may not have, as such, the ability to induce a phenomenon of immunogenicity corresponding to the formation of antibodies specific for a given antigen. An immunogenic composition according to the present invention, comprises in a preferred embodiment, liposomes. In another preferred embodiment, in particular in the context of conjugates containing spacer-phospholipid molecules, the conjugates are combined with an adjuvant, such as lipid A, in particular, monophosphoryl lipid A (MPLA). Particularly preferred are compositions comprising conjugates, liposomes and further comprising an adjuvant such as lipid A, in particular monophosphoryl lipid A (MPLA).
As well known in the art, lipid A comprises a wide variety of compounds. It can be found in and can be isolated from the outer membranes of most Gram-negative bacteria where it constitutes the hydrophobic anchor of lipopolysaccharide (LPS). Lipid A is glucosamine-based, and lacks glycerol. The acyl chains of lipid A are fully saturated and hydroxylated if attached to glucosamine. Preferably, the lipid A of the present invention, such as MPLA, is a detoxified version of the naturally occuring lipid A or a non-toxic lipid A analogue (also referred to herein as “synthetic lipid A”).
In a preferred embodiment, lipid A is incorporated into the liposomes and/or virosomes of the present invention.
Peptides derived from an extracellular loop of the P-170 protein of any species, for example mouse and human, are part of the present invention. Representative extracellular loops of the murine P-170 protein, the full sequence of which is as of December 2004 disclosed under Swiss-Prot accession number PO6795 and is incorporated herein by reference in its entirety, are:
Suitable subsequences of loop 1 are shown in large capital letters in Table III. Other suitable subsequences encompass amino acid residues from position 1 to 18 of SEQ ID NO 1 (loop 1a, see, e.g., mpeg-p1a) and from position 24 to 38 of SEQ ID NO 1 (loop 1c, see, e.g., mpeg-p1c) However, as the person skilled in the art will appreciate other subsequences of SEQ ID 1 and sequences derived therefrom as well as sequences having the homologies or sequence identities with those subsequences as discussed above are within the scope of the present invention. In a preferred embodiment, such subsequences comprise at least 10 amino acid residues, more preferably between about 10 to 25 amino acid residues and even more preferably between about 10 to 20 amino acid residues.
Murine peptides derived from loops 1, 2 or 4 are referred to herein as murine peptide 1, 2 and 4, respectively. The amino acid sequence of these peptides may be extended, in particular at their C and N terminal end, by one or more amino acid residues, including but not limited to, lysine. Amino acid residues that extend the amino acid sequence of a derived peptide are referred herein as “terminal” residues or “terminal amino acid residues.” An “end-standing residue” or “end-standing amino acid residue” is the “terminal” amino acid residue that is located at the very end of an amino acid sequence. In certain embodiments, two lysines (terminal lysines) are added at both ends of the peptide, in other embodiments, a combination of glycines and lysines are added at each end. Those lysines and glycines may act as attachment points for fatty acid molecules or activated spacer-phospholipid molecules to form conjugates according to the present invention.
By way of examples, conjugates according to the present invention such, as murine palmitotylpeptides (mpp), comprise peptides derived from extracellular loop 1 (mpp1, mpp1a (loop 1a) or mpp1c (loop 1c)), from at least one of extracellular loops 2 (mpp2 ) or 4 (mpp4 ) of the murine P-170 protein, which were characterized by their sequence, their molecular mass and their purity after synthesis according to the experimental protocol shown below in Table I. The immunogenic composition of the invention may comprise a combination of conjugates comprising peptides derived from different loops.
The calculated and observed molecular masses of the peptides derived from extracellular loops 1, 2 and 4 of the murine P-170 protein were obtained by analysis using mass spectrometry, namely:
[M+H+] for the calculated molecular mass,
MALDI-TOF and PDMS-TOF for the measured molecular mass, i.e.
(Matrix Assisted Laser Desorption Ionization—Time of Flight)
(Plasma Desorption Mass Spectrometry—Time of Flight).
According to a particularly advantageous embodiment of the invention, the immunogenic composition of the present invention comprises conjugates comprising peptides derived from extracellular loop 1 and, from extracellular loops 4 and 6 of the human P-170 protein and combinations thereof. The conjugates of the immunogenic compositions may therefore comprise amino acid sequences derived from extracellular loops 1 and 4 or from extracellular loops 1 and 6.
Preferably, the conjugates of the immunogenic composition comprise peptides derived from the three loops 1, 4 and 6 of the human P-170 protein.
Given the 47-amino acid length of loop 1 of the P-170 protein, peptides derived from this loop may result from loop 1 being divided up into three fragments, obtained by cleavage at the glycosylation sites. The three derived peptides give rise to three peptides 1a, 1b and 1c, corresponding to sub loops 1a, 1b and 1c which can be synthesized. Other derived peptides according to the present invention may be fragments of any of these three peptides, containing one or more epitopes. Consequently, the conjugates according to the invention comprise all or part of the peptides derived from extracellular loop 1 of the human P-glycoprotein corresponding to the three peptides resulting from cleavage of said loop 1 at the glycosylation sites. Another suitable subsequence of this loop encompasses amino acid residues from position 33 to 47 of SEQ ID NO 7. However, as the person skilled in the art will appreciate other subsequences of SEQ ID NO 7 and sequences derived therefrom as well as sequences having the homologies or sequence identities with those subsequences as discussed above are within the scope of the present invention. In a preferred embodiment, such subsequences comprise at least 10 amino acid residues, more preferably between about 10 to 25 amino acid residues and even more preferably between about 10 to 20 amino acid residues.
The peptide sequence of the conjugates can be used as they are for the preparation of immunogenic compositions according to the invention, in particular, in combination with liposomes. The peptides of these conjugates may be chosen, respectively, from the following amino acid sequences, which stem from human P-170 protein as disclosed by Swiss-Prot accession number PO8183 as of December 2004, which is incorporated herein by reference:
and/or a combination thereof.
For extracellular loop 1, the three peptides 1a, 1b and 1c are advantageously used in the same composition. Alternatively, combinations of peptides 1a and 1b or 1a and 1c or 1b and 1c may be used. However, as the person skilled in the art will appreciate other subsequences of SEQ ID NOs 7, 11 and 13 and other sequences derived therefrom as well as sequences having homologies or sequence identities with those subsequences as discussed above are within the scope of the present invention. In a preferred embodiment, such subsequences comprise at least 10 amino acid residues, more preferably between about 10 to 25 amino acid residues and even more preferably between about 10 to 20 amino acid residues. For example, a subsequence of loop 1 that corresponds or comprises amino acid residues 33 to 48 of SEQ ID NO 7 can be used instead of SEQ ID NO 10. See, for example Hpp1c′ in Table II. It is readily understood that the corresponding Hpp1c conjugate comprising the above specificed sequence can also be used in the context of the present invention.
The amino acid sequence of the human peptides may also be extended, in particular at their C and N terminal ends, by one or more amino acid residues, including, but not limited to, lysine or glycine or combinations thereof. In certain embodiments, one or two lysines are added at both ends of the peptide, in other embodiments a combination of glycines and lysines are added at each end. Those terminal lysines and glycines may act as attachment points for fatty acid molecules or activated spacer-phospholipid molecules to form conjugates according to the present invention.
Table II recapitulates the amino acid sequences of the conjugates corresponding to extracellular loops 1, 4 and 6 of the human P-170 protein. The amino acid sequences corresponding to the sequences of the derived peptides appear in large capital letters, while the small capital letters correspond to the added amino acids to which the fatty acid molecules may be coupled.
The sequence, the molecular mass and the purity of the peptides described in Table II can be controlled by means of the techniques described above for the peptides of Table I.
A peptide derived from an extracellular loop advantageously has a purity equal to or greater than 90%, advantageously between 91% and 98%, measured by HPLC chromatography after synthesis.
In the context of the present invention, the amino acid sequences corresponding to the sequences of the extracellular loops (represented in large capital letters) can be extended, in particular at their end(s), with amino acid residues (represented in the peptides illustrated herein in small capital letters) to which, for example, the fatty acid residues are coupled. Advantageously, the molecules of fatty acid contain a C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 or C24 carbon chain are preferably C16 palmitic acid molecules. The carbon chains of the fatty acid molecules of between C12 and C24 are linear or branched. Preferably, the fatty acid molecules have linear carbon chains. To avoid reaction incompatibility during the peptide synthesis, in particular during the final step of deprotection in the presence of strong acid, the fatty acid molecules of the present invention are, in certain embodiments, neither mono- nor polyunsaturated.
Preferably, each conjugate comprises at least four molecules of fatty acid containing a carbon chain of between C12 and C24, the fatty acid molecules are preferably distributed at the N- and C-terminal ends of the peptides. However, other distributions of the fatty acid molecules are within the scope of the invention such as within the amino acid sequence. The peptides of conjugates may be each coupled to four molecules of palmitic acid; they are therefore tetrapalmitoylated.
Preferably, two molecules of palmitic acid are coupled to the N-terminal end and two molecules of palmitic acid are coupled to the C-terminal end of the peptide.
Preferably, and as illustrated in Table II, the amino acid sequences of the extracellular loops of the peptides of the conjugates are extended in the N- and/or C-terminal position by one or more amino acid residues, to allow combination with the molecules of fatty acid containing a carbon chain of between C12 and C24 or with spacer-phospholipid molecules. The phospholipid moiety of the spacer-phospholipid molecule is preferably phosphatidylethanolamine, wherein the fatty acid moiety of the phosphatidylethanolamine is preferably as described above.
The peptides may be combined with said fatty acids or spacer-phospholipid molecules exclusively in the N-terminal position or, alternatively, exclusively in the C-terminal position. Advantageously, in the conjugates, the combining of the peptides with the fatty acids or spacer-phospholipid molecules is carried out in the N-terminal and C-terminal position of the peptide sequences, in particular so as to give tetrapalmitoylated sequences.
Alternatively or cumulatively, fatty acids or spacer-phospholipid molecules may be attached to internal residues in the peptide sequence.
“Pegylation”, i.e. the process comprising covalently coupling a PEG molecule to a peptide, to increase the immunogenicity/immunogenic nature of a peptide used as antigen, is a technique well known to those skilled in the art (2002. Adv Drug Deliv Rev. 54(4): 459-476).
This process makes it possible in particular to increase the accessibility of the peptide sequence and, in this way, the presentation of the antigen.
In general, with longer peptide sequences, a greater number of molecules of PEG are preferred.
Advantageously, molecule(s) of PEG, from 2 to 8000, preferably PEG having between about 50 and 100 units such as 77 units, is(are) attached to, for example, a lysine (K) residues found in the N- and/or C-terminal position of the amino acid sequences of the extracellular loops of the peptides of the conjugates of the present invention. Each peptide may be covalently attached to at least two molecules of fatty acid containing a carbon chain of between C12 and C24 as described above or to at least two spacer molecules, such as two chains of polyethylene glycol (2-8000) which are each coupled to a phospholipid such as phosphatidylethanolamine to make it possible to reconstitute these antigenic complexes in the lipid bilayer of, e.g., the liposomes. Under suitable administration conditions, these complexes reconstitute, in adjuvant-liposomes, leading to the induction of anti-P-170 antibodies.
A “spacer” according to the present invention is a molecule that can form a bridge between an amino acid residue, such as a terminal lysine or an analogue thereof, and a phospholipid, such as phosphatidylethanolamine, in the conjugates of the present invention. Those molecules include, but are not limited to, PEG having 2 to 8000 units, polyglycine having preferably between 2 and 100 residues and polylysine molecules having preferably between 2 and 100 residues. A spacer molecule may be attached to a phospholipid of the present invention to form a spacer-phospholipid molecule. However any other suitable polymers may be used as spacers. The coupling of one or more peptides according to the present invention to one or more of those spacer-phospholipid molecules results in a conjugate according to the present invention. The peptide may be attached to the spacer-phospholipid molecule via the ε amino group of one of the one or more lysine(s) that have been added to the peptides of the present invention.
Advantageously, the two spacer-phospolipid molecules, especially if the molecular weight of the spacer, e.g., PEG, exceeds 2000 Da (for example, is 3400 Da) are attached onto lysine residues of protected or unprotected peptides by coupling in solution phase (see example below). Attempts to accomplish a simple “dipegylation” of lysine groups by solid-phase synthesis had little or no success (Lu, Y. A. et al., Reactive Polymers 22:221 (1994)).
To form the immunogenic compositions of the invention from the above mentioned conjugates, liposomes are advantageously selected as carrier for presenting the conjugates in the composition of the present invention.
Advantageously, said conjugates are present at the surface of the liposomes.
In certain embodiments, lipid A is incorporated into said liposomes.
For the purposes of the present invention, the term “liposome(s)” is understood to mean an artificial spherical particle comprising or consisting of one or more layers of phospholipids.
The composition according to the present invention advantageously comprises the conjugates and the liposomes in a molar ratio of between 1/10 and 1/1000, preferably between 1/50 and 1/500, advantageously in a molar ratio of 1/250.
Advantageously, the liposomes are prepared by mixing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and cholesterol in a molar ratio of, respectively, 0.9:0.1:0.7. The products used above are preferably of synthetic origin in order to avoid the possibilities of contamination with endotoxins, prions or viruses. For example, the DMPC and DMPG phospholipids are of synthetic origin (Avanti Polar Lipids USA) and the cholesterol, which is 98% pure, is of animal origin. Monophosphoryl lipid A (MPLA), also of synthetic origin, and known to increase the immune response (Fries et al. 1992. Proc Natl Acad Sci 89(1): 358-362) was added to liposomes, and tested, at a concentration of 40 μg per μmol of phospholipids.
Thus, the composition according to the present invention may also comprise alum and/or immunomodulators such as, but not limited to, lipopolysaccharide (LPS) derivative such as lipid A, monophosphoryl lipid A (MPLA) or a synthetic lipid A analogues, including synthetic MPLA, and/or microcapsules of proteins and of polysaccharides and/or muramyldipeptide (MDP) and muramyltripeptide (MTP) and derivatives thereof. Immunostimulating agents have been reviewed by Werner & Jolles (Werner & Jolles, Eur. J. Biochem. 242, 1-9 (1996)). Such immunogenic compositions are generally prepared in the form of liquid solutions, or of injectable suspensions, or else in a solid form suitable for solubilization prior to injection in the context, for example, of a kit for making use of the present composition, as described below.
The invention also relates to peptides which are derived from at least one extracellular loop of the P-170 protein and which cause the induction of anti-P-170 antibodies when they are used in the immunogenic compositions as described. These peptides may, respectively, be chosen from the following amino acid sequences:
SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, and SEQ ID NO 13 and other subsequences of SEQ ID NOs 1 and 7 as discussed herein.
Also included in the present invention are the nucleic acid, DNA or RNA, sequences encoding the peptides as described above. Reference is made hereby to U.S. Pat. No. 5,198,344 to Croop et al and U.S. Pat. No. 5,206,352 to Roninson et al which are incorporated herein by reference in their entirety.
The present invention relates not only to the immunogenic composition and conjugates per se and as defined above, but also to methods for preparing the conjugates and composition.
The experimental results below show that the combining of specific peptides with only two molecules of palmitic acid may not result in the expression of an immunogenic response. This reflects the need to select the fatty acids so as to confer on the conjugates formed, the conformation required for the induction of an immune response.
Particular attention is also given to the synthesis of the conjugates according to the invention, and in particular to their peptide region. In fact, the previous studies by Tosi et al. (1995) demonstrated that the peptide sequence of mpp1, which is the longest of the conjugates, with 43 amino acids, did not bring about an immune response. In the absence of explanations or working hypotheses for this observation, the inventors have revealed, in the context of the present invention, the essential role of a very precise peptide synthesis. The inventors have therefore developed and shown that an improved method of synthesis allowing a more effective production of certain amino acids by avoiding their involvement in early terminating reactions makes it possible to obtain in particular an immunoreactive conjugates. In one embodiment of the invention, conjugates can therefore be obtained in a suitable form for producing the immunogenic composition of the invention by means of a solid-support synthesis according to the Boc/benzyl strategy.
In the case in point, as described in the example section, the peptides corresponding to the sequences of extracellular loops 1, 2 and 4 of murine P-170 were therefore synthesized via an Applied Biosystems 430A peptide synthesizer using the tert-butyloxycarbonyl/benzyl or Boc/benzyl strategy and, in situ, activation with (N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide.
The synthesis of the peptides can be carried out on a peptide synthesizer, for example an Applied Biosystem 430A synthesizer, using (13,14)-tert-butyloxycarbonyl/benzyl and in situ activation with (N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (Schölzer M. et al Science 1992, 256 (5054): 221-225).
According to this method, the synthesis of the conjugates is based on the synthesis of the amino acid sequences of the extracellular loops of the peptides of the conjugates on a solid support according to the Boc/benzyl strategy one or more amino acid residues are introduced at the N and/or C-terminal end so as to allow combination with the molecules of the fatty acid containing a carbon chain of between C12 and C24, followed by a step consisting of deprotection of the amine functions of the N and C-terminal lysines in order to couple them with the fatty acids containing a carbon chain of between C12 and C24. The final step is cleavage with a strong acid, such as anhydrous hydrofluoric acid, of the conjugates synthesized on the solid support, such as a resin.
This improved method of synthesis allows a more effective production of certain amino acids by avoiding their involvement in early terminating reactions.
The combining of said peptides with the fatty acids can be carried out either in the N-terminal position or, alternatively, in the C-terminal position.
Advantageously, the combining of the peptides with the fatty acids is carried out in the N-terminal and C-terminal position of the peptide sequences, in particular to produce tetrapalmitoylated sequences.
Alternatively or cumulatively, it is possible to combine the fatty acids with internal residues in the peptide sequence.
Optionally, once they have been synthesized, the conjugates are purified, for example by RP-HPLC or reverse-phase high performance liquid chromatography. This purification step is made delicate by the presence of a lipid chain, which results in a broadening of the chromatographic peaks and solubility problems. Isolation of the expected product, from the impurities formed, during the elongation of the peptide, can be difficult and can result in low yields. In addition, the purification may be difficult insofar as the fatty acid is located in the C-terminal position. In fact, in this case, the desired product, but also the impurities, carry the lipophilic portion responsible for the chromatographic difficulties. In addition, since this strategy involves, at the end of synthesis, a step of cleavage and of deprotection in strong medium, this treatment limits the choice of the lipophilic portion (Deprez et al. 1996. Vaccine 14(5): 375-382; Stöber et al. (1997) Bioorg. Med. Chem. 5(1): 75-83).
Other strategies that result in a very precise peptide synthesis can also be used. The Fmoc strategy is, in particular, of advantage in the context of the synthesis of conjugates containing spacer-phospholipids. Here, partially side chain protected peptides may be produced, that is, peptides having unprotected terminal amino acids. The production of such partially side chain protected peptides is, in particular, preferred if the peptide contains internal amino acids, such a lysine or histidine, that might react with preactivated non-peptidic moieties which are discussed below. After resin cleavage, those partially side chain protected peptides may be coupled to preferably at least two non-peptidic moieties, that are preferably preactivated, such as preactivated spacer-phospholipid molecules, to form a conjugate according to the present invention. This coupling takes place preferably in solution with at least a solvent and, preferably, in the presence of an excess of amine base. According to this method, the synthesis of a epitope carrying sequence comprising the amino acid sequence of an extracellular loop of a peptide derived from a P-170 molecule on a solid support is advantageously based on orthogonal Fmoc/tert-butyl chemistry. The N- and/or C-terminal position of the sequence contains one or more amino acid residue, preferably lysine residue, more preferably, two lysine residues, that allow site specific conjugation of a, preferably pre-activated, spacer-phospholipid molecule, by, for example, an amide linkage. If internal lysine and/or histidine residues are present, those terminal lysine residues may be protected with an orthogonal butyl, such as 1-(4,4-Dimethyl-2,6-dicyclohexylidene) 3-methyl-butyl (ivDde). In a preferred embodiment of this method, removal of the N-terminal Fmoc group, and optional acetylation of the free amino terminal group, is followed by chemoselective deprotection of the orthogonal butyl and selective resin cleavage. The release of the partially side protected peptides having unprotected terminal amino acid residues is followed by the coupling of at least one unprotected terminal amino acids, preferably at each end, to a pre-activated form of a spacer-phospholipid molecule. Only after the coupling reaction is quenched, the remaining side chain protecting groups are removed with, for example, a standard acidic cocktail. If no internal lysine and/or histidine residues are present, tert-butoxycarbonyl (Boc) groups can be used to protect the terminal amino acids. In this case, deprotection and resin cleavage can be performed simultaneously and is followed by coupling the fully deprotected sequence via its terminal amino acids to a pre-activated form of a spacer-phospholipid molecule.
An “activated” or “pre-activated” spacer-phospholipid molecule is any spacer-phospholipid, which can be coupled to a peptide because it contains a functional group capable of reacting with a functional group in the peptide to produce a conjugate according to the present invention. The activated spacer can be an alkylating reagent, such as PEG aldehyde, PEG epoxide or PEG tresylate, or it can be an acylating reagent, such as PEG ester.
Spacers of the present invention comprise, but are not limited to, PEG, poly-amino acids (e.g., poly-glycine, poly-histidine, poly-leucine, polyglutamic acid) and poly-saccharides (e.g., polygalacturonic acid, polylactic acid, polyglycolide, chitin, chitosan), and synthetic polymers, including but not limited to, polyamides, polyurethanes, polyesters. As the person skilled in the art will appreciate, appropriate co-polymers may also be used such as, but not limited to, a co-polymer of poly(methacrylic acid) and N-(2-hydroxy) propyl methacrylamide. Molecules constituting modifications to PEG such as poly (propylene)glycol, are also within the scope of the present invention. Spacer molecules and activation strategies are also disclosed, for example, in U.S. Pat. No. 5,672,662 to Harris et al, which is incorporated herein by reference in its entirety.
The conjugates so obtained may be purified and characterized by appropriate methods.
Irrespective of the method of production, the conjugates may then be presented at the surface of liposomes.
This presentation may be obtained mechanically. Specifically, the conjugates mixed with the liposomes may fit exactly into the phospholipid membrane of the liposomes by means of their lipid doubles chains.
The present invention also relates to the use of this composition in methods of immunization according to the invention.
The present invention therefore relates to a method of immunization comprising a first administration, in particular by injection, of the immunogenic composition according to the invention and a boost administration of said composition (or booster), for example of two successive injections. The boost injections, exposing the same antigen several times, induce a strong secondary immune response. The repeated exposure of peptides derived from the extracellular loops of the P-170 protein, to the immune system, induces immunological memory and also rapid subsequent secondary responses with a high antibody titer.
More particularly, in the present method of immunization, the injections in humans are carried out 1 month apart.
According to one embodiment, the immunization with the composition according to the invention can be carried out concomitantly with or preceding an anticancer treatment administered to a patient.
Preferably, the immunization precedes the chemotherapy treatment in order to prevent and anticipate the appearance of a multidrug resistance phenotype in the patient. This treatment plan will be preferred to immunization concomitant with the chemotherapeutic treatments when the diagnosis and evolution of the cancer allow the curative therapeutic treatment (chemotherapy) to be delayed by at least 30 days from the date of diagnosis. The late treatment of the cancer does not prevent the method of immunization with the composition of the present invention being used. In fact, combining the anticancer treatment and the immunization with said immunogenic composition at the same time nevertheless induces an immune response for the production of anti-P-170 autoantibodies having a curative or palliative effect on the appearance of the multidrug resistance phenotype. The curative effect of the immunization with the present composition is illustrated by a reversion of multidrug resistance phenotype. In the context of the invention, the expression “reversion” of the multidrug resistance phenotype denotes the change from a phenotype of multidrug resistance to a phenotype “sensitive to the chemotherapeutic treatments”.
For the purpose of the present invention, the term “chemotherapy”, “anticancer” or “chemotherapeutic drugs” is intended to mean any curative or palliative treatment of primary or secondary tumors based on cytotoxic agents. Chemotherapy generally requires several cycles of treatment.
In the context of a method of immunization preceding the chemotherapy treatment, the implementation of the first injection of the composition according to the present invention precedes the start of the chemotherapeutic treatment by at least 60 days, preferably 63 and 67 days.
The compositions according to the invention can be administered topically, systemically (orally, nasally and via other mucosal routes) and/or parenterally (intravenously, subcutaneously, intramuscularly or intraperitoneally) or by combination of these routes, and effectively induce a protective immune response against multidrug resistance. The composition is formulated so as to allow easy administration via the various pathways above. In particular, the choice of the secondary compounds (wetting agent, emulsifier or buffer) is dictated by the chosen mode of administration.
Advantageously, the immunization is carried out by means of intramuscular and intraperitoneal administration, respectively, in humans and in mice.
One of the embodiments of the present invention also relates to the provision of an antibody, in particular an autoantibody, induced against human or murine P-170, which binds specifically to the peptides of the conjugates according to the invention.
This antibody may be a polyclonal or monoclonal antibody, selected from the group comprising the lgG1, lgG2 and lgG3 isotypes and an lgM. In particular the lgG2 antibody may be of the subtype 2a or 2b. Antibodies according to the present invention also include, e.g., multispecific antibodies (for example, bispecific antibodies), or antibody fragments. The antibodies may also be engineered to, for example, improve their affinity or diminish their immunogenicity. Such antibodies and methods of producing them have been described in detail. See, e.g., United States Patent Publication 20060045877, which is incorporated herein by reference in its entirety.
The invention is also directed towards hybridomas producing monoclonal antibodies. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. See, e.g., U.S. Pat. No. 5,703,213 and U.S. Patent Publication and 20040152105, which are incorporated herein by reference in their entirety.
The present invention also relates to an immunogenic composition comprising, firstly, a carrier and, secondly, conjugates comprising at least one peptide derived from extracellular loop 1 of the P-170 protein allowing the induction of appropriate antibodies, wherein said conjugates exhibiting all or part of the conformation of extracellular loop 1 of the P-170 protein, for the reversion of multidrug resistance appearing in a patient suffering from a cancer.
This composition is particularly suitable for treatments of solid tumors expressing the MDR1 gene encoding the human P-170 protein.
The present invention also relates to the use of the immunogenic composition according to the invention, for producing a vaccine intended for the treatment and/or prevention of a multidrug resistance in a patient suffering from a cancer. The cancer as envisioned will be a kidney cancer, liver cancer, colon cancer, cancer of the intestine, prostate cancer, breast cancer, bladder cancer, brain cancer, blood cancer (leukemia) and/or cancer of the medullary tissues (myeloma). It may also be a solid tumor expressing the MDR1 gene encoding the human P-170 protein. The use of the immunogenic composition may also be carried out in combination with an anticancer treatment.
In particular, the vaccines may be used in patients bearing solid tumors, in particular cancers expressing the MDR1 gene encoding the human P-170 protein, such as kidney cancer, liver cancer, colon cancer, cancer of the intestine, prostate cancer, breast cancer, bladder cancer, brain cancer, blood cancer (leukemia) and/or cancer of the medullary tissues (myeloma). However, as the person skilled in the art will realize, the immunogenic composition according to the present invention may be used for any cancer that is susceptible to chemotherapeutic treatment.
Finally, the composition according to the present invention may be used directly in patients spontaneously expressing multidrug resistance.
The present invention also comprises a method of treatment and/or prevention of a multidrug resistance appearing in a patient suffering from a cancer, such as cancers susceptible to chemotherapeutic treatment, in particular a cancer affecting the kidney, the liver, the colon, the intestine, the prostate, the breast, the bladder, the brain, the blood (leukemia) and/or the medullary tissues (myeloma), comprising the administration of the immunogenic composition as described.
The cancer particularly targeted by the treatment as described is a solid tumor expressing the MDR1 gene encoding the human P-170 protein.
The invention is also directed at a kit for making use of the immunogenic composition, comprising the immunogenic composition according to the invention and, optionally, reagents and/or instructions for use.
The synthesis of the peptides was carried out at a peptide synthesizer, for example an Applied Biosystem 430A synthesizer, using (13,14)-tert-butyloxycarbonyl/benzyl and, in situ, activation with (N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (Schölzer M. et al. Science 1992, 256 (5054): 221-225) Advanteously, the peptides were then coupled covalently with four molecules of palmitic acid per molecule of peptide (
An improved method of synthesis allowing more effective production of certain amino acids by avoiding their involvement in early terminating reactions has been developed. This is particularly advantageous in the case of mpp1due to its length (43 amino acids). In particular, the peptides mpp1, mpp2 and mpp4 were synthesized and the results of the synthesis (analysis of the peptide sequence, molecular mass, and purity) were recorded in Table 1 above, each peptide having been controlled for its sequence by amino acid analysis after total acid hydrolysis, for its molecular mass by mass spectrometry analysis and for its purity by HPLC.
According to this method the synthesis of the conjugates was based on the generation on resin of the desired peptide sequence, and then the deprotection of the amine functions of the N- and C-terminal lysines in order to couple them with the fatty acid containing a carbon chain of between C12 and C24. The final step was cleavage with anhydrous hydrofluoric acid. The conjugates were obtained using the Boc/benzyl strategy. The optimal approach consisted of generating the peptide sequence on the solid phase and then in coupling an activated fatty acid to a selectively deprotected amine (or thiol) function. If the fatty acid is introduced at the N-terminal end, the peptide does not require any particular functional arrangements. On the other hand, introduction of the fatty acid in the C-terminal position is generally carried out on the ε-amino function of a lysine side chain. In the Boc/benzyl strategy, it is often necessary to introduce the Boc-L-Lys(Fmoc)—OH amino acid during synthesis of the peptide. After having generated the entire sequence, the amino function was deprotected and then acylated with the fatty acid containing a carbon chain of between C12 and C24. The conjugate is finally deprotected and cleaved from the resin in the presence of a strong acid, anhydrous hydrofluoric acid (
Certain conjugates of the present invention can also be described by the general formula:
Pal-K(Pal)-G-peptide-G—K(Pal)-K(Pal)-NH2
wherein “Pal” stand for the respective palmitic chain.
Solid phase peptide synthesis (SPPS) was advantageously carried out on a 2-chlorotrityl resin preloaded with Gly (H—Gly-2-CITrt, Novabiochem, 0.54 mmol/gr) using the 9-fluorenylmethyloxycarbonyl)/tert butyl (Fmoc/tBU) orthogonal protection strategy. Two Lys residues were incorporated at each N- and C- terminal end of the antigenic peptide sequence produced to allow for the site-specific conjugation of PEG- lipid groups by an amide linkage.
As illustrated in
As illustrated in FIG. 5A′, if the antigenic peptides did not contain internal Lys or His residues, advantegously tert-butyloxycarbonyl (Boc) groups were used to protect the two terminal Lys residues. Totally deprotected peptides were then obtained by sequential steps of Fmoc N-terminal deprotection, acetylation of the N-terminus and finally simultaneous side-chain deprotection and resin cleavage (B′) with TFA:TIPS:H2O (95:2.5:2.5). Coupling of these non-protected peptides to the activated DSPE-PEG-SPA (D) was carried out under similar conditions as outlined above.
Finally, the peptide-PEG-lipid conjugates obtained this way were then purified by HPLC and their identity confirmed by MALDI-TOF mass spectrometry. Pegylated conjugates having a peptide as claimed (i) comprising a partial SEQ ID NO 1 comprising amino acid residues 1 to 18 (mPEGp1a), or (ii)/(iii) corresponding to SEQ ID NO 2 (mPEGp2)/SEQ ID NO 3 (mPEGp4) were prepared according to the above protocol.
Certain pegylated conjugates of the present invention can also be described by the general formula:
AcNH—K(DSPE-PEG)-peptide-K(DSPE-PEG)-G—OH
In order to further understand the degree of immunogenicity of the conjugates according to the invention, two types of conjugates were produced and tested. The first type of conjugate corresponded to the synthetic sequences of the peptides derived from the extracellular loops of the murine P-170 protein, covalently coupled to four molecules of fatty acid containing a carbon chain of between C12 and C24, per molecule of peptide. The second type of conjugate was made up of a peptide coupled only to two molecules of fatty acid. This study is illustrated by means of a specific example of dipalmitoylated and tetrapalmitoylated conjugates.
Table III describes the sequences of the di- and tetrapalmitoylated conjugates corresponding to or derived from loops 1, 2 and 4 of the murine P-170 protein and also their immunogenic capacity measured through the detection of antibodies by the Dot Blot technique and also the antibody titer by fluorescence units. It was observed that the mice which received liposomes containing conjugates with two palmityl residues did not show an immune response, with the exception of mpp′4, derived from loop 4 of the murine P-170 protein. The antibody titer induced by this conjugate was, however, four times lower than the titer induced by mpp4, i.e. the same sequence coupled to four palmityl residues. The mpp4 and mpp2 conjugates engender an immune response with an antibody titer in the region of 400 fluorescence units. In the context of the first peptide synthesis, no antibody was detected following injection of the mpp1 conjugate corresponding to the longest amino acid sequence, which could result from the peptide synthesis. In fact, the synthesis of this loop is very hard and long since there are many possible chemical terminations, or chemical rebridging which could explain the lack of immune response of this first batch of peptides. For the animal immunization trials, a further synthesis of the peptides, in particular of loop 1, was therefore carried out.
In view of these results, the model coupling each molecule of peptides to four molecules of fatty acid containing a carbon chain of between C12 and C24, strengthens the immunogenic capacity of said conjugates incorporated into the membrane of liposomes in the immunogenic composition according to the invention (
I-4 Vaccine Preparations
a. Mpp conjugate based vaccines (Lp vaccines)
The immunogenic compositions prepared comprise:
Lp1: Mpp conjugates, liposomes (DMPC, DMPG, cholesterol)
Lp2: Liposomes (DMPC, DMPG, cholesterol)
Lp3: Liposomes (DMPC, DMPG, cholesterol), MPLA, conjugates
Lp4: Mpp conjugates.
The liposomes present at their surface the three conjugates mpp1, mpp2 and mpp4 added in a molar ratio of 1:250 with the phospholipids. The organic solvents allowing homogenization of this combination were evaporated off, and the resulting film, after hydration with sterile PBS pH=7.4, was adjusted to a final phospholipids concentration of 4 mM. Finally, the liposomes in suspension are, at the time of immunization, mixed with sterile alum (Pasteur Mérieux) in a ratio by volume. The vaccine preparations injected into the animals therefore correspond to the immunogenic compositions Lp1, Lp2, Lp3 and Lp4 in formulations comprising alum as adjuvant for immunity, the alum also being capable of prolonging the absorption time of the vaccine.
b. MPeg conjugate based vaccines (Lpeg vaccines)
The immunogenic compositions prepared comprise:
Lpeg1: MPeg-p1a, mPeg-p2, mPeg-p4, alum, liposomes (DMPC, DMPG, cholesterol)
Lpeg2: MPeg-p1a, mPeg-p2, mPeg-p4, lipid A, alum, liposomes (DMPC, DMPG, cholesterol)
Lpeg3: MPeg-p1a, alum, liposomes (DMPC, DMPG, cholesterol)
Lpeg4: MPeg-p2, alum, liposomes (DMPC, DMPG, cholesterol)
Lpeg5: MPeg-p2, lipid A, alum, liposomes (DMPC, DMPG, cholesterol)
Lpeg6: Mpp2, alum, liposomes (DMPC, DMPG, cholesterol)
Lpeg7: MPeg-p4, alum, liposomes (DMPC, DMPG, cholesterol)
The liposome lipids were admixed with the respective conjugates and, where appropriate, with synthetic MPLA, CHCl3, MeOH and EtOH. The organic solvents allowing homogenization of this combination were evaporated off, and the resulting film, after hydration with sterile PBS pH=7.4, was adjusted to a final phospholipids concentration of 4 mM. Finally, the liposomes in suspension are, at the time of immunization, mixed with sterile alum (Pasteur Mérieux) in a ratio by volume. The vaccine preparations injected into the animals therefore correspond to the immunogenic compositions Lpeg1 to Lpeg7.
Liposome characterization via Fluorescamine
Size exclusion chromatography (SEC) of ultracentrifuged (200 kG for 2 hrs, SORAL DISCOVERY 90 SE) empty liposomes confirmed that essentially all the liposome is in the pellet.
The peptide content was determined by addition of Fluorescamine (FLA) (FLURAM from Fluka) which can label lysine residues resulting in a highly fluorescent product. This FLA assay confirmed that the liposomes presented the peptide portion of the respective conjugate at their outer surface.
In particular, addition of FLA at 1.2 mM to the supernatant fraction gave a signal of 26000 (FLUOROCOUNT microplate fluorometer (ex. 400 nm, em. 485 nm), Packard (Conn., USA)), while background PBS gave a signal of 10000. To determine the ratio of liposome bound and non-bound peptide, an aliquot of the reconstituted pellet containing mPeg-p2 liposomes was solubilised in 2% Triton X-100 (Sigma) and FLA added. The total fluorescence was 65000 while the background from empty liposomes was 20000. Subtracting backgrounds from the supernatant: 26000−10000/65000−20000 gave a ratio of free/bound peptide=1:3.5 or 22% free peptide. In order to determine the percent peptide on the liposome exterior, the assay was repeated on the pellet sample reconstituted in PBS alone. The fluorescence was the same as that of mPEGp2 liposomes which were solubilised in 2% Triton X-100. This implies that of the liposome integrated peptide, all peptide is exposed on the outer surface. In order to ensure that 1.2 mM FLA is optimal, the assay was repeated using 3 other concentrations of FLA. Fluorescence emission was highest for both mPEGp2 in PBS and 2% Triton samples at 1.2 mM FLA and decreased at 1.8 mM presumably due to quenching. The total peptide fluorescence was calculated as (supernatant−PBS)+(pellet in triton X-100−empty liposomes)=(26000−10000)+(65000−20000)=61000 which corresponds to 22 μM. For performing all assays, liposomes were diluted 2-fold, thus a concentration of 44 μM mPEGp2 was detected in this sample which is very close to the theoretical 48 μM concentration expected for no peptide loss during formulation.
The studies were carried out on 6- to 10-week-old female B6D2F1 mice derived from crosses between C57B1/6 females and DBA/2 males (Charles River Laboratories). The mice used in the experiment weigh between 19 and 22 g. Blood samples were taken from the animal 7 to 12 days after immunization, from the retro-orbital sinus.
a. Lp vaccines
The mice were immunized three times, two weeks apart, by intraperitoneal injection with 200 μl of vaccine composition. This experimental protocol is carried out for the four preparations on groups of nine B6D2F1 mice (Iffa Credo, L'Arbresle, France).
To quantify by Dot Blot the various immunoglobulins induced by the immunization, 100 μl of blood were taken, from each mouse, from the retroorbital sinus one day before the booster and 15 days after the final injection. Each blood sample was then centrifuged and the serum isolated was used for the quantification.
b. Lpeg vaccines
The mice are immunized three times, two weeks apart, by intraperitoneal injection with 200 μl vaccine composition with decreased amounts of alum in the second and third immunization (500, 100 and 100 μg, respectively). This experimental protocol is carried out with the all of the above Lpeg preparations on groups of eight B6D2F1 mice (Iffa Credo, L'Arbresle, France).
Doxorubicin (Dox) (Sigma) and vinblastine (VLB) were used as anticancer agents in the protocols for the in vivo model of induction of solid tumors and of chemotherapy after immunization.
Doxorubicin is the leading cytostatic antineoplastic agent of the anthracycline family, it is therefore widely used alone or in combination in the treatment of many tumors. The main mode of action of doxorubicin appears to be on the inhibition of DNA topoisomerase II. However, like all anticancer medicinal products, doxorubicin has side effects, in particular of hematological, digestive and inflammatory type, and especially cardiac toxicity, which limits the use thereof in chemotherapeutic treatments. The doxorubicin solution was used at a concentration of 10−3 mol/l in the present experimentations.
Vinblastine is a vinca alkaloid commonly used in therapy as an agent for blocking cell mitoses in metaphase, hence its name of mitotic spindle poison. Vinblastine therefore preferentially cures rapidly dividing cells; it is therefore particularly suitable for the treatment of testicular cancer and Kaposi's sarcoma. However, there are many and varying toxic manifestations of vinblastine, the main one being blood toxicity. Finally, the vinblastine solution is used at a concentration of 10−2 mol/l in the experimental trials of the present invention.
The B16R cell line, originating from a murine melanoma and selected for its resistance to doxorubicin, was chosen in the present invention for the development of solid tumors. The B16R cells were cultured in vitro, harvested in the exponential growth phase and cleaned with a phosphate buffer saline (PBS) before they were administered by subcutaneous injection into the rear flank of the mouse. The injection volume is 50 μl of a suspension of 1.106 B16R cells in 0.85% NaCl (Candido K A et al. Cancer Res 2001, 61 (1): 228-236). The mice developed a melanoma with an average size of 2.0 g±1.2 g within a period of between 22 and 24 days after inoculation of cancerous cells.
After tumor development, the B16R line was confirmed to be resistant to chemotherapeutic treatments with doxorubicin. Consequently, the preceding experimental conditions served as a model for inducing the solid tumor from murine P388R cells (murine lymphoid neoplasma cells characterized and used as reference cells for their MDR properties—Kohls W D. et al. Cancer Res 1986 September, 46(9): 4352-6), also resistant to doxorubicin.
Other tumor cells can be used provided that they exhibit resistance to the chemotherapy tested.
A protocol for chemotherapy using two anticancer agents, vinblastine and doxorubicin, was established as described in
a. Lp vaccine
The chemotherapeutic treatment of the mice pre-immunized with the vaccine preparations Lp1 and Lp2begins one day after the subcutaneous injection of 106 P388R cancer cells (day 0), by means of a weekly injection of doxorubicin at a dose of 5.5 mg/kg (days 1, 10 and 22) followed by the alternating injection of vinblastine at a dose of 2.5 mg/kg (days 4 and 14). During this period, the food intake, water intake and weight of the mice, and also their survival, were recorded. Before the injection with the P388R cells, samples of mouse serum were taken during a period for between 15 and 45 days after immunization, in order to quantify the anti-P170 antibodies and monitor their activity.
b. Lpeg vaccine
The chemotherapeutic treatment of the mice pre-immunized with vaccine preparations Lpeg1 to Lpeg7 begins one day after the subcutaneous injection of 106 P388R cancer cells (day 0: 45 days after last injection), by means of a weekly injection of doxorubicin at a dose of 5.5 mg/kg (days 1, 9 and 17) alternating with injections of vinblastine at a dose of 2.5 mg/kg (days 5, 13, and 21). During this period, the food intake, water intake and weight of the mice, and also their survival, were recorded. Before the injection with the P388R cells, samples of mouse serum were taken during a period for between 15 and 80 days after immunization, in order to quantify the anti-P170 antibodies and monitor their activity.
The conjugates according to the invention serving as antigenic molecules, diluted in PBS, were firstly deposited at ambient temperature onto nitrocellulose membranes. After 30 minutes, these antigenic molecules are blocked with 3 ml of a solution containing PBS-5% skim milk. The membranes were incubated for 2 hours at ambient temperature, without washing, with 24 μl of murine serum prediluted volume-for-volume with PBS, in 2 ml of PBS containing 1% of skim milk and 0.1% of tween 20. Said murine serum was taken during a period of 15 to 45 days after the third immunization. After three washes in PBS-1% skim milk-0.1% tween 20, the membranes were sequentially incubated for 1 hour at ambient temperature, in 3 ml of PBS-1% skim milk-0.1% tween 20 containing the peroxydase anti-mouse secondary antibody diluted to 1/3000, and then after 2 washes, in 3 ml of PBS-1% skim milk-0.1% tween 20. The membranes were then washed once for 10 minutes in PBS alone and kept in a refrigerator at 4° C. overnight, in 500 μl of PBS. A chemoluminescence peroxydase substrate (ECL™ kit, AMERSHAM Pharmacie Biotech) was deposited at the surface of the membranes (0.125 ml/cm2), and left for one minute, and the membranes are then drained off and placed in a “cold” cassette between 2 films of Saran®. The membranes are immediately exposed by autoradiography, the emitted light resulting from the reaction of oxidation of the luminol by the peroxydase being collected on KODAK X-OMAT films with varying times from a few minutes to 1 hour, depending on the strength of the signal. The antibody titers were estimated using a densitometer suitable for the above system. The sensitivity of the chemoluminescence reaction has a threshold for detection of the induced antibodies evaluated at 0.2 ng/ml under the experimental conditions.
a. LP vaccine
The experiments were designed to evaluate the immune response in vivo in the mice immunized with the vaccine preparations described above used anti-mpp1, mpp2 and mpp4 antibodies and also lg (M, G3, G2a, G2b, G1) specific anti-murine secondary antibodies.
As can be seen from the results obtained and shown in
The mice immunized with the vaccine preparation Lp4 exhibit anti-mpp1, anti-mpp2 and anti-mpp4 lgM antibodies following the first immunization. This quantity of lgM antibodies was shown to decrease until it disappears following the third immunization so as to allow a predominantly lgG1 antibody immune response to emerge (
The results shown in
Immunization of the mice with the Lp1 vaccine preparation induces, as supported by the results shown in
It was observed, by comparing the quantities of lgG1 antibodies, that the mpp2 conjugate is, respectively, 2.6 and 2 times more immunogenic than the mpp1 and mpp4 conjugates. In addition, it is noted that the Lp1 vaccine preparation induces the strongest overall immune response, i.e. the isotypes (lgM, G3, G2a, G2b, G1) corresponding to each of extracellular loops 1, 2 and 4 together.
The antibody titers induced by the mpp1 conjugate are significant and comparable in value to the antibody titers detected for the mpp2 and mpp4 conjugates, unlike the results observed with Tosi et al. (1995. Biochemical and biophysical research communications 212(2): 494-500).
After having determined that the Lp1 vaccine preparation had the best immunogenic capacity, its innocuity was checked over a period of 18 months after the final immunization. The mice immunized exhibit no significant variations in weight compared with the mice immunized with the Lp2 control vaccine. In addition, no behavioral modification, for example with regard to vigilance and appetite, was observed in the animals immunized with the Lp1 vaccine preparation. Finally, histopathological analyses of the organs naturally expressing the P-170 protein (spleen, liver, kidneys, adrenoglands, pancreas, ovaries, heart and lungs) demonstrated a lack of induced toxicity and/or of autoimmunity in the mice immunized with the Lp1 vaccine preparation. Specifically, the only lesions that could be observed in the intraperitoneal position or at the periphery of the organs (liver, pancreas, spleen and ovaries) were attributed exclusively to the use of alum in the immunogenic composition. Complementary analyses to investigate the agent inducing the lesions observed confirmed this result.
b. Lpeg vaccine
The experiments were designed to evaluate the viability of P388R cells treated with different chemotherapeutic agents either in conjunction with or without sera from selected “Lpeg” vaccinated B6D2F1 mice.
As can be seen from the results obtained and shown in
The in vivo study of the evolution of the multidrug resistance phenotype in the mice immunized with the Lp1 and Lp2 (control) vaccine preparations was initiated according to a protocol of induction of solid tumors followed by the chemotherapeutic treatment plan. The anticancer treatment begins 1 day after inoculation of the cancer cells.
Prior to injection of the P388R cells into the immunized mice, the antibody titers in the sera of the mice immunized with the Lp1 vaccine preparation were determined: 100%, 40% and 80%, respectively, of the sera exhibited anti-mpp1, 2 and 4 lgG1-type antibodies, with a mean value of 0.3, 0.21 and 0.33 μg/ml (1 U corresponds to 0.2 μg/ml).
The survival time for the mice immunized with the Lp1 and Lp2 vaccine was represented as a function of time (
The results represented by graph show that the mean survival time for the group of mice immunized with Lp1 is 39 days, whereas, in the group immunized with the Lp2 preparation, it is 22 days. The mice immunized with the Lp1 vaccine preparation in a preventive manner therefore exhibit a 77% increase in mean survival time compared with the control.
In the Lp2 group, a survival time of 70 days was observed for one of the mice.
This 77% increase in survival time is observed even though the chemotherapeutic treatment as such was only administered 22 days starting from the injection of the resistant cancer cells. Now, it is observed that the survival rate falls starting from the end of administration of the anticancer agents; consequently, it can be deduced that a complete reversion would be observed if the chemotherapeutic treatment continued, in the knowledge that only the latter has a curative effect, unlike the autoantibodies obtained in the patient immunized with the composition according to the present invention.
These results are very promising since the best published results obtained in the treatment of multidrug resistance with the same cancer model described a 49% increase in survival in mice treated with S9788 at doses of 100 mg/kg/day (Pierre et al. 1992. Invest New Drug. 10: 137-148). In addition, Yang et al. (1999. BBRC. 266: 167-173) observed, with the same cell line, a 35% increase in survival in mice treated with vincristine and cyclosporin A. Other authors have also demonstrated that certain reverting agents such as trans-flupenthixol can accelerate mortality by means of an increase in the invasive potential of the cancer cells.
This increase in survival time is all the more pleasing since the experimental murine cancer models are very demanding with respect to the effectiveness of the treatment, since, as soon as they are inoculated, the cancer cells have a degree of resistance that is greater than that observed clinically. This observation implies that the reversion agents are active as soon as the cancer cells are injected; now, it is noted that these agents are generally active at cytotoxic concentrations that are reached gradually during treatment.
Immunization with the Lp1 vaccine preparation induces in the mice the formation of active autoantibodies capable of rapidly, and in a long-lasting manner, inhibiting, in vivo, resistance to chemotherapy.
Immunization with the Lp1 vaccine therefore makes it possible to rapidly inhibit chemoresistance and to reestablish in vivo the activity of anticancer agents in patients who have become refractory to the chemotherapeutic treatment. In the course of complementary experiments, the circulating autoantibodies in the mice immunized with the Lp1 preparation induced no cytotoxicity, no development of autoimmune lesions, nor any increase in the evasive potential of the cancer cells.
Thus, Lpeg 1, similar to Lp1, produced on average an about 75% increase of the 50% survival time of vaccinated animals, allowing one to draw, for Lpeg1 similar conclusions as for Lp1.
The above descriptions use in particular peptides of the murine P170 protein. The protocols described are, however, naturally applicable in a similar manner to the synthesis of any other peptide, in particular those which have been described above for the human P170 protein.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR0309188 | Jul 2003 | FR | national |
This application is a continuation in part application of application Ser. No. 11/274,885, filed Nov. 16, 2005 as well as a continuation in part application of application Ser. No. 10/565,904, filed Jan. 25, 2006, both of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP04/08330 | Jul 2004 | US |
| Child | 10902276 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11274885 | Nov 2005 | US |
| Child | 11550788 | US | |
| Parent | 11059633 | Feb 2005 | US |
| Child | 11274885 | US | |
| Parent | PCT/EP04/08330 | Jul 2004 | US |
| Child | 11059633 | US | |
| Parent | 10902276 | Jul 2004 | US |
| Child | PCT/EP04/08330 | US |
