EP2874662A1

EP2874662A1 - System for delivering lectin-based active ingredients

Info

Publication number: EP2874662A1
Application number: EP13747466.4A
Authority: EP
Inventors: Laurent Paquereau; Gregori GROSS; Caroline LADURANTIE
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite Toulouse III Paul Sabatier
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite Toulouse III Paul Sabatier
Priority date: 2012-07-23
Filing date: 2013-07-19
Publication date: 2015-05-27
Also published as: US20150182631A1; FR2993460A1; CN104507502A; JP2015524424A; WO2014016504A1; KR20150046006A; FR2993460B1; CA2879239A1

Abstract

The invention concerns the use of fungal fruiting body lectins or a variant thereof for delivering an active ingredient to a biological target.

Description

DELIVERY SYSTEM OF ACTIVE PRINCIPLES BASED ON LECTINE

Technical area

The present invention relates generally to a delivery system and more particularly to a lectin-based delivery system.

The invention finds applications, in particular, in the treatment of cancer.

Previous Art

Recent developments in the pharmaceutical field have focused on protein delivery systems. These protein-based platforms are very good candidates for use in the medical field. Indeed, these systems show good biocompatibility and biodegradability coupled with low toxicity.

A large number of proteins have been used as a drug delivery system including ferritin / apoferritin, capsids of different viruses, albumin, gliadin etc. These protein delivery systems have various forms such as microspheres, nanoparticles, hydrogels, films and protein cages.

Protein-based delivery systems and especially protein-cages appear as a promising delivery system that avoids some disadvantages of polymer-based delivery systems due to their uniform size, bioavailability and biodegradability (Maham et al., 2009).

There is therefore a need for new protein delivery systems. In addition to their bioavailability and biodegradability properties, these delivery systems must be able to be manufactured simply, reproducibly and in large quantities.

In addition, these delivery systems must be designed to deliver therapeutic agents specifically to the cells to be treated in order to limit treatment-related side effects.

As a result, the delivery systems must confine the therapeutic agents within their protein cage and release them once the target to be treated is reached.

Finally, the delivery systems must be easily characterized and their safety must be proven.

Summary of the Invention

The family of fungal sporocarp lectins, also called group 2 fungus lectins, is a family of fungal lectins with both sequence and structure homology. The sequence homologies of members of this lectin family are widely described in Birck C et al. (2004), Trigueros et al. (2003) and Khan et al. (201 1).

In addition, the analysis of the three-dimensional structures of some members showed a similarity of structure. For example, Xerocomus chrysenteron lectin (XCL) (Birck C et al. (2004)), Amaricus bisporus lectin (ABL), Carrizo M. et al (2004), and Boletus edulis (BEL) (Michele Bovi et al. ((201 1)) all form a tetramer with a central cavity.

This family of fungal sporocarp lectins comprises, in particular, the lectin of Agaricus bisporus (ABL), the lectin of Arthrobotrys oligospora (AOL), the lectin of Boletus edulis (BEL), the lectin of Gibberella zeae (GZL), the Xerocomus chrysenteron lectin (XCL), Pleurotus cornucopiae lectin (PCL) and Paxillus involutus lectin (PIL) (Birck et al., 2004) (Crenshaw et al., 1995).

The inventors have succeeded in confining an active agent within the cavity of a fungal sporocarp lectin multimer and have shown that the thus-contained active agent can then be delivered to a biological target.

The use of fungal sporocarp lectin as a containment complex makes it possible to eliminate, or at least mitigate, all or some of the disadvantages of delivery systems of the prior art.

Indeed, these lectins have an affinity and specificity for an epithelial tumor marker.

Among the members of this family is a lectin, ABL, present in the fungus Agaricus Bisporus, that is to say the mushroom of Paris. This mushroom is one of the most consumed in the world. We can therefore exclude, a priori, toxicity or allergenicity of this protein against the human species.

The lectins of this family and their variants are easily produced recombinantly and thus excessively high levels of production and purification (g / liter) can be achieved.

These lectins are particularly stable over time (several months at 4 ° C, several weeks at room temperature) and are very resistant to harsh physicochemical conditions (SDS, temperature, ionic strength).

Finally, besides their ability to contain a reversibly active agent and its low toxicity, these lectins form a multimer with a cavity that can contain an active agent without the need for a covalent bond between the multimer and the active agent it contains. . A first aspect of the invention therefore relates to the use of a fungal sporocarp lectin or fungal sporocarp lectin variant for delivery of an active agent which is a therapeutic agent or an agent. from diagnosis to a biological target.

In a second aspect, the invention also relates to a complex comprising an active agent which is a therapeutic agent or a diagnostic agent and a fungal sporocarp lectin or fungal sporocarp lectin variant.

In seeking to improve the performance of these lectins in the confinement of active agents, the inventors have discovered that certain variants of XCL, a lectin particularly suitable for use as a delivery system, have improved containment capacity while retaining their capacity. to release the active agent once the target is reached. The invention relates to this type of variant, specifically an XCL variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

Another aspect of the invention also provides:

a pharmaceutical composition comprising a complex according to the invention and a pharmaceutically acceptable excipient,

a fungal sporocarp lectin, a variant thereof or a complex according to the invention for use in a method of treatment of the human or animal body and, in particular, in a method of treating cancer.

detailed description

Use of fungal sporocarp lectin for the deliyrance of an active agent

The inventors have succeeded in controlling the structuring of a multimer of certain fungal sporocarp lectins so that an active agent can be stably inserted into their cavity and released once the multimeric-active agent complex has reached biological target given.

The invention thus relates to the use of a fungal sporocarp lectin or fungal sporocarp lectin variant for delivery of an active agent which is a therapeutic agent or a diagnostic agent to a biological target. The term "therapeutic agent" refers to an agent that has pharmacological activity or a health benefit when administered in a therapeutically effective amount.

In a preferred embodiment, the therapeutic agent is a chemotherapeutic agent.

The chemotherapeutic agent may be a chemotherapeutic cytotoxic agent such as, for example, a DNA damaging agent, an antimetabolite, an antimitotic, or a Vinca alkaloid (Cancer immunotherapy: immune suppression and tumor growth, George C. et al.) (Chemotherapy at Dorland's Medical Dictionary)).

The DNA damaging agents may be alkylating agents such as cyclophosphamide, chlorambucil, chlormethine, busulfan, treosulfan and thiotepa, topoisomerase inhibitors such as camptothecin, irinotecan and topotecan or amsacrine , etoposide, etoposide phosphate and teniposide or platinum-based compounds such as cisplatin, carboplatin, oxaliplatin.

Examples of anti-tumor antibiotics are anthracyclines such as doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin or mitomycin.

Examples of anti-metabolites are folate antagonists such as methotrexate, purine antagonists such as fludarabine and pyrimidine antagonists such as 5-fluorouracil.

Examples of antimitotics are taxanes such as paclitaxel, and docetaxel.

Examples of Vinca alkaloids are vincristine, vinblastine, vinorelbine and vindesine. The term "diagnostic agent" refers to an agent that when administered in an effective amount can identify whether a subject is suffering or likely to develop a particular condition.

In one embodiment, the diagnostic agent may be a radioactive agent or a fluorescent agent.

The diagnostic agent may for example comprise a radioisotope of iodine (I), such as 1231, 1251, 131 1, etc. , barium (Ba), gadolinium (Gd), technetium (Te) including 99Tc, phosphorus (P) including 31 P, iron (Fe), manganese (Mn), thallium (Tl), chromium (Cr), including 51 Cr, carbon (C) including 14C or fluorescently labeled compounds.

In a preferred embodiment, the diagnostic agent is not or only slightly detectable when confined in the lectin multimer and only becomes significantly detectable once it is released at the biological target. In a preferred embodiment, the active agent according to the invention is a therapeutic agent. The term "variant" refers herein to a protein having an amino acid sequence having at least 80% identity to the amino acid sequence of the protein of which it is the variant and which has an ability to confine an agent. active substantially equal to or greater than that of the protein of which it is the variant.

Typically, the variant of a protein has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of the protein of which it is the variant.

Percent identity refers to comparisons of amino acid sequences and is determined by comparing two optimally aligned sequences on a comparison window, wherein the portion of the amino acid sequence in the comparison window may include additions or deletions (i.e. deviations) from the reference sequence (which does not include addition or deletion) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where an identical amino acid residue is found in both sequences to arrive at the total number of positions in the comparison window and multiplying the result by 100 to reach the percentage of the amino acid residue. sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where an identical amino acid residue is found in both sequences or an amino acid residue is aligned with a gap to reach the number of corresponding positions, dividing the number of corresponding positions by the total number of positions in the comparison window and multiplying the result by 100 to reach the percentage of sequence identity. The person skilled in the art knows that there are several known algorithms for aligning two sequences. An optimal alignment of sequences for comparison can be achieved, for example, by the local alignment algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2: 482, by the algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. ScL USA 85: 2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA), or by visual verification (Current Protocols in Molecular Biology, FM Ausubel et al., Eds., (1995 Supplement) (Ausubel )). Examples of algorithms that are suitable for determining percent identity and sequence similarity are the BLAST and BLAST 2.0 algorithms that are described in Altschul et al., 1990, J. Mol. Biol. 5: 403-41 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. The BLAST analysis software is available at the National Center for Biotechnology Information website.

The ability to confine an active agent can be measured according to the fluorescein method described in the examples.

In a preferred embodiment, the fungal sporocarp lectin of the invention is selected from the group consisting of ABL, AOL, GZL, XCL, PCL, BEL and PIL or a variant thereof.

Preferably, the fungal sporocarp lectin is XCL, ABL, BEL or a variant thereof.

XCL, Xerocomus chrysenteron lectin is a protein belonging to a family of sporocarp fungal lectins that has been isolated from an edible fungus by humans, Xerocomus chrysenteron.

This protein, known for its insecticidal activity, has in particular been described by Wang M et al. (2002), Trigueros V et al. (2003) and Birck C et al. (2004).

The amino acid sequence of this protein is SEQ ID NO: 1.

XCL has the isoform XCL2 of SEQ ID NO: 5.

Birck C et al. (2004) showed that XCL appears, in solution, in the form of a tetrameric structure, generating in its center a cavity whose walls are delimited by each monomer. This same structure is found for the lectin of Amaricus bisporus, ABL (Carrizo M. et al (2004)) and Boletus edulis, BEL (Michele Bovi et al. ((201 1)).

The inventors have discovered that certain XCL variants have optimal characteristics for use as a containment complex.

These three preferred variants are mutants of XCL, of which threonine 12 has been replaced by cysteine and / or alanine 38 has been replaced by cysteine. These variants improve the maintenance of the tetrameric assembly of XCL in its closed form while being able to control its opening.

The inventors have, in particular, shown that the inter-chain disulfide bridge of the variant A38C provides two types of structural constraints. On the one hand, this covalent bond reduces the freedom of movement of the loops closing the access to the cavity of the protein cage which prevents leakage of the confined active agent. On the other hand, the addition of the bonds between the monomers implies that the dissociation of the oxidized variant can take place only if the four interfaces are broken. This variant therefore has its highly stabilized tetrameric structure. The invention therefore relates to an XCL variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

Table 1: Summary of sequences

In a preferred embodiment, the invention thus relates to the use of an XCL variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 for delivery of an active agent which is a therapeutic agent or a diagnostic agent to a biological target.

In a preferred embodiment, the XCL variant has the amino acid sequence SEQ ID NO: 2.

In a preferred embodiment, the biological target is a cancer cell.

Indeed, most fungal sporocarp lectins including XCL (Damian et al. (2005)) and ABL (Milton et al. (2001)) specifically recognize Thomsen antigen. Friedenreich (TF) (Damian L et al (2005)). The TF antigen (Ga ^ 1 -3GalNAca ^ 1) or its direct precursor Tn (Gal-NAc-O-serine) are expressed in most carcinomas and especially in bladder cancers (Langkilde NC et al. 992)), colorectal (Itzkowitz SH, et al (1989)), gastrointestinal, prostate, ovarian (MacLean GD, et al (1992)), breast (Wolf BC, et al (1989) and Carraway KL, et al (2005)), lung (Stein R et al., (1989)), melanoma (Heimburg J, et al (2006)).

In contrast, TF antigen is poorly or not expressed in normal tissues (Springer GF (1984) and Cao Y et al (1996)).

Therefore, the specificity of the lectins of the invention for TF allows the targeting thereof to cancer cells.

The invention also relates to a method for delivering an active agent which is a therapeutic agent or a diagnostic agent to a biological target in which the said active agent is brought into contact with a sporocarp fungal lectin or a variant thereof. this.

Preferably, in one embodiment of the invention, the active agent is confined in the sporocarp fungal lectin multimer or a variant thereof.

Complex The present invention also relates to a complex comprising an active agent which is a therapeutic agent or a diagnostic agent and a fungal sporocarp lectin or fungal sporocarp lectin variant.

The active agent is a therapeutic agent or a diagnostic agent as defined above.

Typically, the fungal sporocarp lectin or the lectin variant of the complex according to the invention is in the form of a multimer.

Typically, the lectin multimer has an internal cavity in which the active agent is located.

Preferably, there is no covalent bond between the lectin multimer and the active agent.

In a preferred embodiment, the multimer is a homomultimer.

In another embodiment, the multimer may be a heteromultimer. In a preferred variant of this embodiment, the heterodimer is composed of fungal sporocarp lectin and a variant of this lectin. In the preferred embodiment, the multimer of the complex according to the invention is a tetramer, more preferably a homotetramer.

Preferably, the complex of the invention comprises a fungal sporocarp lectin selected from the group consisting of ABL, AOL, GZL, XCL, PCL, BEL and PIL or a variant thereof.

In a preferred embodiment, the fungal sporocarp lectin of the complex of the invention is XCL, ABL or a variant thereof.

In a preferred embodiment, the fungal sporocarp lectin of the complex according to the invention is XCL or an XCL variant.

In another preferred embodiment, the fungal sporocarp lectin of the complex of the invention is an XCL variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ. ID NO: 4.

The invention also relates to a process for producing a complex according to the invention characterized in that it comprises a step of contacting fungal sporocarp lectin or sporocarp fungal lectin variant with an active agent which is a therapeutic or diagnostic agent.

The method of making the complex may further comprise a step of removing the active agent that has not been unconfined.

The method may comprise a step of reducing a fungal sporocarp lectin or a variant thereof prior to the contacting step with the active agent and an oxidation step after the step of contact with the active agent.

This step allows the creation of disulfide bridges to stabilize the lectin multimer, especially for modified variants with one or more cysteines, to be able to generate such bridges.

Pharmaceutical composition

The invention also relates to a pharmaceutical composition comprising a complex according to the invention and a pharmaceutically acceptable excipient.

The excipients are selected according to the pharmaceutical form and the desired mode of administration from the excipients usually employed.

The composition according to the invention may for example be administered by injection, by spraying or orally.

The pharmaceutical composition according to the invention may for example be in liquid, solid, gel or freeze-dried form. In these compositions, the active agent is advantageously present at physiologically effective doses.

Preferably, these pharmaceutical compositions are intended for non-systemic administration, for example enterally.

Treatment method

The present invention also relates to a complex according to the invention for use in a method of treating the human or animal body.

The terms "treatment or treatment" both refer to curative treatment and preventative or prophylactic measures to prevent or slow down the disease or the targeted disease state.

Those in need of treatment include those already affected by the disease as well as those who may have the disease or who need to be prevented from the disease. Thus, the subject to be treated may have been diagnosed as having the disease or may be predisposed or susceptible to having the disease.

The invention also relates to a method of treating a subject comprising administering to said subject a therapeutically effective amount of a complex of the invention.

The invention also relates to the use of a complex according to the invention for the manufacture of a medicament.

It is known that some lectins of this family penetrate effectively into cancerous epithelial cells. Indeed, after binding to the cell surface, the lectin is internalized via the clathrin-dependent pathway and is transported into the late endosomes or lysosomes of these cells (Francis F. et al, 2003). The acid and protease conditions of these compartments result in the release of the active agent contained in the lectin multimer.

The invention relates to a complex according to the invention for use in a method of treating cancer.

In one embodiment, the cancer is selected from the group consisting of bladder cancer, colorectal cancer, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, melanoma and lung cancer. Preferably, the cancer is selected from the group consisting of colorectal cancer, bladder cancer, gastrointestinal cancer, and ovarian cancer.

The invention also relates to a method of treating cancer in a subject comprising administering to said subject a therapeutically effective amount of a complex of the invention.

The invention also relates to the use of a complex according to the invention for the manufacture of a medicament for treating cancer.

The invention also relates to a method of administering an active agent to a subject comprising the step of administering to said subject a complex according to the invention.

Administration can be done orally or by injection.

Brief Description of Drawings

Other features and advantages of the invention will become apparent on reading the description which follows. This is purely illustrative and should be read in conjunction with the attached drawings in which:

FIG. 1 represents the minimum and maximum distances between two e-amine groups exposed on the surface of XCL and belonging to different protein subunits;

FIG. 2 represents the emission spectra of FITC-XCL and RITC-XCL. A- Continuous curves: Emission spectrum of FITC-XCL (max 520nm) and RITC-XCL

(Xmax 580nm); Discontinuous curves: deconvolution of the em ission spectrum obtained in (B). B- Continuous curve: numerical addition of the spectra obtained in (A) for FITC-XCL and RITC-XCL; Discontinuous curve: emission spectrum obtained by mixing FITC-XCL and R ITC-XCL;

FIG. 3 represents the emission spectra of FITC-A38C and RITC-A38C;

A. Continuous Curves: Emission Spectrum of FITC-A38C (Xmax 520nm) and RITC-A38C (Xmax 580nm); Discontinuous curves: deconvolution of the emission spectrum obtained in (B). B- Continuous curve: numerical addition of the spectra obtained in (A) for FITC-A38C and RITC-A38C; Discontinuous curve: emission spectrum obtained by mixing FITC-A38C and RITC-A38C;

FIG. 4 shows the evolution of the intensity ratio 580 nm / 520 nm as a function of the XCL protein concentration. FIG. 5 represents the variation of the elution volumes of the exclusion chromatography as a function of the XCL concentrations;

FIG. 6 represents the kinetics of the exchange by measurement of FRET at 580 nm for XCL;

FIG. 7 represents the fluorescein confinement in XCL and the variant

A38C reduced and not reduced;

Figure 8 shows the relative fluorescence intensities of XCL Trp at 346 and 330 nm as a function of temperature;

Figure 9 shows the DOSY experiment.

EXAMPLES

Material and methods :

Protein preparation

All mutants were made using the Quickchange mutagenesis kit (Stratagene). The A38C and T12C mutants of XCL were constructed from the cDNA of the native XCL protein. Proteins were produced and purified as described in the literature (Trigueros et al. (2003)). A further purification step was carried out on size exclusion chromatography (Sephacryl S300) equilibrated with 50mM phosphate buffer, 100mM NaCl, pH 7.2 at a flow rate of 3 ml.mn ^-1 . concentrated at 60 μΜ on a Vivaspin 15R 30,000 MWCO (Sartorius stedim) column The mutant A38C was oxidized by incubation for 8 h in 50mM phosphate buffer, 100mM NaCl, pH 8.5 and 10mM oxidized glutathione (GSSG) Excess glutathione was removed on a column of PD-10 sephadex G-25M (GE Healthcare), equilibrated with 50mM phosphate buffer, 100mM NaCl, pH7.2.

Labeling of XCL and A38C proteins at FITC and RITC

XCL or A38C (30 μΜ) are incubated at 25 Ό for 1 h with stirring in a buffered medium consisting of phosphate buffer (50 mM, pH 9) and either FITC (1 mM) or RITC (1 m M). The reaction is stopped by addition of Tris-HCl (10 mM, pH 9). The labeled proteins are purified on PD-10 sephadex G-25M columns (GE Healthcare) previously equilibrated with 50 mM phosphate buffer (pH 8.5). The stoichiometry of the labeling is determined spectrophotometrically at pH 8.5. The concentration of fluorescent moieties bound to proteins is calculated using the following extinction coefficients: ε494 nm = 77000 cm ^{^"1} ^{M" 1} for FITC and ε560 nm = 85000 cm ^"1 ^{M" 1} for the RITC. The concentrations of XCL and of A38C after labeling are calculated with ε278 nm = 120000 cm ^-1 M ^-1 , and taking into account the absorption of the fluorescent probes at this wavelength.

FRET's Experience

FRET at equilibrium

Fluorescence energy transfer was used to highlight the exchange of XCL monomers. The exchange reaction is initiated by mixing the same volume of XCL-RITC (1 μl or buffer only as reference) and XCL-FITC (1 μl) at 25 ° C. in 50 mM phosphate buffer, 100 mM NaCl, pH 8.5. After one hour of incubation, the emission spectrum of the sample excited at 490 nm is recorded by spectrofluorimetry (Photon Technology International QM-4). The same experiments were performed with A38C-RITC and A38C-FITC.

Kinetics of FRET

The exchange rate of the subunits was measured by the same technique. The reaction is initiated by mixing the same volume of XCL-RITC (1 μl) and XCL-FITC (1 μl) at 25 ° C. in 50 mM phosphate buffer, 100 mM NaCl, pH 8.5. The excitation is carried out at 468 nm and the fluorescence emission at 580 nm is recorded every 0.5 seconds. The fluorescence intensity is normalized using the equation:

. . made)

ψ f - r¾

where F (t) represents the experimental fluorescence at the considered time, F ₀ the value of the initial fluorescence, and the value of the final fluorescence when the equilibrium state is reached. Kinetic data were modeled with GOSA software (Czaplicki et al., 2006). The best fit was obtained for a double exponential equation:

F = 1 - [axs ^" ^ + (i - α) xe ^{~ k} ^ 1 where:

a represents the proportion of slow dissociation molecules, the constant kinetics of slow dissociation and the constant kinetics of rapid dissociation.

Size exclusion chromatography

Size exclusion chromatography experiments were performed on a GE-Healthcare Superose 12 PC 3.2 / 30 column. The columns are pre-balanced in the buffer tested, namely 50 mM phosphate buffer, 100 mM NaCl, pH 7.2 or pH 4.4 in the absence or in the presence of 0.003% of SDS. The flow rate is 0.5m L / min. BSA and RNase A are used as a size marker. containment

Confinement experiments are performed by incubating wild-type protein (XCL) or mutants (A38C) with the test molecule. To carry out the control experiments with the mutant A38C, an oxidation step is carried out in the presence of GSSG in a 50 mM phosphate buffer, 100 mM NaCl, pH 7.2, OVN. The GSSG is then removed on a PD-Sephadex G-25M column (GE Healthcare) pre-equilibrated in 50 mM phosphate buffer, 100 mM NaCl, pH 8.5. The eluate is concentrated to 1 ml on Vivaspin 15R 30,000 MWCO (Sartorius stedim). A batch of A38C is reduced to TCEP (argon) with added NaOH for a final pH of 8.5. The proof of concept of confinement was performed with fluorescein at a concentration of 10mM. The incubation is carried out for 1 h at 25 ° C. in 50 mM phosphate buffer, 100 mM NaCl. For mutant A38C reduced a new oxidation step is performed at GSSG, OVN. Free fluorescein is removed on a Sephadex G-25M PD-10 column (GE Healthcare). The sample is then concentrated on Vivaspin 15R 30,000 MWCO (Sartorius stedim). The measurement of the confinement is carried out by spectrophotometry. The amount of fluorescein is measured using an absorption coefficient of ε51 1 nm = 75000 cm ^-1 M ^-1 . The concentrations of XCL and A38C after confinement are calculated with ε278 nm = 120000 cm ^-1 The percentage of confinement is calculated by reporting fluorescein / protein concentrations.

Results:

Previous studies have shown that XCL is organized as a tetramer with an inner cavity. The inventors have demonstrated that it is possible to confine a molecule in this cavity without a covalent bond between the molecule and XCL. In addition, the inventors have designed XCL variants capable of remaining in tetrameric form for a long time. The inventors have also shown that the confined molecule can be released under conditions similar to those encountered in target cells.

Design and production of tetramers stabilized by disulfide bridges.

In order to increase the stability of the tetrameric structure of XCL, the inventors have designed XCL variants whose monomers are covalently bound. The inventors have tested a strategy consisting of the formation of disulfide bridges between the monomers by substituting amino acid residues of wild-type XCL with cysteine. To minimize the number of substitution the inventors had to determine the amino acids that were close to their counterparts in the opposite monomer. As revealed by the 3D structure of XCL, two amino acids have such a property. The distance between the beta carbons of threonine 12 and its counterpart is 3.9 angstroms and that between alanine 38 and its counterpart is 3.4 angstroms. These distances are typical of the range of beta carbons of cysteines involved in disulfide bridges (Galat et al., 2008). In addition, the side chains of these residues are on the surface of the protein and completely accessible to an oxidative agent. An analysis of the energy minimization with the WINCOTT software showed that the orientation of the side chain of threonine 12 and alanine 38 and their structural environment was compatible with the formation of disulfide bridges.

By producing these molecules thus modified, the inventors wish to obtain a tetrameric assembly that can not be dissociated. The particularity of variant A38C is that the presence of two additional disulfide bridges results in stabilization of the entire tetramer. If we consider that the structure of XCL is a square in which each monomer is a wedge, the disulphide bridges A38C will link them diagonally. Dissociation of oxidized A38C tetramers will only be possible if all interfaces are broken. A strong stabilization of the tetramer is envisaged making its dissociation very unlikely.

Clone A38C was obtained by site-directed mutagenesis using the XCL plasmid as a template and the protein produced and purified using the same protocol as that used to produce XCL (Trigueros et al (2003)). A yield of 25 mg of purified protein per liter of culture was obtained. The last part of the protocol consisted of a 12-hour oxidation step in the presence of 10 mM of oxidized glutathione. This step is required to produce disulfide bridges between the monomers. The formation of these covalent bonds was verified by SDS-PAGE. Two SDS-PAGEs have been prepared. Beta- mercaptoethanol was added to the samples in the first gel so no beta-mercaptoethanol was added to the second gel. When XCL is heated to 95 ° C 5 min before deposition, a single band with a molecular weight slightly above 20 kDa is observed on both gels. This band corresponds to the species in the form of monomer. Conversely, when XCL was not heated prior to deposition, a single molecular weight band of approximately 80kDa was observed which implies that XCL remained in tetrameric form despite conditions as hard as 2% SDS. Under reducing conditions, A38C showed exactly the same behavior as XCL. Under non-reducing conditions, when the samples were heated, 3 bands were observed. The most intense band migrates as a 40kDa protein, that is, as a dimeric species. These species correspond to 2 monomers covalently linked by the creation of disulfide bridges. It represents 90% of the species in the case of A38C. Another band which represents 5% of the species and has a molecular weight of 21 kDa is also observed. This band corresponds to monomers from unoxidized species. The third band, which also represents 5% of the species, has an apparent molecular mass of 60 kDa corresponding to the tetrameric species. All these results show that the strategy of rational modification of XCL by genetic engineering made it possible to obtain an oxidation rate of approximately 90%.

A T12C variant was produced in the same manner as A38C by site-directed mutagenesis using the XCL plasmid as a template. Using the same protocol, 10mg of protein per liter of culture was produced. The oxidation rate of the T12C variant was searched using the same method by SDS-PAGE as for A38C. These rates were compared with those obtained with the variant A38C. Several oxidative conditions were tested with different concentrations of reduced glutathione. In fact, the addition of reduced glutathione prevents the formation of incorrect bonds by allowing the reduction of disulfide bridges which could be insufficiently stabilized by other interactions. The oxidation rate of each sample was tested by SDS-PAGE under non-reducing conditions after heating the samples for 5 min at 95 ° C. As for A38C, 3 bands were observed for T12C. The oxidation rate obtained for T12C is therefore comparable to that obtained for A38C. However, when the reduced glutathione is added to the oxidation buffer at a concentration of 10 mM, a sharp decrease in oxidation rate was observed with respect to A38C. This result shows that the additional oxidized T12C disulfide bridge is less stable than the A38C disulfide bridge.

Reversible opening of the tetramer

As indicated previously, XCL is organized in solution in the form of a tetramer. The inventors have determined whether there exists a spontaneous exchange between the tetrameric forms and any dimeric or monomeric minority forms.

Highlighting the spontaneous opening and closing of XCL.

The oligomerization equilibrium of the XCL protein was characterized by measuring the appearance of Forster-type resonance energy transfer (FRET) between two XCL populations labeled separately either with FITC (Fluorescein IsoThioCyanate) or RITC (Rhodamine IsoThioCyanate).

When analyzing the structure of XCL (FIG. 1), the minimum and maximum distances between two e-amine groups exposed to the surface and belonging to different protein subunits are respectively 21 and 67 angstroms and therefore both in the range of distance compatible with the appearance of FRET.

Two samples of XCL were marked separately with either FITC or RITC as described in the material and methods. The stoichiometry of labeling is 4 mol of FTIC per mol of XCL and 0.9 mol of RITC per mol of XCL. The same stoichiometry was obtained for A38C.

The emission spectra of FITC-XCL and RITC-XCL are shown in Figure 2A (continuous curves). The numerical addition of these spectra gives the continuous curve presented in Figure 2B. In this same figure, the spectrum obtained by mixing the labeled protein, either by the donor or by the acceptor (dashed curve) is also presented. This spectrum was deconvolved, giving the spectra shown in dashed lines in Figure 2A.

A decrease in the fluorescence intensity corresponding to the fluorescein emission and at the same time an increase in the fluorescence emission intensity of the rhodamine are observed, which demonstrates the existence of a transfer of energy. between the two fluorophores.

Insofar as the transfer efficiency depends on the donor / acceptor distance at power six and the dimensions of the XCL molecule are of the order of magnitude of the Forster distance, the appearance of this transfer of energy is possible only if the monomers FITC-XCL and RITC-XCL have redistributed and belong at the end of the experiment to the same tetramer. Therefore, access to the cavity is possible spontaneously.

For comparison, the same experiment was performed with the variant A38C. This variant is covalently linked by two additional disulfide bridges. The emission spectra of FITC-XCL and RITC-XCL are shown in Figure 3A (continuous curves). The numerical addition of these spectra gives the continuous curve presented in Figure 3B. A perfectly superimposable spectrum was obtained after mixing the donor and acceptor labeled proteins and incubating this mixture for 1 h at room temperature (dashed lines of Figure 3B). When deconvolved, the spectrum gives a spectrum represented by the dashed lines (Figure 3A) and shows that no FRET is observed. This experiment shows that the oxidized monomers of A38C do not redistribute and remain tetrameric.

Determination of the equilibrium constant of the tetramer dissociation reaction

FRET measurements:

In the previous paragraph, it has been shown that the dissociation of XCL occurs and is reversible. Thus, this equilibrium can be shifted to dissociation and the efficiency of energy transfer should decrease. The inventors have benefited from this phenomenon for the calculation of a dissociation constant (Kd) of the tetramers of XCL.

The mixture of FITC-XCL and RITC-XCL was diluted to different concentrations and incubated for 1 hour at room temperature. The fluorescence spectrum was recorded with 468 nm light excitation. In order to significantly determine the decrease in energy transfer and normalize the fluorescence intensity, a I580 / I520 ratio was calculated for each concentration and read as a function of the XCL concentration (Figure 4). A transition in the intensity of energy transfer occurs at a concentration close to one micromolar. Obviously, the titration curve is not complete until saturation is reached for the points of highest concentration. Due to the solubility limit of XCL and the labeling protocol, protein concentrations can not exceed 10μΜ in this experiment.

Measures in size exclusion chromatoqraphy:

In order to confirm the data obtained in FRET, the displacement of the equilibrium was evaluated by exclusion gel chromatography. Serum albumin (BSA) and ribonuclease A were used to calibrate the column. Elution volumes of 12.6 ml and 15.5 ml were respectively obtained. At a high concentration (here at 30μΜ), XCL has an elution volume of 13.2ml close to the elution volume of BSA as expected for the tetrameric species. At first sight, the existence of an equilibrium between tetrameric and dimeric (or monomeric) species suggested an elution in two peaks by exclusion chromatography. Nevertheless, it has been shown by numerical simulation that, depending on the rate of association, the dissociation rate, the flow rate and the separation characteristics of the column, two populations of exchanging molecules could lead to a single peak by chromatography. size exclusion (Yu et al., (2006)). The inventors have made several injections using variable concentrations of XCL and observed that indeed a only peak was obtained. However, this peak is shifted to longer elution volumes as the concentration is decreased, indicating a smaller molecular weight. When the elution volumes are recorded in relation to the XCL concentrations (FIG 5), a transition at a concentration close to the micromolar is observed which is consistent with the results previously obtained in FRET.

Microcalorimetry experiments

To accurately measure the tetramerization equilibrium constant and also to determine the thermodynamics of the tetramerization of XCL, the inventors used isothermal titration microcalorimetry (Burrows et al., (1994) Lovatt et al., (1996) Barranco- Medina et al (2009). In this method, a concentrated solution of XCL was diluted in a buffer, and the heat of dissociation of the dimers (or monomers) was measured. The amount of heat released for each injection is governed by the enthalpy exchange of the tetramer to the dimer and the tetrameric-dimer equilibrium constant. The modeling of these results makes it possible to extract the dissociation equilibrium constant (Kd = 4.5μΜ) and an enthalpy variation of 16.6 kilocalories per mole of tetramer.

This enthalpy variation is within the range of values typically observed for an oligomeric protein of this size. On the other hand, the equilibrium dissociation constant is low in comparison with those obtained for other polymeric proteins, which demonstrates the remarkable stability of this protein assembly.

The convergence of these results demonstrates that XCL constantly undergoes exchange between the dimeric form and the tetrameric form, even at high protein concentration.

To verify that the opening and closing of the tetramer occur at speeds compatible with confinement experiments, we have determined the kinetic parameters of this exchange.

Determination of the kinetic constant of dissociation of the tetramer

The dissociation kinetics of the tetramer was studied by mixing XCL-FITC and XCL-RITC and measuring the fluorescence emission intensity at 580 nm as a function of time (Figure 6). It is observed that this fluorescence intensity increases until an equilibrium is reached after more than 300 seconds of reaction, which indicates that the exchange between the subunits is slow. The increase of the fluorescence intensity and therefore the transfer efficiency between fluorescein and rhodamine are the result of two successive phenomena: firstly the dissociation of the tetramers into dimers and secondly their reassociation. Considering the shape of the curve (Figure 6), no latency is observed at the beginning of the kinetics, which implies that the reassociation rate is much greater than the dissociation rate. It can therefore be considered that the rate of reassociation is negligible in this case. The kinetic data obtained were modeled using the GOSA software. The model best able to describe the observed phenomenon is an exponential double growth corresponding to two dissociation reactions with different velocities:

F = 1 - [axis ^"k1 (1 -a) ^χ e ^{" k2 t} ]

with:

a: proportion of molecules involved in the slow reaction,

k1: kinetic constant of the slowest dissociation reaction

k2: kinetic constant of the fastest dissociation reaction

The values of the constants obtained after optimization are:

a = 0.39; k1 = 0.72 min-1; k2 = 2.52 min-1

This double exponential increase suggests that XCL can follow two pathways of dissociation. The tetramer ABCD formed by XCL can dissociate in two different ways: to AB and CD dimers or to AD and BC dimers.

Confinement of an agent in the cavity

The inventors used fluorescein to validate the confinement capacity of the XCL tetramer.

Fluorescein (279 to ³ ) has the advantage of being small and very soluble in aqueous solvent (it is therefore possible to place at high concentration during confinement). In addition, it absorbs visible light at 51 1 nm, which allows it to be detected. The inventors have preferred to follow the absorbance of this molecule rather than its fluorescence. Indeed, the Fluorescence of a molecule is dependent on its chemical environment, therefore, once confined, fluorescein could see its fluorescence yield significantly altered.

Containment was performed for XCL and A38C variant.

In the case of XCL, the inventors incubated the protein (15 μl) with fluorescein (10 mM) and then separated the protein from the unconfined fluorescein molecules by two successive gel filtration columns. Previously, the inventors verified that if these two columns are carried out on the fluorescein solution at 10 m, no residual free fluorescein is detected. The fluorescein detected during the confinement is necessarily bound to the protein or contained by the latter.

The A38C protein was also used to carry out the confinement, the protocol being identical except that the protein which is initially oxidized is reduced before being placed in the presence of the fluorescein solution, then in a second oxidized phase. overnight in the presence of an oxidizing agent, oxidized glutathione. This protein is noted A38R in Figure 7.

The confinement experiment was also carried out with protein A38C which was not reduced before being placed in the presence of the fluorescein solution, then which in a second time was oxidized overnight in the presence of an oxidizing agent, oxidized glutathione. This protein is noted A38NR in FIG.

At the end of these confinement and free fluorescein removal steps, the respective concentrations of fluorescein and protein are determined by UV absorbance at 51 nm and 280 nm. For XCL as for A38C, 1 1 independent experiments were carried out. Containment rates of 9% and 14% containment are obtained for XCL and A38R respectively (Figure 7).

The samples were then incubated for 4 days at room temperature, the free fluorescein was again removed and then the confined fluorescein was assayed. The confinement rate then remained unchanged for XCL as for A38C.

Fixation of fluorescein on XCL can be related to two types of interactions. Fluorescein can bind nonspecifically on the surface of the protein or in the cavity. Maintaining containment for 4 days indicates that fluorescein is not bound in a labile manner, otherwise it would dissociate after this time.

These confinement rates demonstrate, on the one hand, that an agent can be contained within the cavity and, on the other hand, that this cavity has a non-covalent binding affinity with respect to this test molecule. Indeed, for passive diffusion confinement, we expect a theoretical confinement efficiency of the order of 0.3%. The use of A38C in oxidized form after confinement has made it possible to multiply the rate of confinement by two. The tetramer engineering strategy proposed by the inventors therefore makes it possible to increase the confinement rate significantly.

Dissociation of the complex at acidic pH corresponding to that of the lysosomes

In order to use XCL to transport and deliver an agent into cells, a thorough knowledge of the dissociation conditions is required.

The conditions of use of a confinement complex in therapy in a homeothermal organism imply that these vectors can maintain their structure in the temperature conditions of the target organism. XCL originating from a poïkilothermal (or heterothermal) organism that is to say non thermally regulated endogenously, the behavior of the tetramer as a function of temperature and in particular its behavior below 45Ό was analyzed.

Thermal dissociation

Fluorescence of trvptophane

Thermal dissociation of XCL was monitored by measuring the intrinsic fluorescence of tryptophans. Each monomer of XCL contains 3 tryptophan residues: one in a hydrophobic cavity (Trp 77) and two located on the same interface between two monomers (Trp 27 and Trp 35). The inventors have used these latter two Trp as probes of the quaternary structure of XCL, taking advantage of their position in the tetramer structure. To increase the sensitivity of the measurement, the ratio of fluorescence intensities 346nm / 330nm was measured as a function of temperature (Figure 8). No modification of this ratio is observed before 50 ° C which indicates that the Trp environment has not changed to this temperature and that the lectin has remained in tetrameric form. Visual analysis of the samples after heating revealed that XCL precipitated in tetrameric form above 45 ° C before unmasking tryptophans.

RMN - DOSY

Thermal dissociation was also followed by DOSY (Diffusion Ordered Spectrometry) experiments. The ratio of hydrodynamic radii of XCL and dioxane were measured as a function of temperature (Figure 9).

If the tetramer dissociates by increasing the temperature, we expect a decrease in this ratio and therefore the hydrodynamic radius of XCL. Conversely, the results reveal a linear increase of this ratio when the temperature increases. This result, counterintuitive, reflects an increase in the XCL hydrodynamic radius probably related to the increase in the movement of unstructured loops. Be that as it may, no transition of the hydrodynamic radius is observed. After 50 ° C., the protein sample precipitates as already described in the fluorescence experiments of tryptophan.

These two experiments demonstrate that this lectin is stable below 45 ° C and therefore it meets thermostability criteria largely sufficient to be used in vivo.

Chemical dissociation

The inventors have studied the behavior of XCL under denaturing chemical conditions close to that encountered in lysosomes. The analysis was carried out by exclusion chromatography at acidic pH (pH 4.4). It appears that the peak elution of XCL in these denaturing conditions is shifting to larger elution volumes reflecting the appearance of a smaller size. When 0.003% SDS is added, the elution peak moves to the elution volume of RNAse A which has a molar mass close to the XCL monomer. The decrease in pH thus leads to the dissociation of the tetramer to dimer, and with a very small amount of detergent these dimers are dissociated into monomers.

The behavior of the oxidized A38C mutant was also analyzed. At neutral pH, A38Cox has an elution volume comparable to that of XCL. When the pH is lowered to 4.4, no change in the elution volume is observed. The addition of the disulfide bridge therefore increases the stability of the protein assembly. The addition of 0.003% of SDS leads to the dissociation of the tetramer into dimers, consisting of monomers connected to each other by the disulfide bridge.

Lysosomes are subcellular organelles of degradation. Their content is acidic, reducing and contains acid hydrolases, protein-degrading proteases that are addressed to them. The results obtained show that under these reducing and acidic conditions, XCL or its mutant A38Cox are dissociated into dimers, allowing the release within the lysosomes of the active agents confined in the cavity.

The present invention has been described and illustrated in the present detailed description and in the Figures. The present invention is not limited to the embodiments presented. Other variants and embodiments may be deduced and implemented by the person skilled in the art upon reading the present description and the appended figures. REFERENCES

During this application, different references describe the state of the art of the invention. The descriptions of these references are hereby incorporated by reference in the present description.

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Claims

1. Use of a fungal sporocarp lectin or fungal sporocarp lectin variant for delivery of an active agent which is a therapeutic agent or a diagnostic agent to a biological target.

2. Complex comprising:

an active agent which is a therapeutic agent or a diagnostic agent and

a fungal sporocarp lectin or a variant of a fungal sporocarp lectin.

3. Complex according to claim 2 characterized in that the active agent is a therapeutic agent.

4. Complex according to claim 2 characterized in that the active agent is a diagnostic agent.

5. Complex according to any one of claims 2 to 4 characterized in that said lectin is selected from the group consisting of ABL, AOL, GZL, XCL, PCL, BEL and

PIL or said variant is a variant of a fungal sporocarp lectin selected from the group consisting of ABL, AOL, GZL, XCL, PCL, BEL and PIL.

6. A method of manufacturing a complex according to any one of claims 2 to 5 characterized in that it comprises a step of contacting a sporocarp fungal lectin or a variant of a sporocarp lectin fungal with an active agent which is a therapeutic agent or a diagnostic agent.

An XCL variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

A pharmaceutical composition comprising a complex according to any one of claims 2 to 5 and a pharmaceutically acceptable excipient.

9. Complex according to any one of claims 2 to 5 for use in a method of treating the human or animal body.

10. Complex according to any one of claims 2 to 5 for use in a method of treating cancer.