WO2004111090A2 - Molecules de ciblage et de liberation de composes therapeutiques et leur utilisation - Google Patents
Molecules de ciblage et de liberation de composes therapeutiques et leur utilisation Download PDFInfo
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- WO2004111090A2 WO2004111090A2 PCT/FR2004/001435 FR2004001435W WO2004111090A2 WO 2004111090 A2 WO2004111090 A2 WO 2004111090A2 FR 2004001435 W FR2004001435 W FR 2004001435W WO 2004111090 A2 WO2004111090 A2 WO 2004111090A2
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4721—Lipocortins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to chimeric molecules for targeting and releasing therapeutic compounds in mammals, especially humans.
- the molecules of the invention mainly comprise three segments or functional domains: a targeting segment, capable of preferentially binding to the surface of the targeted cells, a therapeutic segment, comprising the biologically active compound, and a linking segment between the segment of targeting and the therapeutic segment, the binding segment being cleavable at the target site.
- the invention also relates to the preparation of these molecules, synthesis intermediates or domains thereof, pharmaceutical compositions containing them, and their uses, in particular in the pharmaceutical field.
- the molecules and compositions of the invention are very particularly suitable for targeting pathological cells engaged in an apoptotic pathway, and for the treatment of pathologies or associated tissues, in particular cancers and inflammation.
- Anti-tumor compounds are in principle highly cytotoxic compounds, which are distributed throughout the body when administered systemically. They produce extremely damaging side reactions to the individual. There is therefore a very great interest in distributing these anti-tumor compounds only in tumor tissues, that is to say in targeting these tissues by means of a specific vector. Although this problem of treatment toxicity is particularly acute for cancer, it can be generalized to a large number of treatments in which, in general, a minimum of side effect is sought for maximum therapeutic effect.
- a system called ADEPT Antibody Directed Enzyme Prodrug Therapy
- ADEPT Antibody Directed Enzyme Prodrug Therapy
- an antibody carries an enzyme to the tumor site.
- a prodrug is administered and converted into an active molecule by this enzyme (US 5,760,072; US 5,433,955).
- US 5,760,072; US 5,433,955 the enzyme allowing activation of the prodrug is targeted.
- the pro-drug is therefore distributed throughout the body, which does not entirely exclude side reactions and reduces the dose of pro-drug actually supplied to the tumor site.
- this approach is complex because it requires the use of several types of molecules.
- An analogous method based on targeting the enzyme capable of activating the pro-drug has been described in the applications (WO97 / 26918; WO 98/51787).
- the object of the present invention is to provide molecules capable of targeting tissues presenting a pathology and of specifically liberating therefrom compounds with therapeutic properties for this pathology. Thanks to the molecules of the invention, the therapeutic compounds exert their activity in a localized and more effective manner, and offer risks of side effects much lower.
- the molecules of the invention can be constructed to target different types of pathological cells or tissues, preferably in humans.
- the invention is based on the targeting of apoptotic cells, thanks to a targeting element having the property of binding to cell membranes expressing a negatively charged lipid, in particular phosphatidylserine (PS).
- PS phosphatidylserine
- Apoptosis or programmed cell death, is a normal physiological phenomenon, characteristic of multicellular organisms and present in particular in humans. Under normal conditions, the rate of apoptosis is however low and the apoptotic cells are very quickly phagocytosed either by neighboring cells, or more often by professional phagocytic cells such as macrophages or dendritic cells. In addition, apoptotic sites are often very dispersed and show no synchronization. As a result, physiological apoptosis is generally undetectable.
- Apoptosis is signaled to phagocytic cells by the presence of PS on the surface of apoptotic cells, resulting from the loss of asymmetry in their plasma membrane.
- the presence of detectable apoptosis is a sign of a physiological disorder where the phagocytic functions are overwhelmed and unable to cope with the increase in cells to be eliminated.
- Well-known examples of such disorders are, for example, apoptosis of the heart muscle after a heart attack or hepatic apoptosis following a strong viral infection or other conditions of an acute nature. Apoptotic cells are also found in other tissues or pathological mechanisms, such as inflammation and cancer.
- the pathological tissue contains little or not enough target cells capable of massively retaining the therapeutic molecules of the invention
- these can be used in combination with an agent or a treatment causing or promoting apoptosis in within the pathological tissue in order to increase the therapeutic benefit.
- an agent promoting apoptosis for example a conventional anti-tumor
- the advantage of targeting apoptotic cells in the tumor environment lies in the kinetic effect linked to the administration of the compound anti-tumor: in the first case, the action of the therapeutic compound, if administered continuously, produces an increase in the "target” and therefore in the effective concentration of said therapeutic compound in the tumor environment, while, in the second case, the size of the "target” tends to decrease as well as the effective concentration of the therapeutic compound.
- This cumulative effect resulting from the targeting of apoptotic cells is also very important for controlling the initial stages of metastasis by maintaining an optimal concentration of the therapeutic compound in the final stages of tumor regression.
- the strong decrease in the effective dose of anti-tumor compound and its strong localization in the tumor region has of course also the effect of considerably reducing the secondary toxic effects on the patient.
- the molecules of the invention therefore make it possible to target different pathological tissues and to release a therapeutic or biologically active agent therein in situ, thereby reducing their undesirable cytotoxic effects on healthy tissues.
- a first subject of the invention therefore resides in chimeric molecules comprising a targeting region, an active region and a binding region sensitive to the environment of a pathological tissue or cell.
- the targeting region is preferably a region targeting apoptotic cells.
- the active region can be any therapeutic compound, typically anti-cancer or anti-inflammatory.
- the therapeutic compound is typically less active when it is in the form of a chimeric molecule of the invention, than in a free form.
- Another subject of the invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a chimeric molecule as defined above.
- Another object of the invention relates to the use of targeting molecules as defined above for the preparation of medicaments.
- these drugs are anti-tumor or anti-inflammatory drugs.
- the invention can be used in particular for the treatment of solid or liquid or hematopoietic tumors, in particular of breast, lung, intestine, colon, prostate, brain, head and neck, liver cancers. , skin, lymphoma, melanoma, etc.
- the therapeutic compound is an anti-inflammatory
- the molecules according to the invention can be used for the preparation of medicaments intended for treatment of acute or chronic inflammatory pathologies such as asthma, ulcerative colitis, Crohn's disease, septic shock, collagen diseases and P arthritis.
- Another object of the invention relates to the use of targeting molecules as defined above for the local delivery of active principles in the vicinity of pathological tissues in subjects.
- the present invention also relates to methods of treating a pathology in a subject comprising the administration of a molecule or a composition as defined above.
- the pathology concerned is cancer or inflammation.
- the treatment method may further comprise a preliminary step consisting of a treatment making it possible to generate cells engaged in a process of apoptosis in the pathological tissue. It also relates to methods of local delivery of active principles in the vicinity of pathological tissues in subjects, comprising the administration of a molecule or of a composition as defined above.
- the molecules of the invention typically comprise three parts or functional domains linked to each other, namely a targeting region (C), a biologically active region (A) and a binding region (L) sensitive to the environment of a tissue or pathological cell.
- a targeting region C
- A biologically active region
- L binding region sensitive to the environment of a tissue or pathological cell.
- the arrangement of the different elements can vary, and in particular according to the order A-L-C or C-L-A.
- the region (or function) L can be inserted or included within one of the regions A or C.
- Targeting segment C is a molecule capable of recognizing or preferentially binding cells engaged in a pathological process, preferably apoptosis; 2)
- the binding segment L is a molecule ensuring the binding between A and C, said binding being cleavable at the target site allowing the release of the therapeutic segment A;
- Part A is a biologically active molecule with therapeutic properties.
- Targeting segment C comprises a polypeptide capable of binding to the surface of cells characteristically or specifically present in a tissue presenting a pathology, or generated in this tissue by a prior or combined treatment.
- This targeting segment C is preferably capable of binding to the membranes of cells engaged in a process of apoptosis, these exposing on their surface negatively charged lipids such as phosphatidylserine.
- part C comprises a polypeptide capable of preferentially binding to the surface of tumor cells or present in tumor tissue or generated in these tissues by treatment with an agent promoting apoptosis, for example an anti-tumor (chemotherapy, radiotherapy, etc.).
- an agent promoting apoptosis for example an anti-tumor (chemotherapy, radiotherapy, etc.).
- the targeting segment C comprises a polypeptide capable of preferentially binding to the surface of the cells present in an inflammatory tissue and in particular the neutrophil cells which accumulate in these tissues and die there by apoptosis 24 48 hours after their arrival.
- Neutrophils are "baits" for targeting molecules.
- the term "preferentially bind" indicates that the targeting element has a particular affinity for the cells or tissues considered, even if a non-specific or less important bond with other cells or tissues cannot be completely excluded in vivo.
- the preferential bond nevertheless ensures targeting of the chimeric molecules of the invention towards the pathological sites, reducing the dissemination and the potential side effects.
- the targeting segment C is preferably a peptide molecule.
- peptide molecule is meant any molecule composed or comprising amino acids, natural or not, optionally modified, such as for example any protein or fragment of protein, a polypeptide or peptide, natural or synthetic, modified or not.
- the common property of these elements is to be able to bind preferentially to cells characteristic of pathological situations and, in a more preferred mode, to cell membranes exhibiting negatively charged lipids, in particular phosphatidylserine.
- the present invention provides for the use of any protein, fragment or derivative of protein meeting this criterion.
- proteins capable of binding to membranes exposing negatively charged lipids. Mention may in particular be made of the Annexin family, the families of proteins comprising a Cl or C2 domain, such as factors V and VIII of blood coagulation; families of proteins comprising a PH domain or a FYVE domain; or proteins with an identical domain or homologous to domain 5 of ⁇ 2-Glycoproteins-I ( ⁇ GP-I). These proteins, or domains derived from or derived from their sequences can be used as a targeting element in the chimeric molecules of the invention. For reasons of immunogenicity, the human version of these proteins or protein domains is preferably chosen. In addition, whenever possible, it is necessary to select and use the smallest active domain of these proteins in order to ensure the best diffusion of the molecule through the micro-vascularization towards the targeted tissues and above all a better bio-distribution and elimination by the renal route.
- the targeting element comprises a peptide sequence derived from the proteins or protein fragments mentioned above.
- fragment is meant a sequence of at least 10 consecutive amino acids, preferably between 50 and 500 amino acids, even more preferably around 250 to 350 amino acids.
- These derivatives have at least 50% identity with the initial proteins or the fragments thereof. Preferably, they have 60%, 75%, 90% or 95% identity.
- Some of the protein domains mentioned, in particular the PH and FYVE domains can moreover be mutated so as to modify their lipid specificity to adapt them to the precise needs of targeting cells in the apoptotic phase.
- the targeting segment C can contain one or more sites for binding to the segment L.
- the presence of several sites has the advantage of being able to distribute several molecules therapeutic A in one go and increase in the same proportions the concentration of A in the environment of the tissues affected by the pathology concerned.
- the structure of the molecule according to the present invention is then:
- the targeting segment C can comprise a repetition of several polypeptides or motifs for binding to pathological or target cells (designated CO), in order to increase the efficiency of targeting.
- the general formula of such molecules is as follows:
- n 1 or 2.
- the targeting segment C comprises the sequence of an annexin, or of a fragment or a derivative thereof.
- Targeting segment C preferably comprises the sequence of the “four-domain heart” of a protein of the annexin family.
- the annexin is of type V, a fragment or a derivative thereof.
- Annexin of human origin is preferred.
- the targeting segment C comprises domain 1 of this annexin.
- the targeting segment C comprises a Cl or C2 type domain, a fragment or a derivative thereof. More particularly, the invention relates to targeting segments C comprising the sequence of a Cl domain of a coagulation factor, a fragment or a derivative thereof. Alternatively, the invention relates to targeting segments C comprising the sequence of a C2 domain of human coagulation factor VIII, a fragment or a derivative thereof.
- the targeting segment C is a polypeptide constructed on the basis of a Cl-type domain topology.
- the targeting segment C comprises a polypeptide of sequence chosen from SEQ ID Nos 1-8, preferably SEQ ID Nos 2-4, and 6-8, or a fragment thereof.
- C1F8-S1 (SEQ ID No 6) KCQTPMGLAS GHIRDFQITA SGQYGQWAPK LARLHYSGSI NAWSTKEPFS WLKIDLLAPM IIHGIKTQGA RQKFSSLYIS QYIIMYSLDG KKWQTYRGNS TGTLMVFFGN VDSSPHRMMYIR
- C1F8-S2 (SEQ ID No 7) KCQTPMGLAS GHIRDFQITA SGQYGQWAPK LARLHYSGSI NAWSTKEPFS WIKVDLLAPM IIHGVKTQGA RQKFSSLYIS QFIIMYSLDG KKWQTYRYNS TGTLMVFFGN VDSSGIKILRMPLYLRMPLYRMNP
- C1F8-S3 (SEQ ID No 8) KCQTPLGMAS GHIRDFQITA SGQYGQWWPK LARLHYSGSI NAWSTKEPFS WLKIDLLAPM IIHGIKTQGA RQKFSSLYIS QFIIMYSLDG KKWQTYRGNS TGTLMVFFGN VDLPGIRI
- the targeting segment C is a polypeptide constructed on the basis of a C2 type domain topology.
- the targeting segment C comprises a polypeptide of sequence chosen from SEQ ID Nos 9-16, preferably SEQ ID Nos 10-12, and 14-16, or a fragment thereof.
- C2F5-S1 (SEQ ID No 10) CSTPLGMENG KIENKQITAS SFKKSWWGDY WEPFRARLNA QGRVNAWQPK ANNNKQWLEV DLLKIKKITA VITQGCKSLS SEMYVKSFTI HYSEQGVEWK PFRLKSSMVD KINEGNTNTK GHVKNFPNPP RISRFIRVIP KTWNQSITLR LELFGCDIY
- the targeting segment C is a polypeptide constructed on the basis of a domain 5 topology of ⁇ 2-Glycoproteins-I ( ⁇ 2GP-I) (SEQ ID No 17).
- the targeting segment C comprises a polypeptide of sequence chosen from SEQ ID Nos 17-22, preferably SEQ ID Nos 18-22, or a fragment thereof.
- the targeting segment C comprises a polypeptide whose general sequence is as follows: TJ 2 ASCKU 7 PU 9 KJ 11 U 12 TU 14 U 15 U 16 J 17 GERU 21 J 22 U 23 QEKU 27 J 28 NGML
- the targeting segment C comprises a polypeptide of sequence chosen from SEQ ID Nos 19-22 or a fragment thereof:
- the targeting segment C comprises a polypeptide whose general sequence is as follows: jl. D 2d3. J 4_ J 5_ J 6. z 7_ u 8_ J 9_ J 10. u ll_ R _ J 13_ J 14_ u l 5 _ ⁇ _ G _ ⁇ 18_ G _ ⁇ _ J 21_ E _j23_ J 24_ u 25_ J 26_ J 27 . J 28_
- amino acids J are chosen independently of each other from natural amino acids, or derivatives thereof, such that at least 50% of them are polar residues chosen from R, N, D , C, Q, E, G, H, K, Orn, P, S, T and Y,
- amino acids U are chosen from A, C, G, I, L, M, F, W, Y, and V
- - amino acid X 18 is chosen independently of the other amino acids of the sequence from A, N , C, Q, G, H, I, L, M, F, S, T, W, Y and V,
- amino acid B 37 is chosen independently of the other amino acids of the sequence from R, A, C 5 G, I, L, M, F, W, Y, and V,
- amino acid Z 7 is chosen independently of the other amino acids of the sequence from D and E,
- the amino acids Z 59 and Z 65 are chosen independently from E, D, K, and R, the exponents indicating the position of the amino acids in the sequence.
- the amino acids J can be chosen independently of each other from the set of residues A, R, N, D, C, Q, E, G, H, I, L, K, M, Orn, F , P, S, T, W, Y, and V, and in such a way that at least 50% of them are polar residues chosen from R, N, D, C, Q, E, G, H, K, Om, P, S, T.
- the peptide of formula S1 can advantageously be a peptide sequence chosen from the peptide sequences SEQ ID No 23-32.
- the sequence S1 represents a type of peptide in its shortest form. It is understood that this sequence can also comprise one or more additional amino acids at one or the other of the ends, for example from 1 to 15 amino acids, in general from 1 to 10 amino acids to obtain additional functionalization. .
- a small sequence called a functionalization sequence, can be linked to the peptide allowing attachment to the L segment.
- This functionalization sequence can be located at the N-terminal end of the sequence S1. It can be around 3 amino acids preferably chosen from GSC-, GST-, GSP-, GSS-, GSG-, and GSQ-.
- These functionalization sequences are advantageous in that they allow in particular easy labeling by radio-tracers such as 99m Tc or 18 F and allow, by injection into humans at a tracer dose, to follow in vivo the fate perfectly. of the drug, its bio-distribution and to control its good localization.
- sequence S1 can be duplicated within the same peptide to produce a molecule exhibiting an even higher affinity for the apoptotic sites.
- the segment L is a binding molecule cleavable at the target site, and connecting part A and part C.
- Segment L can be any molecule or chemical bond, of covalent type, including molecules in part or totally of peptide nature , modified or not, natural or not.
- the L segment advantageously contains a recognized chemical function which can be cleaved in the environment of the tissue or pathological cells, for example by an enzyme or a set of enzymes specific to the environment of the targeted cells.
- linker segment L which can be cleaved preferentially in the environment of the targeted cells allows a local and targeted release of the active principle and gives the molecules of the invention a behavior of the targeted pro-drug type. Indeed, the molecule is not very active as long as it does not reside in the environment of the targeted cells possessing the enzymes or other cleavage factors.
- the linking segment L can be simple or branched.
- the advantage of a branching is to be able to bring and distribute several therapeutic molecules with a single targeting molecule C.
- the L (or LO) segment can be of a varied nature, such as a bond or chemical molecule, and in particular of a peptide nature.
- the L segment is a cleavable peptide link sensitive to proteases (or other enzymes), called in the following text "intervening proteases", more specifically overexpressed either on the surface of cells presenting the pathology, either released into the environment of the target cells, or even released by the cells in the apoptotic phase.
- proteases or other enzymes
- Tumor and inflammatory cells are known to excrete a variety of intervening proteases, in particular metallo-proteases of the extracellular matrix (MMP) , urokinases, specific proteases for the cleavage of the extracellular segment of membrane cytokines or their receptors (ADAM), etc.
- MMP extracellular matrix
- urokinases metallo-proteases of the extracellular matrix
- ADAM membrane cytokines or their receptors
- sequences cleaved by these proteases are given in Table 2.
- These intervening proteases play an important role in the evolution of the tumor or of the inflammatory tissue and in particular in the invasion of the surrounding tissues and the formation of metastases, or l invasion by specialized inflammatory reaction cells.
- the level of MMP in the tumor or inflammatory environment is much higher than that present in normal tissue because, in particular, the control of the expression of these MMPs depends on the action of certain cytokines. It is therefore possible to take advantage of this expression differential to amplify the principle of targeting of the anti-tumor or anti-inflammatory compound by only allowing activation of this compound at the targeting sites.
- Simple pro-drugs have already been designed but they do not solve the problem of uniform distribution of the drug throughout the body because there is no accumulation of the pro-drug within the cancer tissue or inflammatory.
- the present invention a means is proposed to accumulate the antitumor or anti-inflammatory principle only in tumor or inflammatory tissue already comprising cells in the process of apoptosis, and to make the drug active essentially in this tissue.
- the CL or LC assembly constitutes a vectorization-activation system of anti-tumor or anti-inflammatory compounds. This together therefore meets the following double imperative: reduce the average effective concentration of the drug in the body, that is to say reduce the secondary toxic effects, and restrict the action of the drug to the only tissue of interest, that is to say increase its efficiency.
- the cleavable linker segment L is therefore an at least partially peptide link comprising a sequence recognized and cleaved by an intervening protease mainly present in the targeted tissue.
- the L or LO link can therefore be represented as comprising two parts, L1-L2, designed such that the intervening proteases cleave the peptide bond of the link between L1 and L2 and that the released molecule L2-A or A-L1, depending on the molecules chosen, ie a molecule which is therapeutically active, preferably at least as much as the initial molecule A.
- the length of the parts L1 and L2 can be optimized as a function of the accessibility necessary to the active site of the intervening proteases, with the constraint limit as much as possible the final size of the final molecule for the reasons mentioned above.
- L (L1-L2) n -D (F5B) where D, Ll, L2 and n have the definition given above.
- connection between the different functional elements of the molecules of the invention can be carried out by any chemical, enzymatic or genetic coupling method known per se to those skilled in the art.
- they can be chemical, peptide, nucleic bonds, etc.
- the groups can be coupled together by maleimide, succinimide, intein, biotin, amino, amide, carboxylic, phosphate, ester, ether, etc. bonds.
- the link L is linked to the molecule C (and / or A) by a maleimide group, known for its rapid and total reaction with a thiol group carried by an accessible cysteine residue from the targeting segment C (and / or of molecule A).
- the link L is linked to the molecule C (and / or A) by reaction of the terminal carboxylic group of the peptide L with an amino group carried by the therapeutic segment A (or targeting C).
- the linking segment L is coupled to the C-terminal (in the case of an H 2 NLA molecule) or N-terminal (in the case of an AL-COOH molecule) end of the segment C by means of an intein.
- the segment L (or the cleavage function) is part of the element C or A. It can be for example an N-terminal or C-terminal extension of the segment C or A, especially when the latter is peptide in nature.
- the molecules of the invention can be assembled in one or more steps, depending on the coupling techniques used.
- a CL type molecule can be produced first, then coupled with one or more therapeutic molecules A.
- the connections between the functional segments call for different chemical reactions, simultaneous synthesis or assembly is possible .
- the purely peptide version of the L link is preferably obtained by direct synthesis, using in particular the common peptide synthesizers and conventional methods of synthesis, especially in solid phase.
- the advantage of the direct synthesis of the link is that it allows the use of non-natural amino acid residues thus allowing better adaptation of the sequence to the intervening proteases.
- This recognition sequence can also be modified with respect to the natural recognition sequence by an enzyme or a cleavage factor, for example to give it a better affinity or specificity towards the protease in question.
- the length of the link segment can be adapted by a person skilled in the art according to the needs and the nature of the segments C and A. In general, a Fairly short and essentially non-immunogenic linker. It is typically a segment of peptide nature comprising from 3 to 20 amino acid residues, preferably from 3 to 15, even more preferably from 4 to 12. In certain cases, as indicated below, it can be interesting to modify the length of the segment L, in particular to distance the peptide part from the segment C or A, or to create molecules with particular function (penetration in the cell, action on neighboring cells, etc.).
- L or LO can take one of the following two structures:
- L'1 is a non-peptide spacing group and L "l is the peptide part proper;
- L'2 is a non-peptide spacing group and L "2 the peptide part proper.
- the non-peptide spacer group can be chosen from any synthetic chemical molecule, preferably chemically stable and not very immunogenic. It may especially be a polymer, for example of the polyoxyethylene, dextran, polyethyleneimine, etc. type. A preferred example is polyoxyethylene, functionalized or not.
- L “l-L2 and L1-L” 2 can represent the same peptide. Furthermore, the number of residues of the peptide segments L “l-L2 or Ll-L” 2 can vary from four to more than twenty, the optimal value being of the order of six.
- the cleavable link segment corresponds to the following formula:
- n and c can vary from 0 to around 10 and depend on the end chosen for the connection to segment A; for the end linked to A, the value of n or c will be low and preferably equal to 0 (J 3 only present) and for the opposite end the value of n or c is not limited and the corresponding residues will be chosen according to the specificity of the targeted intervening proteases.
- the sequences J- 1 -J 0 and J 3 -J 4 which frame the cleavage site B 1 -B 2 are involved in the interactions of the link with the targeted protease and therefore on the speed of the enzymatic reaction to cut the link B 1 -B 2 .
- Jo GIy, AIa, Leu, Ile, Val, Phe and any hydrophobic non-natural amino acid residue.
- J 3 GIy, AIa, Leu, He, Val, Phe
- J 4 GIy, AIa, Leu, Ile, Val, Phe or any hydrophobic or absent non-natural residue
- the other residues, J-2 ⁇ J-n and J5 ⁇ Jc can be any natural or unnatural amino acid residue as required.
- the length and / or the properties of the linking segment can be adjusted, for example to construct a molecule allowing the therapeutic compound A to interact with or to penetrate into a cell neighboring the target cell on which the segment C leads her.
- the link segment L: - must be long enough to reach neighboring cells.
- a chemical oligomer for example of the polyoxyethylene type, is preferably chosen, comprising a sufficient number of monomers so that the length of the link is approximately 80 to 200 angstroms, typically 130 to 150 angstroms; and / or - comprises a domain allowing or facilitating the passage of A through the cell membrane and, where appropriate, a cleavage region sensitive to intracellular enzymes.
- the link advantageously has one of the following two structures:
- L or L0 A-L3-LMT-LE (F9B) where LE is an essentially extracellular part of the link, LTM a transmembrane part and L3 a function or element cleavable by proteases or intra-cellular esterases (eg, cytosolic).
- the LE part is preferably sufficiently hydrophilic to allow suitable solvation in an aqueous medium in order to obtain a sufficiently extensive structure.
- the LTM part of a length at least equal to about 40 ⁇ , preferably be sufficiently amphiphilic so that, on the one hand, its structure in the extracellular aqueous medium remains weakly compact and that, on the other hand, it can cross the hydrophobic medium of the plasma membrane.
- LMT - [O- (CH2) q -O] - (Fl 1) where q and p are whole numbers different from 0, q> p so that, for LMT, the lipid environment of the membrane is at more favorable energy plan than the aqueous environment.
- the values p 2 and 5>q> 3 are preferable.
- the targeting element can be synthesized by techniques known per se from chemistry or biology.
- the element C-L also constitutes a particular object of the invention.
- the invention can be implemented with any type of therapeutic molecule capable of being associated with the C segment. They may be chemical compounds, drugs, small molecules, etc.) of peptide, nucleic, lipid, etc. compounds. .
- therapeutic segment A is a molecule exhibiting biological activity.
- this biological activity is reduced (or nonexistent) when the therapeutic segment is linked to the L-binding and targeting C segments. Therefore, the biological activity is expressed mainly in the environment of pathological tissues, after targeting. and cleavage in vivo.
- Therapeutic compounds can have various properties, in various therapeutic fields. These may be already known drugs, or new or developing molecules. They may be compounds whose mode of action involves penetration into cells or whose action only involves interaction with the surface of the plasma membrane.
- the preferred compounds A of the invention are anti-tumor or anti-inflammatory.
- the biologically active molecule has anti-tumor properties.
- A can be any anti-tumor molecule or an active derivative of these same molecules. The only constraint is that these anti-tumor compounds can be linked chemically to the rest of the vectorization molecule.
- A can be released into the extracellular medium and passively or actively diffuse inside neighboring cells or else be brought into these cells by a link of the LE-LMT type and then released by the action of endogenous proteases or esterases.
- An example of an anti-tumor compound is constituted by the molecules of the antracycline family and their derivatives.
- antacycline molecules contain an amino sugar.
- the amino group is advantageously used for the link to the L segment (for example to the L2 or LMT part) of the link defined above.
- an anti-tumor compound is constituted by molecules of the TNF ⁇ family or derivatives thereof. These molecules act on cell surface receptors and induce apoptosis.
- the factor TRAIL or A ⁇ o2L (P_W19777) is probably the most interesting since normal cells seem to be protected from its action whereas tumor cells are sensitive to it and can be selectively eliminated by it. action of this pro-apoptotic cytokine.
- This factor can therefore be very useful in so-called “solid” cancers, which implies effective targeting to avoid as much as possible the side effects due to the presence of these molecules in the blood.
- TRAIL is initially a membrane protein associated in trimer and only the extracellular part is active.
- TRAIL-Do This extracellular part, TRAIL-Do, or a set containing TRAIL-Do, can therefore be targeted thanks to the C segment and released in the inter-cellular space thanks to the action of proteases intervening on the cleavable link L which connects C and TRAIL-Do.
- the therapeutic molecule thus constituted has the following structure:
- trimer assembly of the molecules of the TNF family necessary for binding to their receptors, is hampered by the presence of the targeting and binding segments.
- the molecule remains weakly active as long as the link is not cleaved by an intervening protease.
- the cleavable link must have a sufficiently short length while retaining suitable accessibility to the intervening proteases.
- cytokine Another example of a cytokine, which is very interesting for the treatment of certain cancers and in particular melanoma and intracranial glioma, is Interleukin-4 (IL4, 1310839).
- IL4, 1310839 Interleukin-4
- This protein cannot be used without being properly targeted.
- the binding to a targeting segment C of human IL4 or one of its isoforms or of an assembly containing one of these proteins, via a cleavable segment makes it possible to constitute a therapeutic molecule interesting in oncology:
- anti-tumor compounds are in particular:
- Methotrexate is an anti-tumor compound commonly used for the treatment of cancerous tumors. It is an analogue of folic acid which first acts as a false substrate by inhibiting dehydrofolate reductase (DHFR). It also acts by indirect inhibition of thymidilate synthetase (TS).
- DHFR dehydrofolate reductase
- TS thymidilate synthetase
- Methotrexate contains an amino acid, glutamic acid, which can be inserted into the N-terminus of a cleavable link peptide according to the formula (F6B) or (F4B).
- 2-methoxyestradiol (1,3,5 (10) -oestratriene-2,3,17 ⁇ -triol 2-methyl ether) called 2ME 2 , is a byproduct of the metabolism of estrogens having the property of blocking the growth of cells rapidly dividing endothelial cells and tumor cells.
- the molecules of this family have the effect of blocking the cell cycle in G2 and M by their action on the microtubular cytoskeleton. This results in an inhibition of the normal reorganization necessary for the progress of the interphase of mitosis.
- Taxanes and in particular docetaxel, contain functions which can be used for modifications allowing its incorporation at the end of a peptide cleavable by the proteases intervening in the tumor environment.
- R 1 tBuOCO
- R 2 H (docetaxel)
- R, C 6 H 5 CO
- R 2 Ac (paclitaxel)
- cytosine arabinoside or cytarabine or Aracytine is the main representative of this family.
- Other molecules in this family are difluoro-deoxy-cytidine (Gemcitabine).
- these agents Derived from "nitrogen mustards”, these agents have given rise to various anti-tumor compounds acting on nucleic acids and therefore in the intra-cellular space. These are in particular Melphalan and Chlorambucil, two bifunctional alkylating agents of interest for easy bonding to a cleavable peptide bond.
- Melphalan is Phenylalanine-Mustard (L-PAM) is an unnatural amino acid which can be introduced easily at the beginning or at the end of the peptide synthesis of a cleavable link by intervening proteases:
- Chlorambucil only has a carboxylic function and can only be introduced directly into a peptide at the end of synthesis of the peptide link cleavable by the intervening proteases and at the N-terminal end.
- this compound can also be introduced at the C-terminal end of the cleavable peptide link.
- the molecule released in the tumor environment is a hydrophobic amino acid derivative such as, for example, Leu-Melphalan or Chlorambucil-Leu.
- hydrophobic amino acid derivative such as, for example, Leu-Melphalan or Chlorambucil-Leu.
- the biologically active molecule has anti-inflammatory properties.
- NTAl N-terminal segment of annexin I
- the anti-inflammatory properties of this peptide probably result from its action on the receptor of the fMLP thus inhibiting the chemotaxis and the activation of phagocytic cells and in particular their degranulation and their production of toxic oxygen metabolites.
- the sequence of the N-terminal segment of human annexin I is as follows:
- the anti-inflammatory peptide derived from the N-terminal segment of annexin I will advantageously be chosen from the following sequences:
- the underlined sequence represents a so-called consensus sequence having the desired anti-inflammatory properties. Under each variable residue of this sequence is indicated a list of residues which can replace that indicated in the consensus sequence: an acid residue; h hydrophobic residue; p polar residue; o Thr or Ser preferably.
- protease capable of specifically cleaving the NTAl segment at the level of one of the two residues Lysine 25 or 28 present in its N-terminal part -Thr-Val-Lys-Ser-Ser-Lys -Gly-Gly-, These proteases are normally responsible for the in vivo release of the N-terminal segment of annexin I.
- the NTA1 peptide or a judiciously mutated version is simply integrated into the N-terminal part of the C segment for the
- segment C is taken here in its widest sense and defined above.
- the NTA1 peptide or a judiciously mutated version is linked to the C segment via a simple link cleavable by an intervening proteases according to the formula F6A or F6B or a multiple link comprising an element of branch D as defined above (F5A, F5B).
- part A can also contain a repetition of one of the sequences chosen for the NTA1 segment in order to increase the local concentration of the anti-inflammatory peptide and therefore its effectiveness.
- NTAl 1n -C (F2)
- NTAl segment it may be advantageous to link the NTAl segment to the C-terminal end of the C segment.
- a bifunctional link will be used so as to connect the C-terminal ends of C and NTAl: CL-NTAl
- Chronic inflammatory diseases are caused by a large imbalance in the production in the cellular environment of a number of signaling molecules.
- the amplification and sustainability of the inflammatory phenomenon in these diseases results from a complex balance between a large number of proteins with opposite influences, pro-inflammatory molecules and anti-inflammatory molecules.
- interleukin 10 (SwissProt, P22301), or on any of its isoforms, is the most interesting by the regulatory effect it produces on the inflammatory reaction .
- IL10 has multiple functions including functions of stimulation of the immune system. It is therefore very advantageous to target this protein at the inflammatory site proper so as to strictly localize its action.
- IL10 acts as a homodimer on its hetero tetrameric receptor.
- the three-dimensional structure of PILlO and the complex with its receptor, PILlOR offers an additional advantage.
- the structure of the IL10-IL10OR complex shows that the N and C-terminal ends of PIL10 are relatively close in structure.
- the N-terminal end is buried in the heart of the receptor and the C-terminal end is located in the dimerization region of this cytokine. Therefore, blocking by the C-L or L-C segment of one of the N or C-terminal ends of PIL10, prohibits the formation of the complex and finally blocks its action.
- PIL10 can therefore only be done by the action of proteases intervening in the inflammatory environment which have the effect of releasing this cytokine exclusively in this environment.
- P35225 another anti-inflammatory cytokine
- P35225 another anti-inflammatory cytokine
- the therapeutic molecule based on PILlO or PILl 3 has one of the following general structures, depending on the choice made for the positioning of PILlO or PIL13: CL '1 -L "1 -L2- (IL10 / IL13) (according to the formula F6A) (ILl 0 / IL13) -Ll -V '2-L'2-C (according to formula F6B)
- the cleavage site of the intervening proteases is provided between L “l and L” l or between L “2 and L'2.
- the length of the residual segment L” l-L2 or Ll-L “2 is such that it does not interfere not the formation of the IL10-1L10R or IL13-IL13R ⁇ l / 2 complex It is easy to adjust the effective sequence of PIL10 to satisfy this constraint In either case the L1 or L2 segment may be absent.
- Therapeutic segment A can be selected from non-activating inhibitors of membrane receptors for pro-inflammatory cytokines.
- IL1 interleukin 1
- PILlR soluble inhibitor of the PIL1 receptor
- sILIRa Small protein
- the efficacy of sILIRa has been tested in various diseases and in particular in rheumatoid arthritis and has been shown to be relatively active.
- the doses used in patients under subcutaneous injections are enormous, from 30mg to 150mg.
- the pharmacokinetics is also unfavorable since the half-life time in the circulation is only 21 min, which is very short for therapeutic use in the case of rheumatoid arthritis.
- the C-L- (ILlRa) or (ILlRa) -L-C group is completely inactive.
- the structure of the ILlRa-ILlR complex shows that the N and C-terminal ends of PILlRa are very close in structure and are buried at the heart of the receptor. Therefore, the activation of the inhibitor can only be done by the action of proteases intervening in the inflammatory environment which have the effect of releasing the inhibitor exclusively in this environment.
- the therapeutic molecule based on PILlRa or on any of its isoforms has one of the following general structures, depending on the choice made for the positioning of PILlRa:
- the cleavage site of the intervening proteases is provided between L “l and L” l or between L “2 and L'2.
- the length of the residual segment L" l-L2 or Ll-L “2 is such that it does not interfere not the formation of the IL1-ILlRa complex.
- PILlO it is easy to adjust the effective sequence of PILlRa to satisfy this constraint.
- the L1 or L2 segment may be absent.
- Glucocorticoids There are many non-peptide molecules with important anti-inflammatory properties such as Glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs), Methotrexate. a) Glucocorticoids:
- Glucocorticoids are natural hormonal molecules produced by the body and are involved in many physiological processes. Their action is strictly intracellular and intervenes by their binding to nuclear receptors inducing the transcription of a certain number of genes, hence the complex role of these hormones at multiple stages of the inflammatory reaction.
- the use of Glucocorticoids and their non-natural derivatives as a medicine is therefore always very delicate, while their effectiveness can be very great. Their targeting and strict release into the inflammatory environment is therefore crucial.
- Glucocorticoids are steroid molecules, therefore quite hydrophobic, and their action takes place at the level of the cell nucleus after passive diffusion through the plasma membrane. For use as a targeted drug, Glucocorticoids can therefore simply be released into the inflammatory environment.
- All of these molecules have chemical functions, for example hydroxyl groups, allowing their grafting on peptide molecules and in particular on an L segment cleavable by intervening proteases as for anti-tumor compounds.
- the Glucocorticoids-based therapeutic molecule has one of the following general structures, depending on the choice made for positioning the molecule with respect to the targeting segment: CL '1 -L "1 -L2- (0-Glucocorticoid) (according to formula F6 A) (Glucocorticoid-0) -Ll-L '' 2-L'2-C (according to formula F6B)
- the action of the intervening proteases releases either the L “l-L2- (O-Glucocorticoid) molecule or the (Glucocorticoid-O) -Ll-L” 2 molecule, that is to say a pro-drug which must passively diffuse in the surrounding cells where they will be treated with endogenous proteases and esterases to finally release the active molecule.
- the segments L “l-L2 or Ll-L” 2 be hydrophobic and as short as possible, taking into account the cleavage capacities of the intervening proteases. It is advantageous to limit the number of residues to two or even to one the number of residues in L "l or L" 2. It may also be advantageous not to introduce the segments L1 or L2.
- Nonsteroidal anti-inflammatory drugs are mostly inhibitors of cyclooxygenases (COX-I and COX-2), important enzymes involved in the metabolism of arachidonic acid. These are drugs generally reserved for the treatment of severe inflammatory diseases and used in generally very high doses and therefore generating undesirable side effects.
- COX-I and COX-2 cyclooxygenases
- these drugs generally reserved for the treatment of severe inflammatory diseases and used in generally very high doses and therefore generating undesirable side effects.
- NSAIDs there is a class of compounds having a carboxylic function which can be used for their grafting on peptide molecules and in particular on an L segment cleavable by intervening proteases as for the anti-tumor or anti-inflammatory compounds mentioned above.
- Methotrexate also has anti-inflammatory properties and can be used in the same way as for its anti-tumor properties described above.
- PCRDX a peptide, here called PCRDX, comprising at least the CRDl domain of any TNFR X to block the trimerization of the latter in a very specific manner and therefore block its function: by binding to a membrane subunit of TNFR (monomer), the free PCRDX peptide, in the form of monomer or dimer, blocks the trimerization of this membrane subunit without activating the receptor since the latter can no longer acquire its trimeric structure.
- PCRDX peptide or a mutated version of this domain, linked to the C segment by an L1-L2 link cleavable by one of the intervening proteases generally present in the inflammatory environment so as to release the inhibitor only at the targeted inflammatory site.
- the general structure of the therapeutic molecule of the present invention is therefore: C-L1-L2-PCRDX or PCRDX-Ll -L2-C Where the cleavage takes place between Ll and L2.
- the C-L1-L2-PCRDX structure is directly active because it leaves the C-terminal end of CRDl free, allowing it to bind to CRDl of a cellular receptor.
- the PCRDX-L1-L2-C structure has the additional advantage of only making the CRDl segment active when it is released by cleavage of the L1-L2 segment, thereby limiting the risk of side effects accordingly.
- the C-terminal end of the PCRDX is congested by the presence of the L1-L2-C segment, unsuitable for any interaction with an extracellular domain of a TNFR, thus making its interaction more difficult.
- the activity is recovered by the cleavage of the link L1-L2.
- sequences can be mutated, in particular in the region of interaction with the original TNFR1 and TNFR2 receptors, in order to increase their affinity with these receptors.
- Analogous sequences from any TNF family cytokine receptor can be used to produce specific inhibitors for these receptors.
- the cleavable link L1-L2 can be advantageously chosen from the peptide sequences recognized and cleaved by specific proteases whose role is to release the extracellular part of the membrane TNFRs or of the membrane precursors of the TNFs.
- proteases belong to the family of ADAMs already mentioned.
- ADAM-17 or TACE protease specific for the release of TNF ⁇ and TNF ⁇ (LT ⁇ ) will be chosen and the sequence of the L1-L2 segment will therefore be chosen so that it contains one of the following sequences:
- L1 * L2 SPLAQA * VRSSSR (SEQ ID No 38) or fragments thereof, PLAQ A * VRSSS (SEQ ID No 39), LAQA * VRSS (SEQ ID No 40), AQA * VRS (SEQ ID No 41) , QA * VR (SEQ ID No 42), or any combination of the groups of sequences located on either side of the cleavage site marked with an asterisk, for example.
- the molecules of the invention can be constructed to target different types of pathological cells or tissues, preferably in humans. They can be used for the preparation of medicaments and / or in methods of therapeutic treatment.
- a particular object of the invention consists of a pharmaceutical composition comprising a chimeric molecule as defined above.
- Another object of the invention relates to the use of a chimeric molecule as defined above for the preparation of a medicament.
- these drugs are anti-tumor or anti-inflammatory drugs.
- the molecules according to the invention can be used for the preparation of medicaments intended for acute pathologies such as asthma, ulcerative colitis, Crohn's disease, septic shock, diseases collagen and arthritis.
- the invention can be used for the treatment of various tumors, in particular solid or liquid or hematopoietic tumors, in particular breast, lung, intestinal, colon and rectum, brain, meninges, stomach, esophagus, liver, pancreas, bladder, head and neck, male or female reproductive systems, skin, etc.
- tumors in particular solid or liquid or hematopoietic tumors, in particular breast, lung, intestinal, colon and rectum, brain, meninges, stomach, esophagus, liver, pancreas, bladder, head and neck, male or female reproductive systems, skin, etc.
- compositions of the invention can comprise any pharmaceutically acceptable excipient or vehicle, such as salt, solutes, etc. It can be saline, buffered, isotonic, water, etc.
- the compositions may further comprise other active agents, used in combination, simultaneously, separately or spaced over time.
- Another object of the invention relates to the use of targeting molecules as defined above for the local delivery of active principles in the vicinity of pathological tissues in subjects.
- the present invention also relates to methods of treating a pathology in a subject comprising the administration of a molecule or a composition as defined above.
- a pathology concerned and cancer or inflammation Preferably, the pathology concerned and cancer or inflammation.
- the treatment method may further comprise a preliminary step consisting of a treatment making it possible to generate cells engaged in a process of apoptosis in the pathological tissue. It also relates to methods of local delivery of active principles in the vicinity of pathological tissues in subjects, comprising the administration of a molecule or of a composition as defined above.
- treatment designates preventive, curative, palliative treatment, as well as the management of patients (reduction of suffering, reduction of the size of a tumor or of the progression of pathology, improved lifespan, slower disease progression, decreased inflammatory site), etc.
- the treatment can also be carried out in combination with other agents or treatments (chemotherapy, radiotherapy, gene therapy, etc.).
- the treatments and medicaments of the invention are very particularly intended for humans.
- the therapeutic compound can be used at different doses and according to different protocols.
- the administration can be carried out by any method known to those skilled in the art, preferably by injection, typically by intraperitoneal, intra-tumor, intradermal, intra-cerebral, intravenous, intra-arterial or intramuscular route.
- the doses administered can be adjusted by those skilled in the art. Typically, from about 0.01 mg to 100 mg / kg are injected. It is understood that repeated injections can be carried out.
- the invention can be used in mammals, in particular in humans.
- Example 1 Example of the production of cleavable L segments for the release of anti-tumor and anti-inflammatory compounds:
- L segment is only peptide and contains only natural residues, it will preferably be integrated into the C segment at its N or C-terminal end by the conventional methods of molecular biology. H can however be integrated if necessary into segment A if that is peptide and obtained by molecular biology.
- the L segment or the LA unit in a reactive form for a later connection to the C segment.
- An interesting example occurs in the case where a thiol group brought by a Cysteine residue is present in segment C, preferably far from the site of binding of C to negatively charged membranes. This thiol group makes it possible, by a simple, rapid and total chemical reaction, to link the L-A assembly provided with the maleimide functional group.
- the synthesis protocol is described in FIG. 1.
- the fragment L consists of a protected peptide linked to an acid-labile resin rink via the FMOC strategy well known to those skilled in the art.
- the reactive fragment has the property of forming a covalent bond with a nucleophilic group - here the SH group of a Cysteine - of protein C.
- the reactive group can be a bromoacetamide or, as here and more advantageously, a maleimide group .
- the spacer group between the maleimide and the peptide L can be an alkyl, alkoxy or poly alkoxy group terminated by a carboxylic function for coupling to L.
- FIG. 1 The fragment L consists of a protected peptide linked to an acid-labile resin rink via the FMOC strategy well known to those skilled in the art.
- the reactive fragment has the property of forming a covalent bond with a nucleophilic group - here the SH group of a Cysteine - of protein C.
- the therapeutic segment is an anti-tumor compound of the antracycline family, doxorubicin.
- AA represents any amino acid sequence which may constitute a cleavable link. The example described below corresponds to the sequence AA ⁇ Gly-Ser-Gly-Val-Leu.
- the filtered resin is washed with 40 ml (for 30 s each time) with the following solvents: 3 times methanol, 3 times DCE, 3 times DMA.
- the unreacted hydroxyl groups of the resin are blocked with acetic anhydride (13.5 mmol) in 3 ml of pyridine and 15 ml of DMA and then the resin is washed as follows (40 ml each time): 2 times isopropanol, 3 times DMA, 2 times isopropanol, 6 times DCE, 2 times isopropanol, 3 times DMA, 3 times isopropanol.
- the resin is then dried under vacuum and has an Fmoc content of approximately 0.35 mmol / g.
- DMA 2 times DMA, 1 time isopropanol, 4 times DMA.
- the resin is dispersed in DCM and treated with 20 ml of AcOH / DCM cleavage mixture (10/90 v / v) for one hour then is filtered, washed 3 times with 20 ml of cleavage mixture then 3 times 20 ml of DMA to extract the peptide from the resin. Hexane (15 times the volume) is added to the filtrate to remove acetic acid in azeotropic form. The resulting protected peptide is dried under vacuum and purified by "flash" chromatography.
- Example 2 Synthesis of a mixed peptide and non-peptide link for the release of anti-tumor compounds.
- the non-peptide part of the link is introduced using the compound (12), obtained according to the diagram in FIG. 2, replacing the N-maleoyl- ⁇ -alanine used in the previous synthesis (FIG. 1).
- the compound (12) is then used as the compound N-maleoyl- ⁇ -alanine for the synthesis of a mixed cleavable link according to the protocol described in Figure 1 for the compounds (5) and (6).
- Example 2b Synthesis of an arm comprising a spacer group and a cleavable peptide group and coupling on the one hand to a targeting molecule C and on the other hand to doxorubicin.
- the lyophylisate was taken up in a H2O / DMSO 50/50 mixture (2 ml) and was purified by HPLC (Source column 15RPC 10 ⁇ 250 mm). The fraction corresponding to the final compound C-S- (12) was collected and then lyophilized.
- the final compound C-S- (12) was subsequently subjected to the action of the various proteases (MMP2, MMP3 and MMP9) according to the following protocol:
- NTAIl Two constructions are proposed, one corresponding to the long version of NTAl named NTAIl, according to the sequence S7 (Seq ID 33) and the other to the short version, named NTAIc, according to the sequence S8 (Seq ID 35).
- the pGEX 6Pl plasmids used are commercial (Amersham biosciences) as well as the enzymes (Biolabs) used: BamH I, EcoR I, Ban II, T4 DNA Ligase, CIP, T4 kinase.
- the expression vector used is the vector pGEX 6Pl (Amersham-Biosciences). This vector allows the expression of the protein of interest fused to the GST at its Nter end. The protein of interest is recovered without fusion protein after digestion by the PreScission.
- the coding sequence of segment C (from the vector pGEX2T, constructed in the laboratory) will be inserted into this vector between the BamH I and EcoR I sites.
- the NTAII or NTAIc segment will then be inserted in the form of cassettes in the vector pGEX 6P containing the coding sequence of the C segment between the Ban II and BamH I sites.
- the vector pGEX 6P has two Ban IL sites.
- the first step therefore consists in making this site unique in order to use it as a cloning site for the NTA1 segment.
- the Ban II site located at position 3890 is removed by a silent directed mutagenesis step (Quick Change kit, Stratagene, Ban II + and Ban II - oligos) following the supplier's recommendations.
- the plasmid "pGEX-6P-mut" is then obtained.
- the coding sequence of segment C is extracted from the plasmid pGEX2T by enzymatic digestion using the restriction enzymes BamH I and EcoR I (Biolabs). Briefly, 20 ⁇ g of DNA is digested sequentially with 200 U of enzyme, at 37 ° C. overnight. After each digestion, the DNA is purified after migration on agarose gel using the “GFX PCR DNA and Gel Band Purification kit” (Amersham-Biosciences) kit. 20 ⁇ g of plasmid "pGEX-6P-mut" is also digested under the same conditions with BamH I and EcoR I in order to obtain the vector in which the coding sequences will be inserted.
- BamH I and EcoR I Biolabs
- the latter is dephosphorylated by an incubation of 2 h at 37 ° C. in the presence of 2 U of CIP (Biolabs).
- the vector “pGEX-6P-mut”, opened in BamH I / EcoR I and dephosphorylated, is then purified using the Amersham kit.
- Ligation of the coding sequence of segment C (10 moles of insert for 1 mole of vector) in the vector “pGEX-6P-mut” is then carried out by incubation for 2 h at room temperature in the presence of 400 U of T4 DNA ligase (Biolabs).
- the pGEX-6P-mut plasmids containing the C segment coding sequence are then obtained.
- NTA11 and NAlc cassettes are obtained by hybridization of 100 pmol of the complementary oligos (NTAlc + / NTAlc - and NTAIl + / NTAIl -) at 95 ° C for 5 min in a 20 mM Tris HCl buffer ⁇ H7.5; 300 mM NaCl; 1 mM EDTA.
- the 5 ′ ends of the cassettes are then phosphorylated by an incubation at 37 ° C. for 2 h in the presence of 50 U of T4 polynucleotide kinase (Biolabs). The enzyme is inactivated by incubation at 65 ° C for 20 min.
- the last step consists in digesting the plasmids pGEX-6P-mut the coding sequence of segment C by Ban II and BamH I in order to insert the cassettes.
- 20 ⁇ g of DNA is digested sequentially with 50 U of Ban II and 100 U of BamH I, at 37 ° C. overnight.
- the DNA is purified after migration on agarose gel using the Amersham kit.
- the dephosphorylation of the vectors is carried out by incubation at 37 ° C for 1 hour in the presence of 10 U of CIP, in order to avoid their recircularization during the ligation step.
- the ligation of the cassettes in the vectors is carried out as described above.
- the NTAIc-C and NTAII-C sequences cloned in the vector pGEX-6P-mut are therefore obtained.
- DNA sequences are checked with the Big Dye Terminator sequencing kit, Perkin-Elmer Applied Biosystems, on a Perkin-Elmer Abiprism 310 sequencer, according to the supplier's protocol.
- the expression is carried out in a strain of E. coli BL21 gold (Stratagene) at 30 ° C.
- the bacteria are cultured in a Luria Berthani medium (Gibco) containing 150 mg / L of ampicillin. When the turbidity of the cultures reaches an optical density at 600 nm
- binding buffer 50 mM Tris-HCl pH 7.5; 150 mM NaCl.
- the proteins are then purified by affinity chromatography on a GSTrap Fast Flow column (Amersham-Biosciences). A 5 mL column is prepared according to the manufacturer's instructions. The protein sample (30 mL) is loaded onto the column and the column is washed with 10 volumes of binding buffer.
- a HiLoad 26/60 Superdex 75 column (300 mL) (Amersham-Biosciences) is balanced using 2 volumes of buffer A (150 mM ammonium bicarbonate pH 7.9).
- the protein resulting from the purification by GSTrap is then injected, and its elution is carried out with 2 volumes of buffer A.
- the protein thus purified is aliquoted, lyophilized and stored at 20 ° C.
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EP04767301A EP1631592A2 (fr) | 2003-06-10 | 2004-06-09 | Molecules de ciblage et de liberation de composes therapeutiques et leur utilisation |
US10/560,025 US20060154854A1 (en) | 2003-06-10 | 2004-06-09 | Molecules for targeting and releasing therapeutic compounds, and the use thereof |
CA002528186A CA2528186A1 (fr) | 2003-06-10 | 2004-06-09 | Molecules de ciblage et de liberation de composes therapeutiques et leur utilisation |
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FR0306944A FR2856069A1 (fr) | 2003-06-10 | 2003-06-10 | Molecules de ciblage et de liberation de composes therapeutiques et leur utilisation |
FR03/06944 | 2003-06-10 |
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US8461311B2 (en) | 2010-06-08 | 2013-06-11 | Washington University | TRAIL trimers, methods and uses therefor |
WO2013063086A1 (fr) * | 2011-10-24 | 2013-05-02 | Intellect Neurosciences, Inc. | Compositions et méthodes de traitement de protéinopathies |
US9498540B2 (en) | 2013-03-15 | 2016-11-22 | Novartis Ag | Cell proliferation inhibitors and conjugates thereof |
US20190134231A1 (en) * | 2016-04-21 | 2019-05-09 | The Board Of Regents Of The University Of Texas System | Methods and compositions for detecting aneurysms |
WO2023161323A1 (fr) * | 2022-02-23 | 2023-08-31 | Resother Pharma A/S | Analogues polypeptidiques dérivés de l'annexine a1 |
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WO1991009953A1 (fr) * | 1989-12-29 | 1991-07-11 | Zymogenetics, Inc. | Procedes permettant d'obtenir des proteines hybrides de fixation de phospholipides |
WO1992019279A1 (fr) * | 1991-05-09 | 1992-11-12 | Board Of Regents Of The University Of Washington | Agents thrombolytiques cibles sur des phospholipides |
WO1999019470A2 (fr) * | 1997-10-09 | 1999-04-22 | The Regents Of The University Of California | Proteines de fusion de la gfp et de l'annexine |
WO1999060135A1 (fr) * | 1998-05-21 | 1999-11-25 | Cancer Research Ventures Limited | Generation de produits geniques multiples a partir de proteines chimeres de fusion, par clivage a l'aide d'endoproteases ubiquistes |
FR2784106A1 (fr) * | 1998-10-02 | 2000-04-07 | Commissariat Energie Atomique | Structure chimique ayant une affinite pour un phospholipide, et compose de marquage, trousse de diagnostic, et medicament comprenant cette structure |
WO2003008594A1 (fr) * | 2001-07-20 | 2003-01-30 | Gene-Life Biotechnology (Shanghai) Limited | Construction du plasmide pour l'expression de la proteine de fusion thrombolytique ciblant de thrombus |
US20030059432A1 (en) * | 2000-02-11 | 2003-03-27 | Dillon Davin C. | Lipophilin complexes for use in cancer diagnosis and therapy |
WO2004003016A2 (fr) * | 2002-07-01 | 2004-01-08 | Commissariat A L'energie Atomique | Peptides marques ayant une affinite pour un phospholipide et utilisations |
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2003
- 2003-06-10 FR FR0306944A patent/FR2856069A1/fr active Pending
-
2004
- 2004-06-09 EP EP04767301A patent/EP1631592A2/fr not_active Withdrawn
- 2004-06-09 CA CA002528186A patent/CA2528186A1/fr not_active Abandoned
- 2004-06-09 US US10/560,025 patent/US20060154854A1/en not_active Abandoned
- 2004-06-09 WO PCT/FR2004/001435 patent/WO2004111090A2/fr not_active Application Discontinuation
Patent Citations (8)
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WO1991009953A1 (fr) * | 1989-12-29 | 1991-07-11 | Zymogenetics, Inc. | Procedes permettant d'obtenir des proteines hybrides de fixation de phospholipides |
WO1992019279A1 (fr) * | 1991-05-09 | 1992-11-12 | Board Of Regents Of The University Of Washington | Agents thrombolytiques cibles sur des phospholipides |
WO1999019470A2 (fr) * | 1997-10-09 | 1999-04-22 | The Regents Of The University Of California | Proteines de fusion de la gfp et de l'annexine |
WO1999060135A1 (fr) * | 1998-05-21 | 1999-11-25 | Cancer Research Ventures Limited | Generation de produits geniques multiples a partir de proteines chimeres de fusion, par clivage a l'aide d'endoproteases ubiquistes |
FR2784106A1 (fr) * | 1998-10-02 | 2000-04-07 | Commissariat Energie Atomique | Structure chimique ayant une affinite pour un phospholipide, et compose de marquage, trousse de diagnostic, et medicament comprenant cette structure |
US20030059432A1 (en) * | 2000-02-11 | 2003-03-27 | Dillon Davin C. | Lipophilin complexes for use in cancer diagnosis and therapy |
WO2003008594A1 (fr) * | 2001-07-20 | 2003-01-30 | Gene-Life Biotechnology (Shanghai) Limited | Construction du plasmide pour l'expression de la proteine de fusion thrombolytique ciblant de thrombus |
WO2004003016A2 (fr) * | 2002-07-01 | 2004-01-08 | Commissariat A L'energie Atomique | Peptides marques ayant une affinite pour un phospholipide et utilisations |
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DATABASE EBI [Online] Annexin A4 1 October 1996 (1996-10-01), XP002269417 Database accession no. P09525 * |
Also Published As
Publication number | Publication date |
---|---|
US20060154854A1 (en) | 2006-07-13 |
WO2004111090A3 (fr) | 2005-09-09 |
FR2856069A1 (fr) | 2004-12-17 |
CA2528186A1 (fr) | 2004-12-23 |
EP1631592A2 (fr) | 2006-03-08 |
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