Polypeptides isolated from thermobrachium celere and their use for the treatment of cancer

Abstract

Inventors describe herein the functional and structural characteristics of the MGL from the anaerobic alkaliphilic thermophile Thermobrachium celere (TcMGL), which was compared to that from Pseudomonas putida (PpMGL). Catalysis was limited to L-Met, L-homocysteine, and L-cysteine, with the highest catalytic efficiency (keat / KM) towards L-Met. The half-life of active TcMGL in human plasma at 37°C was 9.2 hour. Half-life increased from 11.3 hour to 66.7 hour with increasing PLP concentration from 2 to 200 µM, in much greater magnitude than in PpMGL case. TcMGL crystal structure resolution revealed that the enzyme is a tetramer of two catalytic dimers containing a mobile region with open and closed conformations resulting in differences for access to the active site. TcMGL possesses favourable characteristics which makes it a suitable candidate for treatment of cancer. The present invention relates to a polypeptide comprising an amino acid sequence as set forth by SEQ ID NO: 1 or a function-conservative variant thereof.

Description

[0001] POLYPEPTIDES ISOLATED FROM THERMOBRACHIUM CELERE AND THEIR USE FOR THE TREATMENT OF CANCER FIELD OF THE INVENTION:

[0002] The invention is in the field of oncology. More particularly, the invention relates to a polypeptide isolated from Thermobrachium celere and its use for the treatment of cancer.

[0003] BACKGROUND OF THE INVENTION:

[0004] L-Methionine (L-Met) deprivation induces growth arrest and death of cancer cells, whereas normal cells are more resistant to restriction of this essential amino acid1’2,3. Depletion of L-Met in extracellular fluids for cancer treatment can be achieved using methionine gammalyases (MGL, methioninases). MGLs belong to fold Type-I (a Family) pyridoxal 5 ’-phosphate (PLP)-dependent enzymes. They catalyze the a -elimination of L-Met, resulting in the production of methanethiol, a-ketobutyrate, and ammonia4. Various MGLs have been obtained by extraction or recombination of genes from bacteria, protozoa, and plants5. L-Met elimination obtained with the MGL from Pseudomonas putida (PpMGL), which possesses high catalytic efficacy towards L-Met7, exerts cytotoxicity in most cancer cell lines in vitro6. It enhances the effect of 5 -fluorouracil by increasing inhibition of thymidylate synthase7and decreases genomic DNA methylation levels8. However, attempts at reducing the prohibitive anaphylaxis caused by the native PpMGL given by parenteral route in monkey through polyethylene glycol (PEG) conjugation resulted in substances with increased instability, preventing its development in humans.

[0005] To avoid potential immunogenicity of MGLs derived from parasitic and potentially pathogenic microorganisms, we previously characterized the recombinant MGL from the cheese-ripening Brevibacterium aurantiacum (BaMGL, named BL-929 in previous publications), which is abundantly present in food. BaMGL-induced depletion of Met in vitro produced cytotoxicity on a vast panel of human cancer cell lines, and spared normal cells11. Exposure of the human colorectal carcinoma HT29 cells to the BaMGL led to profound changes in methylation-sensitive epigenetic abnormalities associated with gene silencing involved in oncogenesis. These changes included reduced methyl-CpG levels of the hypermethylated tumor suppressor cyclin-dependent kinase inhibitor 2A (CDKN2A) gene promoter, the mRNA expression of which was greatly increased, together with a decrease in the global level of histone H3 dimethyl lysine-9n. However, rapid decrease of enzyme activity occurred in the presence of human serum albumin or plasma. This was partially avoided by supplying PLP inaqueous solutions, but not in plasma. The estimated half-life of BaMGL in human plasma supplemented with any concentration of PLP up to 200 pM was under one hour. In the case of PpMGL, it was possible to extend activity in plasma through PLP addition, but this effect was only partial and limited in time. Moreover, concentration of exogenous cofactor required was much greater than that found in cells and plasma under natural conditions. From these findings, we concluded that intrinsic capacity for cofactor retention of any MGL is essential to attain the intense and durable L-Met depletion required for treatment of cancer. In order to obtain a methioninase tightly retaining its cofactor, we explored the putative carbon-sulfur lyase sequences present in genomes of extremophile bacteria or archaea living in harsh environmental conditions of pH and temperature. These microorganisms have adapted the stability of their enzymes to compensate for the extreme physicochemical conditions in which they live13,14. Their PLP-dependent enzymes carry features suggestive for strong cofactor retention capacity. Of these enzymes, the aromatic aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus requires the presence of PLP for optimum activity at high temperature15. Similarly, PLP bound to the SHMT from the hyperthermophile archaeon Methanocaldococcus jannaschii is necessary for maintaining optimum activity at high temperature, and the resistance to denaturing agents found in this enzyme16.

[0006] SUMMARY OF THE INVENTION:

[0007] The invention relates to a polypeptide comprising an amino acid sequence as set forth by SEQ ID NO: 1 or a function-conservative variant thereof.

[0008] In particular, the invention is defined by claims.

[0009] DETAILED DESCRIPTION OF THE INVENTION:

[0010] Inventors describe herein the functional and structural characteristics of the MGL from the anaerobic alkaliphilic thermophile Thermobrachium celere (TcMGL)17, whose features are compared to those of the enzyme from the mesophile P. putida, whose stability in plasma is greater than that of the BaMGL. TcMGL and PpMGL share 53.9% sequence identity. T. celere is a proteolytic clostridiaceae found in geothermally and anthropogenically heated environments, the optimum growth rate of which occurs at 66°C, and pH 8.216. Present findings demonstrate that TcMGL possesses favorable substrate specificity characteristics and is highly stable in human plasma, which makes it a suitable candidate for cancer treatment development.Definitions

[0011] As used herein, the term “methionine” (also called as Met or M; encoded by the codon AUG) refers to an a-amino acid that is used in the biosynthesis of proteins. It contains a carboxyl group (which is in the deprotonated COO form under biological pH conditions), an amino group (which is in the protonated -NH3 form under biological pH conditions) located in a-position with respect to the carboxyl group, and an S-methyl thioether side chain, classifying it as a nonpolar, aliphatic amino acid.

[0012] As used herein, the term “L-methionine” refers to one of the proteinogenic amino acids. In addition to its role in protein biosynthesis, L-methionine is required for a number of important cellular functions, including the initiation of protein synthesis, the methylation of DNA, rRNA, histones and xenobiotics, and the biosynthesis of cysteine, and cystathionine, phospholipids and polyamines. In all metazoa L-methionine is an essential amino acid (an indispensable amino acid that cannot be synthesized de novo) and must be supplied in the diet.

[0013] L-Methionine (L-Met) deprivation induces growth arrest and death of cancer cells. L-Met restriction to treat cancer requires sustained elimination of this essential amino acid. Pyridoxal 5 ’-phosphate (PLP)-dependent L-methionine y-lyases (MGLs) can efficiently achieve this.

[0014] As used herein, the term “methionine gamma-lyase” or “MGL” belongs to fold Type-I (a Family) pyridoxal 5 ’-phosphate (PLP)-dependent enzymes. They catalyze the a,y-elimination of L-Met, resulting in the production of methanethiol, a-ketobutyrate, and ammonia4. Various MGLs have been obtained by extraction or recombination of genes from bacteria, protozoa, and plants5. This enzyme belongs to the family of lyases, specifically the class of carbon-sulfur lyases. The systematic name of this enzyme class is L-methionine methanethiol-lyase (deaminating; 2-oxobutanoate-forming). This enzyme employs one cofactor, pyridoxal phosphate (PLP). Inventors studied the recombinant MGL from the anaerobic alkaliphilic thermophile Thermobrachium celere (TcMGL). Transformation of an Escherichia coli strain with the putative MGL gene from T. celere optimized for E. coli expression led to production of the purified TcMGL, which characteristics were compared to that of the MGL from Pseudomonas putida (PpMGL). The purified TcMGL enzyme had a maximal activity at 75°C, and was active and stable at 37°C.

[0015] The purified recombinant MGL from the alkalithermophile Thermobrachium celere described herein is characterized by optimum levels of activity at high pH, and at high temperature levels. Under physiologic conditions of temperature and pH found in mammalianenvironments, TcMGL degrades methionine with a high catalytic efficiency (kC!IKmat 37°C is 5-fold that of PpMGL).

[0016] Polypeptides isolated from Thermobrachium celere

[0017] In a first aspect, the invention relates to a polypeptide comprising an amino acid sequence as set forth by SEQ ID NO: 1 or a function-conservative variant thereof.

[0018] SEQ ID NO: 1: Protein sequence

[0019] MDREF AKNMGF STKAIHAGNHKNEFGTLATPIYQTATF VFD S AQQGGNRF AGKEEG YIYTRLGNPT VTVLEEKIAILEGGEAC VATS SGMGAIS S ALWTALK AGDHVVAADTL YGCTFAYLSHGLTRYGVEVTFVDASNPENIKEAMRPNTKAIYIETPANPNLKLIDIKK VAEIAHSKEDCILIVDNTFCTPYIQRPIELGADVVVHSATKFLNGHGDVIAGFVVGRKS FIDQVRLYGIKDMTGACLSPFDAYLILRGLKTLEIRMERHCSNAMKVAKFLEEHKAV KKVYYPGLESFEQYELAKEQMKLPGAIIAFELNGGVEEGIKVLNSCKVCTLAVSLGD AETLIQHPASMTHSPYTREERLKAGISDGLIRLAVGLENAEDIIADLKQALDAII

[0020] SEQ ID NO: 2: DNA sequence Optimized for Escherichia coli expression ATGGATAGGGAATTTGCTAAAAATATGGGATTCAGCACCAAGGCGATCCATGCA GGCAATCATAAAAACGAATTTGGTACACTGGCAACCCCGATCTACCAGACCGCA ACCTTCGTGTTTGATAGCGCGCAGCAAGGTGGCAACCGTTTTGCCGGTAAAGAGG AGGGTTATATCTACACTCGCCTGGGCAACCCGACCGTGACGGTCCTGGAGGAGAA AATCGCCATCTTAGAAGGTGGTGAAGCCTGCGTTGCGACGTCCAGCGGTATGGGT GCGATTAGCTCGGCGCTGTGGACCGCGCTGAAGGCGGGTGACCACGTGGTGGCG GCAGATACCTTGTACGGCTGCACGTTCGCGTACCTCTCCCACGGCTTGACCCGTTA TGGCGTGGAAGTTACGTTCGTAGACGCGAGCAATCCGGAAAACATTAAGGAAGC AATGCGTCCGAACACCAAGGCTATCTACATTGAAACTCCGGCCAATCCAAATCTA AAACTGATCGACATCAAAAAGGTGGCAGAGATTGCGCATTCCAAAGAGGACTGT ATTCTGATCGTGGACAACACCTTCTGCACTCCGTACATACAAAGACCTATTGAAC TGGGTGCTGATGTTGTGGTGCATTCTGCTACGAAATTCCTGAATGGTCATGGCGA CGTTATTGCTGGCTTTGTTGTTGGTCGTAAATCGTTCATTGACCAGGTTCGTCTGT ACGGCATCAAGGATATGACCGGTGCATGTCTGTCCCCGTTTGACGCGTATCTGAT TCTGCGTGGTTTGAAGACCCTGGAGATCCGCATGGAACGCCACTGCAGCAACGCT ATGAAGGTTGCTAAGTTCTTGGAGGAGCACAAAGCCGTCAAAAAGGTGTATTACC CGGGTCTTGAGAGCTTTGAACAGTATGAACTTGCGAAAGAGCAAATGAAGCTGC CGGGTGCGATCATTGCGTTTGAATTGAACGGCGGCGTTGAAGAGGGCATCAAAGT CTTAAACAGCTGTAAAGTGTGCACCTTGGCGGTCAGCCTGGGAGACGCGGAAAC CCTCATCCAACACCCGGCTAGCATGACCCACTCTCCGTATACCCGCGAAGAGCGT CTGAAGGCGGGGATTAGTGATGGTTTAATTCGTTTAGCCGTTGGCTTGGAGAACG CCGAGGATATCATCGCGGATCTGAAGCAGGCACTGGATGCTATCATTTAAIn one embodiment, the polypeptide or peptide of the invention consists in the amino acid sequence as set forth in SEQ ID NO:1 comprising at least 75%, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with SEQ ID NO:1.

[0021] In one embodiment, the polypeptide or peptide of the invention consists in the amino acid sequence as set forth in SEQ ID NO: 1 comprising at least 90% identity with SEQ ID NO: 1.

[0022] The invention provides an isolated, synthetic or recombinant methionine gamma-lyase polypeptide comprising an amino acid sequence as set forth by SEQ ID NO: 1 or a functionconservative variant thereof.

[0023] The present invention thus encompasses function-conservative variants of the methionine gamma-lyase polypeptides as set forth by SEQ ID NO:1. The functionconservative variants may result from modifications and changes that may be made in the structure of the polypeptides of SEQ ID NO:1 (and in the DNA sequences encoding it), while still producing a functional molecule with desirable characteristic (maximal activity at 75°C, and active and stable at 37°C). Typically, the polypeptide according to the invention has maximum activity at 75°C and keeps: substantial activity under physiologic conditions ; substrate specificity for L-Met presents in wild-type TcMGL and high level of stability in plasma.

[0024] As used herein, the term “peptide” corresponds to the chemical agents belonging to the protein family. A peptide is composed of a mixture of several amino acids. Depending on the number of amino acids involved, peptides are categorized as dipeptides, composed of 2 amino acids, tripeptides, made up of 3 amino acids, and so on. Peptides composed of more than 10 amino acids are called polypeptides. Thus, the peptide of the invention can be considered as a polypeptide.

[0025] The peptides according to the invention, may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art.

[0026] Accordingly, "function-conservative variants" are those in which a given amino acid residue, or several amino acid residues, in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). More particularly, said function-conservative variants have little or no impact on the function of the encoded protein. These variants are often found in regions of the gene that are not critical forthe protein's function or structure. Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70 % to 99 % as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A "function-conservative variant" also includes a polypeptide which has at least 60 % amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75 %, more preferably at least 85%, still preferably at least 90 %, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared. Two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 80 %, preferably greater than 85 %, preferably greater than 90 % of the amino acids are identical, or greater than about 90 %, preferably greater than 95 %, are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of sequence comparison algorithms such as BLAST, FASTA, etc.

[0027] The amino acid changes may be achieved by changing codons in the DNA sequence, according to Table 1.

[0028] Table 1

[0029] Amino acids Codons

[0030] Alanine Ala A GCA, GCC, GCG, GCU Cysteine Cys C UGC, UGU Aspartic Acid Asp D GAC, GAU Glutamic acid Glu E GA A, GAG Phenylalanine Phe F UUC, UUU Glycine Gly G GGA, GGC, GGG, GGU Histidine His H CAC, CAU Isoleucine He I AUA, AUC, AUU Lysine Lys K AAA, AAG Leucine Leu L UUA, UUG, CUA, CUC, CUG,

[0031] CUU

[0032] Methionine Met M AUG Asparagine Asn N AAC, AAU

[0033] Proline Pro P CCA, CCC, CCG, CCUGlutamine Gin Q CAA, CAG Arginine Arg R AGA, AGG, CGA, CGC, CGG,

[0034] CGU

[0035] Serine Ser S AGC, AGU, UCA, UCC, UCG,

[0036] UCU

[0037] Threonine Thr T ACA, ACC, ACG, ACU Valine Vai V GUA, GUC, GUG, GUU Tryptophan Trp W UGG

[0038] Tyrosine Tyr Y UAU

[0039] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of methionine gamma-lyase capability or homocysteinase capability. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and, of course, in its DNA encoding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the polypeptide sequences of the invention, or corresponding DNA sequences which encode said polypeptides, without appreciable loss of their biological activity.

[0040] Said methionine gamma-lyase activity can be assessed by different techniques well-known in the art as described hereinafter.

[0041] In the context of the invention, the enzymatic activity of PpMGL and TcMGL was measured with the MBTH (3-methyl-2-benzothiazolone hydrazone) endpoint colorimetric assay well known in the art18. Results from catalysis of amino acids and substituted amino acid were fitted to the Michaelis-Menten kinetics equation using GraphPad Prism (vlO.O.O., GraphPad Software, Boston, MA). One unit of enzyme is defined as the amount that catalyzes formation of 1 pmol of a-ketobutyrate per minute from L-Met.

[0042] In a further embodiment, the enzyme activity according to pH (pH range from 5.5 to 9.5 ) has been performed using colorimetric assay well known in the art.

[0043] In a further embodiment, the thermal stability of TcMGL and PpMGL has been measured using the SUPR-DSF microplate-based spectrofluorometer device.

[0044] In making the changes in the amino-acid sequences of polypeptide, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, PLP cofactor, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0045] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein.

[0046] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. In a particular embodiment, any amino-acid substitution that preserves the enzymatic activity can be performed. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0047] The methionine gamma-lyase activity and homocysteinase activity of the functionconservative variants may be assessed according to any method of assaying methionine gammalyase activity and homocysteinase activity known in the art, such as for instance the assays referred in the instant application.

[0048] The polypeptides of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.

[0049] Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions.

[0050] Alternatively, the polypeptides of the invention can be synthesized by recombinant DNA techniques as is now well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryoticor prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.

[0051] In the context of the invention, a synthetic gene coding for TcMGL sequence was cloned into E. coli to express an N-terminal histidine-tagged recombinant protein to facilitate purification. The product was purified by immobilized metal affinity chromatography, providing an UV-visible absorption spectrum with a 422 nm peak characteristic of lysine-bound PLP. Purification of TcMGL was achieved to homogeneity using a second step of sizeexclusion chromatography. TcMGL was purified to homogeneity from a 0.25-liter scale cell pellet yielding approximately 15 to 20 mg of enzyme. The major band observed on SDS-PAGE is in accordance with the calculated molecular weight of 45.65 kDa. A second band with apparent MW close to 90 kDa is consistent with a dimer.

[0052] In a particular embodiment, the invention relates to a nucleic acid encoding an amino acid sequence comprising SEQ ID NO: 1.

[0053] In a particular embodiment, the nucleic acid according to the invention comprising a sequence as set forth by SEQ ID NO: 2.

[0054] In a particular embodiment, the invention relates to vector comprising the nucleic acid as described above.

[0055] In a particular embodiment, the invention relates to a host cell comprising the expression vector.

[0056] A further object of the present invention relates to a nucleic acid that encodes for a polypeptide of the present invention.

[0057] As used herein, the term “polynucleotide” or “nucleic acid” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogues thereof, or mixtures thereof. This term refers to the primary structure of the molecule.

[0058] Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration whichallows for base pairing and base stacking, such as is found in DNA and RNA. In some embodiments, the polynucleotide comprises a DNA or RNA. In some embodiments, the nucleic acid comprises a mRNA. In other aspect, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a nucleic acid disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, the nucleic acid (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, G, and U in the case of a synthetic RNA.

[0059] As used herein, the terms “coding sequence” or “a sequence which encodes a particular protein” or “encoding nucleic acid”, denotes a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0060] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0061] In particular embodiment, the nucleic acid is an RNA molecule, and more particularly a mRNA molecule.

[0062] In a particular embodiment, the invention relates to a method of producing a polypeptide according to the invention, which method comprises the steps consisting of: (i) culturing a transformed host cell according to the invention under condition suitable to allow expression of said polypeptide; and (ii) recovering the expressed polypeptide.

[0063] As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a polypeptide of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides.

[0064] A variety of expression vector / host systems may be utilized to contain and express the peptide or protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expressionvectors; yeast transformed with yeast expression vectors (Giga-Hama et al., 1999); insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., 2000); or animal cell systems. Those of skill in the art are aware of various techniques for optimizing mammalian expression of proteins, see e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are known to those of skill in the art and a briefly described herein below. Mammalian host systems for the expression of recombinant proteins also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and / or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.

[0065] In the recombinant production of the single domain antibodies and polypeptides of the invention, it would be necessary to employ vectors comprising polynucleotide molecules for encoding the single domain antibodies and polypeptides of the invention. Methods of preparing such vectors as well as producing host cells transformed with such vectors are well known to those skilled in the art. The polynucleotide molecules used in such an endeavor may be joined to a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host. These elements of the expression constructs are well known to those of skill in the art. Generally, the expression vectors include DNA encoding the given protein being operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect genes. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation.The terms "expression vector”, “vector”, "expression construct" or "expression cassette" are used interchangeably throughout this specification and are meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.

[0066] The choice of a suitable expression vector for expression of the peptides or polypeptides of the invention will of course depend upon the specific host cell to be used, and is within the skill of the ordinary artisan.

[0067] Expression requires that appropriate signals be provided in the vectors, such as enhancers / promoters from both viral and mammalian sources that may be used to drive expression of the nucleic acids of interest in host cells. Usually, the nucleic acid being expressed is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the protein of interest (e.g., a single domain antibody). Thus, a promoter nucleotide sequence is operably linked to a given DNA sequence if the promoter nucleotide sequence directs the transcription of the sequence.

[0068] Encapsulation of the polypeptide according to the invention

[0069] The polypeptide of the invention can be used in an isolated (e.g., purified) form or encapsulated in a delivery system.

[0070] Accordingly, in a second aspect, the invention relates to a delivery system comprising the polypeptide of the invention.

[0071] As used herein, the term “delivery system” refers to a carrier or a vector to transport and protect the polypeptides according to the invention until it reaches its target site such as tumour site.

[0072] In another embodiment, the polypeptide of the invention is inserted in any delivery vehicle able to encapsulate a compound.

[0073] In a particular embodiment, the polypeptide of the invention is contained in a a delivery system such as : an erythrocyte, a nanoparticle, a liposome (for example a polymeric liposome or a lipidic liposome), a Virus-Like-Particle (VLP), a dendrimer, a micelle, a nanoemulsion, extracellular vesicle, nanosuspension or a bioreactor.

[0074] In another embodiment, the polypeptide of the invention is vectorised within a bioreactor.As used herein, the term “bioreactor” refers to an apparatus able to immobilize enzymes onto a support material (vector) and then using this setup within a bioreactor to catalyze reactions. This approach offers several advantages: enhances stability, reusability, improves efficiency, simplification of separation. Typically, in the context of the invention, the polypeptide according to the invention can be used in a bioreactor, where conditions such as temperature, pH, and nutrient supply are carefully controlled to optimize enzyme function.

[0075] In a particular embodiment, the delivery system or bioreator according to the invention is a living cell such as an erythrocyte or another cell type or cell ghost to build a bioreactor carrying MGL activity.

[0076] In a particular embodiment, the living cell is any type of cell able to deliver the polypeptide of the invention.

[0077] In a further embodiment, the living cell is eukaryotic cells, more preferably animal cells, notably bird, mammal or human cells.

[0078] Typically, such living cell can be cells to be cultivated for themselves, such as NK cells, lymphocytes, in particular chimeric antigen receptor T cells (CAR T cells), erythroid cells, in particular erythroblasts, cultured red blood cells or cultured meat cells, or cells to be cultivated for producing molecules of interest, in particular proteins, more particularly antibodies or antibody derivatives, notably monoclonal antibodies.

[0079] In a particular embodiment the living cell is an erythroid cell, in particular erythroblasts, cultured red blood cells or cultured meat cells.

[0080] In a particular embodiment the living cell can be stem cells, progenitor cells, or cells of an immortalized cell line of the erythroid lineage.

[0081] In a particular embodiment, the polypeptide according to the invention is contained in an erythrocyte.

[0082] As used herein, the term “erythrocyte” also called as Red blood cells (RBCs), red cells, erythroid cells, or haematids, is the most common type of blood cell and the vertebrate's principal means of delivering oxygen (02) to the body tissues via blood flow through the circulatory system. Erythrocytes are naturally designed to transport oxygen throughout the body, making them an excellent candidate for delivering therapeutic proteins.

[0083] In the context of the invention, the polypeptide according to the invention can be attached to the surface of erythrocytes through chemical or genetic modifications. This allows the erythrocytes to carry and deliver the polypeptide according to the invention to tumour site. Erythrocytes have a long circulation time in the bloodstream, which can be advantageous for delivering the polypeptide according to the invention over an extended period.The polypeptide according to the invention can be encapsulated within erythrocytes according to the method as described in Machover et al 2019, J Pharmacol Exp Ther . 2019 Jun;369(3):489-502. doi: 10.1124 / jpet.119.256537. Epub 2019 Apr 2.

[0084] It is well known to produce RBCs in the art. Typically, the stem cells, progenitors, or cells of an immortalized cell line of the erythroid lineage as the cell source.

[0085] The stem cells may be embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or hematopoietic stem cells and / or progenitors (HSCs / HPs).

[0086] In a particular embodiment, the cell source is hematopoietic stem cells (HSCs).

[0087] Cells of an immortalized cell line of the erythroid lineage can be immortalized at the stage of an erythroid progenitor or an erythroid precursor. In addition, hematopoietic stem cells (HSCs) can also be immortalized.

[0088] Immortalization is preferably performed conditionally. These immortalized cells can then be passaged indefinitely in vitro, cryopreserved and recovered, and, conditionally, produce fully differentiated red blood cells from a defined and well characterized source. Conditional immortalization can be achieved by any method well known to the person skilled in the art.

[0089] Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent stem cells. These cells are both capable of differentiation into many cell types and capable of self-replication. They can maintain this pluripotency of differentiation even after undergoing proliferation by division. Embryonic stem cells refer to pluripotent stem cells derived from blastocyst stage embryos, which is the early stage of animal development. Induced pluripotent stem cells (iPSCs) are produced by introducing several types of transcription factor genes into somatic cells such as fibroblasts.

[0090] The embryonic stem cells (ESCs) are obtained by any means not requiring the destruction of human embryos. For example, by using the technology described by Chung et al (Chung et al, Human Embryonic Stem Cell lines generated without embryo destruction, Cell Stem Cell (2008)).

[0091] The hematopoietic stem cells (HSCs) used in the context of the invention are multipotent cells. They are capable of differentiating into all blood cell differentiation lineages and are capable of self-replicating while maintaining their multipotency.

[0092] Cells of an immortalized cell line of the erythroid lineage are cells already committed to the erythroid lineage but capable of self-replication and under external control of differentiating into erythroid lineage cells.The hematopoietic stem cells and / or progenitors (HSCs / HPs) used in the method according to the invention can be derived from any source, including, being derived from bone marrow, umbilical cord / placental blood or peripheral blood with or without prior mobilization.

[0093] The origin of stem cells and cells of an immortalized cell line of the erythroid lineage is not particularly limited as long as it is derived from a mammal. Preferred examples include humans, dogs, cats, mice, rats, rabbits, pigs, cows, horses, sheep, goats and the like, humans being most preferred.

[0094] In a further embodiment, the erythrocytes are obtained from the subject suffering from a cancer. Typically, said erythrocytes are loaded as following:

[0095] i) Obtain a blood sample from a subject suffering from a cancer,

[0096] ii) Separate erythrocytes from plasma and other blood components,

[0097] iii) Encapsulate the polypeptide according to the invention (electroporation, chemical conjugaison with linkers etc);

[0098] iv) Administrate said erythrocyte to the subject.

[0099] In a particular embodiment, the polypeptide of the invention is vectorized, for example in nanoparticles, such as virus-like particles or liposomes.

[0100] In a particular embodiment, the nanoparticle is PLGA Nanoparticles: Polylactic-co-glycolic acid (PLGA) nanoparticles are well known in the art for drug delivery due to their biodegradability and biocompatibility.

[0101] In a particular embodiment, the polypeptide of the invention is encapsulated in PLGA Nanoparticle.

[0102] In a particular embodiment, the polypeptide of the invention is encapsulated in a pegylated nanoparticle such as PEGylated Liposomes.

[0103] In specific embodiments, it is contemplated that polypeptides according to the invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.

[0104] A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

[0105] For example, Polyethylene glycol-conjugated (said PEGylation) is a well-established and validated approach for the modification of a range of polypeptides (Chapman, 2002). The benefits include among others : (a) markedly improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and / or through evasion of cellular clearance mechanisms; (b) reduced antigenicity and immunogenicity of the molecule to which PEG is attached; (c) improved pharmacokinetics; (d) enhanced proteolytic resistance of the conjugated protein (Cunningham-Rundles et.al., 1992); and (e) improved thermal and mechanical stability of the PEGylated polypeptide.

[0106] Therefore, advantageously, the polypeptide of the invention is covalently linked with one or more polyethylene glycol (PEG) group(s).

[0107] Accordingly, one aspect of the invention provides modified polypeptides, wherein the modification comprises a single polyethylene glycol group covalently conjugated to the polypeptide. Other aspects provide modified polypeptides covalently conjugated to one, two, three, or more polyethylene glycol groups. The one or more PEG may have a molecular weight ranging from about 1 kDa to about 100 kDa, and will preferably have a molecular weight ranging from about 10 to about 60 kDa or about 10 to about 40 kDa. One skilled in the art can select a suitable molecular mass for PEG, based on how the pegylated polypeptide will be used therapeutically by considering different factors including desired dosage, circulation time, resistance to proteolysis, immunogenicity, etc.

[0108] In one embodiment, the PEG of the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH3 ("methoxy PEG"). In addition, such a PEG can consist of one or more PEG side-chains which are linked together. PEGs with more than one PEG chain are called branched PEGs. Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol. For example, a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide. One form of PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini, et al., 1995).

[0109] To effect covalent attachment of PEG groups to the polypeptide, the hydroxyl end groups of the polymer molecule must be provided in activated form, i. e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidylproprionate (SPA), succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenyl carb onate (NPC), and tresylate (TRES)). Suitable activated polymer molecules are commercially available, e. g. from Shearwater Polymers, Inc., Huntsville, AL, USA, or from PolyMASC Pharmaceuticals pic, UK. Alternatively, the polymer molecules can be activated by conventional methods known in the art, e. g. as disclosed in WO 90 / 13540. Specific examples of activated linear or branched polymer molecules for use in the present invention are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Specific examples of activated PEG polymers include the following linear PEGs : NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM-PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDLPEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-NHS.

[0110] The conjugation of the polypeptide and the activated polymer molecules is conducted by use of any conventional method. Conventional methods are known to the skilled artisan. The skilled person will be aware that the activation method and / or conjugation chemistry to be used depends on the attachment group(s) of the polypeptides as well as the functional groups of the PEG molecule (e.g., being amine, hydroxyl, carboxyl, aldehyde, ketone, sulfhydryl, succinimidyl, maleimide, vinylsulfone or haloacetate).

[0111] In one embodiment, polypeptides are conjugated with PEGs at amino acid D and E (for COOH), T, Y and S (for OH), K (for NH2), C (for SH if at least one cysteine is conserved) or / and Q and N (for the amide function).

[0112] In one embodiment, additional sites for PEGylation can be introduced by site-directed mutagenesis by introducing one or more lysine residues. For instance, one or more arginine residues may be mutated to a lysine residue. In another embodiment, additional PEGylation sites are chemically introduced by modifying amino acids on polypeptides of the invention.

[0113] In one embodiment, PEGs are conjugated to the polypeptide through a linker. Suitable linkers are well known to the skilled person. A preferred example is cyanuric chloride (Abuchowski et al., 1977; US 4,179, 337).

[0114] Conventional separation and purification techniques known in the art can be used to purify pegylated polypeptides of the invention, such as size exclusion (e.g. gel filtration) and ion exchange chromatography. Products may also be separated using SDS-PAGE.

[0115] In another embodiment, the polypeptide of the invention is covalently linked with one or more residues of sialic acid (Polysialylation).As used herein, the term “polysialylation” refers to a biochemical modification in which multiple residues of sialic acid are covalently linked together to form a linear or branched polysialic acid (polySia) chain that is attached to a protein, peptide, or glycan structure. This process occurs naturally in certain glycoproteins most notably the neural cell adhesion molecule (NCAM) and can also be performed artificially for therapeutic purposes. The attached polysialic acid chains are highly hydrophilic and negatively charged, which can increase molecular size, improve solubility, reduce immunogenicity, protect against proteolytic degradation, and prolong circulating half-life in vivo.

[0116] In another embodiment, the polypeptide according to the invention is encapsulated in chitosan nanoparticles.

[0117] As used herein, the term “chitosan” refers to a fibrous compound derived from chitin, which is the second most abundant natural polysaccharide and is produced by crustaceans, including crabs, shrimps, and lobsters.

[0118] Typically, such chitosan naonparticles are described in Rejeena Jha et al 2023 : Nanomaterials 2023, 13(8), 1302; https: / / doi.org / 10.3390 / nanol3081302.

[0119] In another embodiment, the polypeptide according to the invention is encapsulated in Polybutylcyanoacrylate (PBCA) Nanoparticles.

[0120] As used herein, the term “Poly(butyl cyanoacrylate) (PBCA)” refers to a biodegradable and biocompatible homopolymer. Typically, such PBCA naonparticles are described in Benjamin-Luca Keller et al 2022, Polymers 2022, 14(5), 998; https: / / doi.org / 10.3390 / polyml4050998.

[0121] In a particular embodiment, the polypeptide or the nucleic acid according to the invention is encapsulated in a viral vector.

[0122] In another embodiment, the invention relates to a vector which comprises the polypeptide of the present invention.

[0123] Typically, the polypeptide or the nucleic acid according to the invention can be delivered in association with a vector. The polypeptide or the nucleic acid of the present invention is included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. So, a further object of the invention relates to a vector comprising polypeptide or the nucleic acid of the invention. Typically, the vector is a viral vector, which is an adeno-associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" means a vector derived from anadeno- associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and / or env genes but without the LTR and / or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses are then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g.. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene intothat packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein that allows transduction of cells of human and other species. Typically, the nucleic acid molecule or the vector of the present invention include "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”.

[0124] Drug conjugate

[0125] The polypeptide according to the invention is conjugated to another molecule or vehicle for a targeted delivery of the polypeptide of the invention to specific cells or tissues, enhancing its therapeutic efficacy and reducing side effects.

[0126] Accordingly, in a third aspect, the invention relates to a drug conjugate comprising the polypeptide according to the invention linked to a heterologous moiety.

[0127] In another embodiment the polypeptide of the invention is covalently coupled to a tumor targeting agent as well known in the art.

[0128] As used herein, the term “conjugation” has its general meaning in the art and means a chemical conjugation. Techniques for conjugating heterologous moiety to polypeptides, are well-known in the art (See, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO 89 / 12624.) Typically, the nucleic acid molecule is covalently attached to lysines or cysteines on the antibody, through N-hydroxysuccinimide ester or maleimide functionality respectively. Methods of conjugation using engineered cysteines or incorporation of unnatural amino acids have been reported to improve the homogeneity of the conjugate (Axup, J.Y., Bajjuri, K.M., Ritland, M., Hutchins, B.M., Kim, C.H., Kazane, S.A., Halder, R., Forsyth, J.S., Santidrian, A.F., Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, J.R., Flagella, K.M., Graham, R.A., Parsons, K.L., Ha, E., Raab, H, Bhakta, S., Nguyen, T., Dugger, D.L., Li, G., et al. (2010). Engineered thio-trastuzumab-DMl conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res.16, 4769-4778.). Junutula et al. (2008) developed cysteine-based site-specific conjugation called “THIOMABs” (TDCs) that are claimed to display an improved therapeutic index as compared to conventional conjugation methods. In particular the one skilled in the art can also envisage a polypeptide engineered with an acyl donor glutamine-containing tag (e.g., Gin-containing peptide tags or Q- tags) or an endogenous glutamine that are made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). Then, a transglutaminase can covalently crosslink with an amine donor agent (e.g., a small molecule comprising or attached to a reactive amine) to form a stable and homogenous population of an engineered Fc-containing polypeptide conjugate with the amine donor agent being site- specifically conjugated to the Fc-containing polypeptide through the acyl donor glutamine-containing tag or the accessible / exposed / reactive endogenous glutamine (WO 2012059882). The term “transglutaminase”, used interchangeably with “TGase” or “TG”, refers to an enzyme capable of cross-linking proteins through an acyl-transfer reaction between the y-carboxamide group of peptide-bound glutamine and the s-amino group of a lysine or a structurally related primary amine such as amino pentyl group, e.g. a peptide-bound lysine, resulting in a s-(y-glutamyl) lysine isopeptide bond. TGases include, inter alia, bacterial transglutaminase (BTG) such as the enzyme having EC reference EC 2.3.2.13 (protein-glutamine-y-glutamyltransferase). In some embodiments, the single domain antibody of the present invention is conjugated to the heterologous moiety by a linker molecule. As used herein,the term “linker molecule" refers to any molecule attached to the peptide of the present invention. The attachment is typically covalent. In some embodiments, the linker molecule is flexible and does not interfere with the binding of the peptide of the present invention.

[0129] As used herein, the term “drug conjugate” refers to a complex molecule composed of a drug (such as the polypeptide of the invention) linked to another molecule, such as an antibody, peptide, or any compound which is cytotoxic agent for a cancer, etc .

[0130] As used herein, the “heterologous moiety” refers to a delivery system such as a bioreactor (e.g. erythrocyte) as described above).

[0131] In a particular embodiment, the polypeptide according to the invention which is covalently coupled to a tumor targeting agent (such as an agent against cancer).

[0132] In a particular embodiment, the drug conjugate comprises a bioreactor loaded with the polypeptide according to the invention (=heterologous moiety) and a cytotoxic agent.

[0133] In a particular embodiment, the drug conjugate comprises a pegylated nanoparticle with the polypeptide according to the invention (=heterologous moiety) and a cytotoxic agent.

[0134] A "fusion" or "chimeric" protein or polypeptide comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences which normally exist in separate proteins can be brought together in the fusion polypeptide. A fusion protein is created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the polypeptide regions are encoded in the desired relationship. "Fusion" or "chimeric" polypeptides and proteins includes a combination of a first polypeptide chain, e.g., the polypeptide according to the invention, with a second polypeptide chain, e.g., a therapeutic agent.

[0135] Therapeutic methods and uses

[0136] The purified recombinant MGL from the alkalithermophile Thermobrachium celere described herein is characterized by optimum levels of activity at high pH, and at high temperature levels. In human plasma under natural conditions of temperature and pH, TcMGL degrades methionine with a high catalytic efficiency (Zrcat / ATn is 5-fold that of PpMGL).

[0137] The polypeptide comprising an amino acid sequence as set forth by SEQ ID NO: 1, a function-conservative variant thereof or the drug conjugate as described above is suitable to be used in therapeutic methods.

[0138] In a third aspect, the invention relates to a polypeptide having SEQ ID NO: 1, a function-conservative variant thereof or the drug conjugate for use as drug.In another aspect, the invention relates to a drug conjugate according to the invention for use as drug.

[0139] In a further embodiment, the drug conjugate for use according to the invention wherein the drug conjugate comprises a bioreactor encapsulating the polypeptide according to the invention (=heterologous moiety) and a cytotoxic agent.

[0140] In a further embodiment, the drug conjugate for use according to the invention wherein the drug conjugate comprises a pegylated nanoparticle with the polypeptide according to the invention (=heterologous moiety) and a cytotoxic agent.

[0141] In another aspect, the invention relates to a polypeptide having SEQ ID NO: 1, a function-conservative variant thereof or the drug conjugate and at least one another therapeutic agent, as a combined preparation for use in the treatment of cancer.

[0142] According to the invention, the polypeptide of the invention or a drug conjugate of the invention, is administered to a subject in need thereof with a therapeutically effective amount.

[0143] In a particular embodiment, the invention relates to a method of treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the polypeptide according to the invention or the drug conjugate according to the invention.

[0144] In a further embodiment, the method for treating a cancer according to the invention, comprising:

[0145] i) Obtain a blood sample from a subject suffering from a cancer, ii) Separate erythrocytes from plasma and other blood components, iii) Encapsulate the polypeptide according to the invention (electroporation, chemical conjugaison with linkers etc);

[0146] iv) Administrate said erythrocyte to the subject.

[0147] In another embodiment the polypeptide of the invention is covalently coupled to a tumor targeting agent as well known in the art.

[0148] As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in theabsence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide drugs to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0149] As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. More particularly, the subject according to the invention has or is susceptible to have a cancer. In another embodiment, the subject according to the invention has or is susceptible to have metastatic cancer.

[0150] As used herein, the term “cancer” refers to a malignant growth or tumour resulting from an uncontrolled division of cells. The term “cancer” includes primary tumors and metastatic tumors.

[0151] In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophilcarcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous feature; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal sarcoma; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative smallintestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia, pleural cancer or colorectal cancer.

[0152] In a particular embodiment, the cancer is selected from the group consisting of, but not limited to: Colorectal carcinoma, Cervix adenocarcinoma, Vulvar epidermoid carcinoma, Hepatocellular carcinoma, Hepatoblastoma, Pre-B-cell acute lymphocytic leukemia, T-Cell acute lymphocytic leukemia, CML blast crisis, Erythroleukemia, Acute promyelocytic leukemia, Acute myelocytic leukemia, Burkitt’s lymphoma, Osteosarcoma, Fibrosarcoma, Prostate adenocarcinoma, Ovarian adenocarcinoma, Pancreas adenocarcinoma, Mammary adenocarcinoma, Melanoma, Renal cell carcinoma, Lung adenocarcinoma, Neuroblastoma or Glioma.

[0153] In a particular embodiment, the invention relates to i) a polypeptide or the drug conjugate according to the invention and ii) a classical treatment as a combined preparation for use by simultaneous, separate or sequential administration in the treatment of cancer and / or metastatic cancer in a subject in need thereof.

[0154] As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication.

[0155] As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

[0156] As used herein, the term “classical treatment” refers to treatments well known in the art and used to treat cancer. In the context of the invention, the classical treatment refers to targeted therapy, radiation therapy, immunotherapy, cyclin-dependent kinase inhibitors (CDKi), hormonal therapy or chemotherapy.

[0157] In a particular embodiment, the invention relates to i) the polypeptide or the drug conjugate according to the invention and ii) a radiation therapy used as a combined preparation for use in the treatment of cancer and / or metastatic cancer in a subject in need thereof.

[0158] As used herein, the terms “radiation therapy” or “radiotherapy” have their general meaning in the art and refer to the treatment of cancer with ionizing radiation. Ionizing radiationdeposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.

[0159] In a particular embodiment, the invention relates to i) the polypeptide or the drug conjugate according to the invention and ii) a chemotherapy used as a combined preparation for use in the treatment of cancer and / or metastatic cancer in a subject in need thereof.

[0160] As used herein, the term “chemotherapy” refers to use of chemotherapeutic agents to treat a subject. As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth.

[0161] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide,triethylene thiophosphorarnide and trimethylol melamine; acetogenins (especially bullatacin and bullatacinone); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin y1! and calicheamicin 01!, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, floxuridine and 5 -fluorouracil (5-FU), either as a single agent or in combination with modulators such as folates and B6 vitamers ; folic acid analogues such as denopterin, methotrexate, pteropterin, pemetrexed, raltitrexed, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine gemcitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; vinca alcaloids and semisynthetic compounds (vincristine, vindesine, vinblastine, vinorelbine), folic acid replenisher such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine;trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vinca alcaloids and semisynthetic compounds (vincristine, vindesine, vinblastine, vinorelbine); dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; mitoxantrone; teniposide; daunomycin; aminopterin; capecitabine, ibandronate; CPT-1-1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0162] In a particular embodiment, the invention relates to i) the polypeptide according to the invention, ii) fluoropyrimidines combined with folates and iii) B6 vitamers in tandem as a combined preparation for use in the prevention and / or treatment of cancer and / or metastatic cancer in a subject in need thereof.

[0163] In a particular embodiment, the invention relates to i) the polypeptide according to the invention and ii) an anti-angiogenesis compound, as a combined preparation for use in the prevention and / or treatment of cancer and / or metastatic cancer in a subject in need thereof.

[0164] As used herein, the term “angiogenesis” refers to a physiological process involving the growth of new blood vessels from preexisting vessels. Angiogenesis is a combinatorial process that is regulated by a balance between pro- and anti -angiogenic molecules. Angiogenic stimuli (e.g. hypoxia or inflammatory cytokines) result in the induced expression and release of angiogenic growth factors such as vascular endothelial growth factor (VEGF) or fibroblast growth factors (FGF).

[0165] As used herein, the term “anti-angiogenesis” refers to any molecule which can inhibit angiogenesis that means the creation of new blood vessels. Typically, anti-angiogenesis compound is well known in the art and refers to the following compounds but not limited to bevacizumab (Avastin, anti-VEGF), itraconazole (anti-VGFR), carboxyamidotriazole, TNP-470 (an analog of fumagillin), CM101, IFN-a, IL-12, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiostatic steroids + heparin, Cartilage-Derived Angiogenesis Inhibitory Factor, matrix metalloproteinase inhibitors, angiostatin, endostatin, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactin, aVp3 inhibitors, linomide, ramucirumab, tasquinimod, ranibizumab, sorafenib (Nexavar), sunitinib (Sutent), pazopanib (Votrient), everolimus (Afinitor), cabozantinib.

[0166] In a particular embodiment, the invention relates to i) the polypeptide or the drug conjugate according to the invention and ii) an immune checkpoint inhibitor, as a combined preparation for use in the treatment of cancer and / or metastatic cancer in a subject in need thereof.

[0167] As used herein, the expressions "immune checkpoint inhibitor", "checkpoint inhibitor" or "checkpoint blockade cancer immunotherapy agent" are used interchangeably and have its general meaning in the art and refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. The immune checkpoint inhibitors include peptides, proteins, antibodies, nucleic acid molecules and small molecules. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. Examples of immune checkpoint inhibitors are provided here below under the associated paragraph.

[0168] In a particular embodiment, the immune checkpoint inhibitor is an antibody.

[0169] Typically, antibodies are directed against PD-1, PD-L1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, CD277, IDO, KIR, LAG-3, TIM-3 or VISTA.

[0170] In a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).

[0171] In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech / Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS).In a particular embodiment, the anti-PD-1 or anti-PD-Ll antibody is atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, pidilizumab, cemiplimab, camrelizumab, sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), dostarlimab (TSR-042, WBP-285), BMS 936559, MPDL3280A, MSB0010718C, MEDI4736 and any combination thereof.

[0172] In a particular embodiment, the immune checkpoint inhibitor is an antibody directed against CTLA-4. Antibodies directed against CTLA-4 are also known such as ipilimumab, tremelimumab, MK-1308, AGEN-1884, XmAb20717 (Xencor), MEDI5752 (AstraZeneca).

[0173] In some embodiments, the monotherapy is performed with an an-anti PD-1. More particularly, the anti-PD-1 is nivolumab.

[0174] In some embodiments, the bi-therapy (as a combined therapy) is performed with anti-PD-1 inhibitor and anti-CTLA-4 inhibitor, and in particular with anti-PD-1 antibody and anti-CTLA-4 antibody. More particularly, the anti-PD-1 is nivolumab and anti-CTLA-4 is ipilimumab.

[0175] In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.

[0176] In some embodiments, the immune checkpoint inhibitor inhibits Tim-3 or its ligand. In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and W02013006490.

[0177] In a particular embodiment, the immune checkpoint inhibitor is a monoclonal antibody directed against LAG-3. Antibodies directed against LAG-3 are also known such as: Relatlimab (BMS-986016), Favezelimab (MK-4280, Merck), LAG3-Ab (Protheragen), TJA-3(I-Mab), LBL-007(BeiGene), LAG525 (Novartis), Tesaro (GSK) (TSR-033), Sym022 (Symphogen), GSK2831781 (GlaxoSmith), INCAGN02385 (Incyte Biosciences International), IMP321 (Prima BioMed / Immutep), MGD013 (MacroGenics), FS118 (F-Star), RO7247669 (Hoffmann-La Roche), EMB-02 (Shanghai EpimAb Biotherapeutics), XmAb841 (Xencor), Fianlimab (REGN3767) or IBI323 (Innovent Biologies).

[0178] In a particular embodiment, the immune checkpoint inhibitor is a bispecific antibody directed against LAG-3 and another immune check point (PD-1, PDL-1, PD-2 etc). Bispecific antibodies directed against LAG-3 are also known such as: Tebotelimab (MGD013, Macrogenetics), FS118 (F-star Therapeutics), RO7247669 (Hoffmann-La Roche), EMB-02 (EpimAb Biotherapeutics).In some embodiments, the bi-therapy (as a combined therapy) is performed with anti-PD-1 and anti-LAG-3. More particularly, the anti-PD-1 is nivolumab and anti-LAG-3 is Relatlimab.

[0179] In some embodiments, the monotherapy is performed with an-anti PD-L1 inhibitor, and in particular with anti-PD-Ll antibody.

[0180] In some embodiments, the bi-therapy (as a combined therapy) is performed with anti-PD-Ll inhibitor and an-anti LAG-3 inhibitor, and in particular with anti-PD-Ll antibody and anti-LAG-3 antibody.

[0181] In some embodiments, the immune checkpoint inhibitor is a small organic molecule. The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0182] Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

[0183] In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.

[0184] In a particular embodiment, the small organic molecules interfere with Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al. 2010, Vacchelli et al. 2014, Zhai et al. 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P-(3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6-fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5 -hydroxy -tryptophan, indole 3-carbinol, 3,3'-diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9- vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P-carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1-methyl-tryptophan, P-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-amino-naphtoic acid and P-[3-benzo(b)thienyl] -alanine or a derivative or prodrug thereof.

[0185] In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4-fluorophenyl)-N' -hydroxy -4-{[2-(sulfamoylamino)-ethyl]amino}-l, 2, 5-oxadiazole-3-carboximidamide :

[0186]

[0187] In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-l,2,4-Triazole-3,5-diamine, l-(6,7-dihydro-5H-benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)-5H-benzocyclohepten-2-yl]- and has the following formula in the art:

[0188]

[0189] In a particular embodiment, the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA-170 are presented by Curis Collaborator and Aurigene on November 2015 at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

[0190] In some embodiments, the immune checkpoint inhibitor is an aptamer.

[0191] Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

[0192] In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al. 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al. 2015.

[0193] In a particular embodiment, the invention relates to i) the polypeptide or the drug conjugate according to the invention and ii) a cyclin-dependent kinase inhibitor (CDKi), as acombined preparation for use in the treatment of cancer and / or metastatic cancer in a subject in need thereof.

[0194] As used herein, the term “cyclin-dependent kinase" refers to a group of protein kinases playing an essential role in regulating the cell cycle. Their direct interaction with cyclins allow progression through G1 phase, transitions to S and G2 phase and finally through mitosis (M).

[0195] As used herein, the term “cyclin-dependent kinase inhibitor" refers to a compound which inhibits the CDKs whose activation drives the cell cycle forward. Therefore, such inhibitor targets CDK function to prevent unregulated proliferation of cancer cells.

[0196] Examples of such inhibitor are well known in the art. In a particular embodiment, the cyclin-dependent kinase inhibitor is selected from the group consisting of but not limited to: Palbociclib (PD-033299, Ribociclib (LEE01), Abemaciclib (LY2835219), Trilaciclib (V03AF12), Dalpiciclib, Roscovitine or Dinaciclib.

[0197] In a particular embodiment, the invention relates to i) the polypeptide or the drug conjugate according to the invention and ii) an hormonal therapy, as a combined preparation for use in the treatment of cancer and / or metastatic cancer in a subject in need thereof.

[0198] As used herein the terms "Hormone therapy" also known as hormonal therapy or endocrine therapy refers to a treatment that adds, blocks, or removes hormones to reduce or inhibit the growth of cancer cells which are hormones dependent.

[0199] In a particular embodiment, the hormonal therapy comprises the following group but not limited to: Androgen deprivation therapy, Estrogen deprivation therapy, High-dose estrogen therapy, Chemical castration, Growth hormone therapy, Thyroid hormone replacement, Antithyroid therapy, Glucocorticoid and / or mineralocorticoid replacement, Antiglucocorticoid therapy, Insulin therapy, Oral contraceptive pills, Menstrual suppression, Bioidentical Hormone Replacement Therapy or Hormone replacement therapy (HRT).

[0200] As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., the polypeptide or the drug conjugate according to the invention) into the subject, such as by oral, parenteral, intrathecal, mucosal, intradermal, intravenous, subcutaneous, intramuscular, in coelomic cavities, delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.In a particular embodiment, the polypeptide or the drug conjugate according to the invention is administered orally.

[0201] In a particular embodiment, parenteral administration is performed with the polypeptide or the drug conjugate according to the invention.

[0202] By a "therapeutically effective amount" is meant a sufficient amount of the polypeptide (or the nucleic acid encoding for the polypeptide or the drug conjugate ) to prevent for use in a method for the treatment of acute exacerbation of chronic obstructive pulmonary disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0203] In a further embodiment, the therapeutically effective amount depends of the type of vector used to encapsulate the polypeptide of the invention.

[0204] In a particular embodiment the therapeutically effective amount refers to a sufficient amount which is needed to maintain methionine depletion or sufficient durably to deplete the methionine and thus to increase the cytotoxicity in the tumor site.Pharmaceutical composition

[0205] In a fourth aspect, the invention relates to a pharmaceutical composition comprising the polypeptide or the drug conjugate according to the invention and a pharmaceutically acceptable carrier.

[0206] In a particular embodiment, the pharmaceutical composition according to the invention for use in the prevention or treatment of a cancer as described above.

[0207] In a particular embodiment, the pharmaceutical composition according to the invention comprising an erythrocyte encapsulated with the polypeptide according to the invention.

[0208] In a particular embodiment, the pharmaceutical composition according to the invention may include any further cytotoxic agent which is used in the prevention or treatment of cancer.

[0209] In a particular embodiment, the pharmaceutical composition according to the invention for use to increase therapeutic efficacy of another agent therapeutic against cancer. Modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.

[0210] In one embodiment, said additional active agents may be contained in the same composition or administrated separately.

[0211] In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the prevention and treatment of cancer.

[0212] The polypeptide of the invention (or the nucleic acid encoding thereof) may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. As used herein, the terms "pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0213] In the pharmaceutical compositions of the invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gelcapsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0214] The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases,it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

[0215] Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0216] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

[0217] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0218] The polypeptide or the chimeric polypeptide according to the invention (or nucleic acid encoding thereof) may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered. The invention will be further illustrated by the following figures and examples.

[0219] Finally, the invention also provides kits comprising at least a polypeptide or the drug conjugate of the invention.In a particular embodiment, the invention relates to a kit comprising the polypeptide or the drug conjugate of the invention for use in therapeutic methods as described above.

[0220] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0221] FIGURES:

[0222] Figure 1: Effect of pH and temperature on the activity and thermostability of MGLs. A) Methioninase activity at 37°C of TcMGL (black) and PpMGL (grey) as a function of pH. Circles: MES buffer. Triangles: Tris buffer. Results are the mean of three determinations + / - SD. B) Activities as a function of temperature. C) Thermal denaturation for TcMGL or PpMGL without PLP (black) and with 1 mM PLP supplementation (grey). Curves are the derivative of the ratio of the intensity of fluorescence at 350 and 330 nm.

[0223] Figure 2: PLP binding to apo-TcMGL and apo-PpMGL assessed by Isothermal Titration calorimetry at 37°C. A) Net heat released for 19 automatic injections of 2 pl of PLP solution into the sample cell containing 800 pl of TcMGL (left) or PpMGL (right). DP: differential power. B) Plot of processed data. C) Histogram of the variations observed for AG (free energy), AH (enthalpy), and calculated -TAS, S being the entropy. The binding parameters and constants determined for the two methioninases towards their PLP cofactor were:

[0224]

[0225] TcMGL 0.93 ± 0.25 -8.56 9.01 ± 0.38 1 0.68 PpMGL 0.58 ± 0.16 -8.84 -12 ± 0.58 -3.16 0.53 * Number of sites accessible for binding per monomer.

[0226] Figure 3: Activity of TcMGL and PpMGL in human plasma in vitro as a function of time and PLP concentration. A) Changes in activity half-life (Z1 / 2) of Pseudomonas putida (PpMGL), and Thermobrachium celere (TcMGL) L-m ethionine y-lyase activity in human plasma according to supplemental PLP concentration. Histogram shows mean A / 2± SD in hours of both enzymes. Data are from three separate experiments, each in triplicate. Data of TcMGL are represented in black, those of PpMGL in grey. B) Normalized half-lives for each enzyme versus supplemented PLP concentration in plasma (pM). Symbols represent mean t / values obtained from each experiment. TcMGL data are shown with black circles, and PpMGL with grey triangles.Figure 4: Elimination of plasma methionine by TcMGL. Residual methionine in percentage of the initial value (15.8 pM) after incubation with 0.1 (triangle), 0.01 (square), or 0.001 (circle) U / ml of TcMGL in the absence of PLP supplementation. Error bars are the standard deviations of three different measurements.

[0227] Figure 5: Cytotoxicity against human cancer cell lines in culture under exposure to TcMGL at various concentrations of PLP. (A & B) Concentration-effect analysis including IC50 of TcMGL against the human colorectal carcinoma HT29 (A), and the ovarian carcinoma SKOV3 (B) cell lines in culture according to concentration of PLP in medium was assessed in cells growing in customized DMEM without B6 vitamer, and in DMEM without B6 vitamer supplemented with five PLP concentrations from 5 pM to 80 pM. Cells were exposed for 72 hours to TcMGL at nine increasing concentrations of enzyme ranging from 0.004 U / ml to 1.0 unit / ml. The boxed plots represent changes of IC50 (with the low 95% confidence interval) of TcMGL according to PLP concentration. Comparisons between plots in HT29 cells using the condition with 80 pM PLP as reference to those with 40 pM, 20 pM, 10 pM, 5 pM, and no additional PLP, resulted in p values of 0.061 (not significant, ns); 0.019; 0.003; 0.00013; and 1.34 x 10'8, respectively. Comparisons between plots obtained in SKOV3 cells using the condition with 80 pM PLP as reference to those with 40 pM, 20 pM, 10 pM, 5 pM, and no additional PLP, resulted in p values of 0.055 (ns); 0.015; 0.011; 0.001; and 0.0001, respectively.

[0228] Figure 6: Percent decrease in L-Methionine concentration in human plasma incubated with unloaded human erythrocytes and with a bioreactor of human erythrocytes loaded with the MGL from Thermobrachium celere. Incubation was done without supplemental PLP (-) or supplemented (+) with 20 pM PLP during 48 hours. L-Met was measured at tO, and 3, 24, and 48 hours from start of incubation. Fresh human plasma was supplemented with 1 mM L-Met (Note: concentrations naturally present in plasma are ~ 20 pM). Mean L-Met concentration measured immediately after start of incubation with the erythrocytes ( / 0) was 925 ± 24,9 pM. RBC (red blood cells) encapsulation conditions by hypotonic dialysis consisted in 800 pl packed RBCs + 200 pl TcMGL (8 mg / 97.6 Unit) + 20 pM PLP. Mean values ± SE were from three separate measurements.

[0229] Figure 7: Percent decrease in MGL activity within human erythrocytes loaded with the MGL from Thermobrachium celere (TcMGL) incubated in human plasma without supplemental PLP (-) or supplemented (+) with PLP (20 pM) during 48 hours. Activity was measured at tO, 24 hours, and 48 hours after start of incubation. (A-D) Assessment of activity with the MBTH method was conducted with supplemental PLP (Charts A and B), andwithout any supplemental PLP (Charts C and D). The mean baseline MGL activity in assays with supplemental PLP was 0.327 Unit / well at Z0, and 0.163 U / well at Z24h; in the absence of PLP in assay mixture mean activity was 0.117 Unit / well at Z0, and 0.068 U / well at Z24h. RBC TcMGL loading was done using encapsulation solution including 800 pl packed RBCs + 200 pl TcMGL (8 mg / 97.6 Unit) + 20 pM PLP.

[0230] EXAMPLE 1:

[0231] Materials & Methods

[0232] Chemicals and reagents

[0233] Chemicals were purchased from Sigma-Aldrich ((St. Louis, MA, USA). Penicillamine was from Interchim (Montlugon, France). Human plasma was obtained from the Centre de Transfusion Sanguine des Armees, 92140 Clamart, France.

[0234] Cloning, Enzyme Production and Purification

[0235] Codon-optimized constructs for the expression of TcMGL and PpMGL in Escherichia coir cloned into the pET28b(+) vector, were obtained from Genscript (www.genscript.com). The protein sequences are available at the Uniprot database (https: / / www.uniprot.org) under accession codes R7RTI8 for TcMGL and Pl 3254 for PpMGL. The proteins carried an N-terminal 6xHis tag and a thrombin cleavage site. Plasmids were transformed into E. coli T7 Express (DE3) competent cells (C2566I, https: / / www.neb.com). Cells were grown in Luria-Bertani medium at 37 °C and stirred at 250 r.p.m. until absorbance at 600 nm (A600 nm) reached 0.8. Temperature was lowered to 18 °C, then 1 mM isopropyl-P-d-thiogalactopyranoside (IPTG) was added to induce protein expression. Cells were grown overnight. They were harvested by centrifugation at 6,000 / for l 5 min and washed with 0.9% (w / v) NaCl. Cell pellets were frozen and stored at -80 °C. All purification steps were performed at 4 °C. Cell pellets were thawed and resuspended in a lysis buffer (50 mM HEPES (4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid) pH 7.5, 100 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 10 mM imidazole, 20 pM PLP) containing protease inhibitor (cOmplete Mini EDTA-free, Roche, Indianapolis, Indiana, USA). Cells were lysed with a One-Shot cell disruptor (Constant Systems Ltd, Daventry, UK) using a pressure of 29,000 psi . Extract was spun at 12,000 g for 10 min at 4 °C. The supernatant containing the N-terminally Histidine tagged recombinant protein was purified by immobilized metal affinity chromatography (IMAC). It was loaded onto a HisTrap HP column (Cytiva, Velizy-Villacoublay, France) connected to an NGC™ medium-pressure liquid chromatography system (Bio-Rad laboratories, Marnes-La-Coquette, France) and pre-equilibrated in lysis buffer.Unbound proteins were washed using the same buffer, and bound proteins were eluted with a linear gradient of 0 to 500 mM imidazole following the absorbance at 280 nm and 422 nm, the latter being typical for lysine-bound PLP. Purified proteins were concentrated using an Amicon Ultra- 15 device (Merck Millipore, Darmstadt, Germany) with a molecular weight cut-off of 10 kDa. TcMGL was further purified for crystallization trials and cytotoxicity assays by gel filtration chromatography on a Superdex 200 10 / 300 GL column (https : / / www.cytivalifesciences.com) in 50 mMHEPES pH 7.5, 100 mMNaCl, 0.5 mM TCEP and 20 pM PLP. Purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was calculated using the extinction coefficient s280nm of 26,360 M'1cm’1for TcMGL, and 24,870 M'1cm’1for PpMGL.

[0236] Enzyme Activity Assay, Michaelis-Menten kinetics

[0237] Enzymatic activity of PpMGL and TcMGL was measured with the MBTH (3-methyl-2-benzothiazolone hydrazone) endpoint colorimetric assay18. Each enzyme was pre-heated to 37°C, and PLP was added to a concentration of 200 pM in the reaction mixture. The reaction was started by adding the stock enzyme to substrate (L-Met, L-cysteine (L-Cys), L-homocysteine (L-HCy), or L-cystathionine in concentrations from 0.025 mM to 50 mM) in 100 mM potassium phosphate buffer pH 8.0, at 37°C. After incubating for 5 mn, the enzymatic reaction was terminated by adding trichloroacetic acid 5% (w / v). The reaction mixture was then incubated at 50°C with 0.02% MBTH and 0.5 M sodium acetate buffer pH 5.0. MBTH reacts with the a-ketoacid reaction product to generate a compound absorbing at 320 nm (r = 15,740 M1cm '). Results from catalysis of amino acids and substituted amino acid were fitted to the Michalis-Menten kinetics equation using GraphPad Prism (vlO.0.0., GraphPad Software, Boston, MA). One unit of enzyme is defined as the amount that catalyzes formation of 1 pmol of a-ketobutyrate per minute from L-Met.

[0238] Enzyme activity according to pH

[0239] Catalysis of L-Met was determined in MES (2-(A-morpholino)ethanesulfonic acid) or Tris (tris(hydroxymethyl)aminomethane) buffer solutions covering a pH range from 5.6 to 8.8 using the MBTH colorimetric assay. Results were expressed as a percentage of the highest activity measured.

[0240] Thermal Stability

[0241] TcMGL and PpMGL thermal stability was measured using the SUPR-DSF microplatebased spectrofluorometer device (Protein Stable Ltd., UK). Proteins were diluted to 0.5 mg / ml (11 pM) in 50 mM HEPES, 100 mM NaCl pH 7.5, with no additional PLP, or with 1 mM PLP.

[0242] 10 pL of each protein solution was dispensed in triplicate in 384-well, black polypropylenePCR-type microplates (Bio-Rad: #HSP3866). Microplates were sealed and centrifuged (1,000 g, 30 sec). The thermal ramp settings were configured using the SUPR-Suite software and ramped the temperature from 10°C to 105°C, at l°C / min rate. Intrinsic fluorescence spectra were measured from 310 nm to 420 nm and were excited at 280 nm. Fluorescence data were processed within the SUPR-Suite software. To quantify the spectral change, the barycentric mean was calculated between 310 nm and 390 nm for each fluorescence spectrum, and plotted against measured temperature values (melt curve). The first derivative of the melt curves was generated, and non-linear least-squared regressing fitting was used to fit Gaussian curves to the derivative data. From the fitted Gaussian curves, the mid-point of inflexion (Tm) and onset of melting (Tonset) are quantified (data not shown).

[0243] Thermal stability of TcMGL was also assessed by differential scanning calorimetry (DSC) at the Macromolecular Interaction measurement Platform (MIP, Gif-sur-Yvette, France), allowing the measurement of enthalpy variation with unfolding (AH). Purified TcMGL at 1 mg / ml (22 pM) in 50 mM Hepes buffer pH 7.5 with 100 mM NaCl was incubated in an Auto PEAQ DSC device (Malvern Panalytical, Palaiseau, France) with temperatures from 25 to 120°C, at 60°C / h pace. Measurements were done without PLP or in the presence of 200 pM PLP.

[0244] Isothermal titration microcalorimetry (ITC)

[0245] Preparatory removal of PLP. Cofactor was removed from purified PpMGL using D,L-penicillamine19. The enzyme was incubated with one-hundred-fold molar excess of D,L-penicillamine in HEPES 50 mM, NaCl 100 mM pH 7.5. PLP -linked D,L-penicillamine was removed by dialysis against buffer. Apo-TcMGL was retrieved from affinity purification since apo- and holo-forms of the enzyme were differentially eluted at distinct imidazole concentrations (data not shown). Procedures to collect enzyme fractions with low PLP occupancy were repeated until the absorbance ratio 422 nm / 280 nm was less than 25% of that observed for PLP -bound MGL.

[0246] ITC measurements. Experiments were performed with a PEAQ ITC isothermal titration calorimeter from MicroCai (Malvern Panalytical, Malvern, UK) at the MIP platform. The experiments were carried out at 37°C in HEPES 50 mM, NaCl 100 mM pH 7.5. Two pl of PLP solution at 800 pM were injected 19 times into the calorimeter cell containing 200 pL solution of 120 pM TcMGL, or 200 pM PpMGL, at intervals of 600 sec for TcMGL, and 180 sec for PpMGL, under stirring at 500 rpm. Time intervals between injections were adapted to each enzyme to allow complete return of thermogram to baseline. Three separate experiments were performed for each enzyme. Data analysis was performed using the MicroCai PEAQ-ITCsoftware provided by the manufacturer. Software uses the relationship between the heat generated by each injection and AT / (enthalpy change in kcal. Mol'1), Kr> (the dissociation constant in mol.L'1), n (the number of sites accessible for binding, per monomer), total protein concentration and free and total ligand concentration20. Variation of Gibbs free energy (AG) and entropy ( A,S') are calculated using the equation AG = -RTlnAV = AH - T S, where R is the gas constant, and T the temperature.

[0247] Crystallization and Structure Determination of TcMGL in Complex with Norleucine TcMGL was purified by two steps of column chromatography (IMAC, then sizeexclusion) and concentrated. Crystallization conditions were initially screened using Qiagen PEGS II suite (Courtaboeuf,_France), and then optimized manually. Crystal was obtained at 20°C in a hanging drop containing the protein solution (13.5 mg ml'1TcMGL, 100 mM NaCl, 50 mM HEPES pH 7.5, 0.5 mM TCEP, 1 mM PLP, 5 mM norleucine) mixed with an equal volume of well solution (100 mM MgCh, 100 mM MES buffer pH 6.5, 26% v / v polyethylene glycol 400) against which it was equilibrated. For data collection, 30% PEG 400 was used as a cryoprotectant and the crystal was flash-cooled in liquid nitrogen. Diffraction data were collected at 100 K on the PROXIMA 1 beamline at the SOLEIL synchrotron (Saint- Aubin, France) using EIGER-X 16M detector. Intensities were integrated using AUTOPROC21and XDS22and further reprocessed by STARANISO23. Data quality was assessed using the correlation coefficient CCI / 2 (data not shown). Initial phases were determined using the model structure produced in silico with Alphafold224as a search model for molecular replacement with PHASER25. Structures were manually inspected and corrected using Coot26, and refined with non-crystallographic symmetry restraints and TLS (i.e. translation, libration and screwrotation) using BUSTER 2.1027. Molecular graphics images were generated using PyMOL28.

[0248] Assessment of MGL Stability in human plasma

[0249] Each enzyme was diluted to 1 U / ml and incubated in human plasma obtained from two different healthy individuals, without PLP supplementation, or in plasma supplemented with eight increasing concentrations of PLP from 2 pM to 200 pM and then incubated in vials at 37°C under an atmosphere with 5% CO2. Samples were obtained at regular intervals up to 32 hours (twelve time points per experiment) for assessment of MGL activity, without addition of any supplemental PLP, using the MB TH colorimetric assay18. Three separate experiments were done, each in triplicate.

[0250] Enzyme activity half-life (6 / 2) data of PpMGL, and TcMGL was analyzed using R software environment v4.4.1. A linear regression was performed on the neperian logarithm of activity data. For each PLP concentration, half-lives were calculated using the formula 6 / 2 =ln(2) / k, where k is the speed coefficient29. Then, half-lives of enzyme activity were computed by modeling with order 1 equation and transformed according to the Box & Cox Method for Gaussian distribution30. Transformed normal half-life distribution was tested with the Shapiro-Wilk normality Test31. Data were modelized with locally weighted scatterplot smoothing (LOESS). Graphs were designed with ggplot2 R-Package v3.5.132. Statistical analysis of normalized 6 / 2 changes was done with Two-way Fisher analysis of variance (ANOVA) regarding factors defined as (1) enzyme type (i.e., PpMGL and TcMGL) and (2) concentration of PLP, in pooled data from three separate experiments each in triplicate. The effect on t / between the two MGLs was tested according to concentrations of PLP in plasma. Concentration of PLP and type of enzyme were tested for interaction during the Two-way ANOVA.

[0251] L-Met elimination from human plasma in vitro

[0252] Human plasma was incubated at 37°C with TcMGL at 0.001, 0.01, or 0.1 U / ml for 1 hour, and 8 hours. L-Met was measured using liquid chromatography coupled to tandem mass spectrometry (UPLC-MS / MS). For accurate quantification, a stable isotope internal standard (purchased from Eurisotop, Saint Aubin, France) was added to the sample before protein precipitation. Samples were first derivatized using the AccQ-Tag™ Ultra kit (Waters Corporation, Milford, MA, USA) according to manufacturer recommendations. Amino acid separation was performed with an Acquity™ UPLC system using a CORTECS™ UPLC Cl 8 column (1.6 pm, 2.1 x 150 mm) coupled to micro TQ-S tandem mass spectrometer operating with the MassLynx software (version 4.2, Waters Corporation, Milford, MA, USA). Internal standard for quantification was labelled on all carbons and nitrogen. The mean baseline concentration of L-Met ± SE in plasma from two different sources was 15.8 ± 0.9 pM. Measurements were in triplicate.

[0253] Results

[0254] Production, characterization and Michaelis-Menten kinetics of the recombinant L-methionine y-lyase from Thermobrachium celere

[0255] Expression and purification. A synthetic gene coding for TcMGL sequence was cloned into E. coli to express an N-terminal histidine tagged recombinant protein. The product was purified by immobilized metal affinity chromatography, providing an UV-visible absorption spectrum with a 422 nm peak characteristic of lysine-bound PLP (data not shown).

[0256] Purification of TcMGL was achieved to homogeneity using a second step of size-exclusion chromatography (data not shown). TcMGL was purified to homogeneity from a 0.25-liter scale cell pellet yielding approximately 15 to 20 mg of enzyme. The major band observed onSDS-PAGE is in accordance with the calculated molecular weight of 45.65 kDa. A second band with apparent MW close to 90 kDa is consistent with a dimer.

[0257] Michaelis-Menten kinetics. Catalytic activity of TcMGL was assessed at 37°C. It is limited to a,y-elimination of L-methionine and L-homocysteine, and a,P-elimination of L-cysteine (Table 1). Mean Kmfor L-methionine was 0.24 ± 0.05 mM, and specific activity was 9.86 ± 0.46 pmol mn'1mg'1. The enzyme had no detectable catalytic activity towards L-cystathionine or D-methionine. TcMGL possesses higher catalytic efficiency (kcat / Km) towards L-Met (125 M'1s'1), and L-Cys (2.26 M'1s'1) than PpMGL whose kcat / Kmwere 23.7 mM'1s'1and 0.88 mM'1s'1towards L-Met, and L-Cys, respectively. Differential catalytic efficiency (kcat / Km) ratios of both enzymes between L-Cys and L-Met (L-Cys / L-Met ratio) were 3.7%, and 1.8% for PpMGL, and TcMGL, respectively, which indicates that catalysis of L-Met predominates in TcMGL. Catalytic kinetics values of PpMGL found herein are similar to that previously reported33,34.

[0258] Influence of pH and temperature.

[0259] Activity on L-Met catalysis of TcMGL according to pH was maximum between 7.0 and 8.5 and decreased under pH values below 7 (Figure 1A). Optimum temperature of the TcMGL for L-Met catalysis was comprised between 70°C and 85°C (Figure IB). At 37°C, activity of the highly potent TcMGL was measured at approximately 10 % from optimum. The plot of activity of PpMGL according to temperature grossly paralleled that of the TcMGL with about 10°C shift towards lower temperatures. TcMGL exhibits marked thermostability. Using SUPR-DSF in the absence of PLP supplementation, Tm values were 79.6°C, and 73.7°C for TcMGL, and PpMGL, respectively ( Figure IB). Addition of 1 mM PLP increased Tm values of TcMGL to 88.3°C (AT = 8.7°C), and in lesser magnitude of PpMGL to 75.7°C (AT = 2.1°C). Similarly, differential scanning calorimetry (DSC, data not shown) assessment of TcMGL thermostability found Tm values of 84.1°C in the absence of additional cofactor, and 91.0°C with addition of 200 pM PLP (AT = 6.9°C). Under these conditions, enthalpy of unfolding was 154 kcal / mol in the absence of PLP, and 210 kcal / mol in the presence of additional PLP (AH = 56 kcal / mol), which demonstrates greater resistance of TcMGL to denaturation at high temperature in the presence of the cofactor.

[0260] Isothermal titration calorimetry (ITC) data for binding of PLP to apoenzyme.

[0261] The dissociation constants (KD) of PLP binding to apo-MGL were slightly different in both enzymes. KD of TCMGL was 0.93 ± 0.25 pM, and that of PpMGL was 0.58 ± 0.16 pM.Unexpectedly, results of the reaction thermodynamics indicate a radically different mechanism of cofactor binding to apoenzyme between the two proteins (Figure 2). Both proteins bind to cofactor with similar Gibbs free energy change (AG CMGL = -8.56 kcal / mol, and AGPPMGL = -8.84 kcal / mol). However, binding of apo-PpMGL to PLP is an exothermic reaction with negative change in enthalpy (AH = -12 ± 0.58 kcal / mol), and small entropy change (TAS = -3.16 kcal / mol), whereas binding of PLP to apo-TcMGL is an endothermic reaction with positive enthalpy (AH = 9.01 ± 0.38 kcal / mol), and positive entropy change (TAS = 17.6 kcal / mol). KD and thermodynamic parameter values were in conformity in all three experiments performed for each enzyme.

[0262] Crystal structure of TcMGL in complex with the inhibitor norleucine.

[0263] To approach structurally the interaction between enzyme and cofactor by X-Ray crystallography, we first grew crystals of PLP-containing TcMGL. Yellow crystals were obtained, but they diffracted X-rays with too low resolution. Conversely, crystals grown in the presence of norleucine, a known inhibitor of methioninases previously used for crystallography35, allowed TcMGL structure determination at 2.6 A resolution. Data collection and refinement statistics were obtained (data not shown).

[0264] Overall structure. The final model is a tetramer (data not shown), as for previously published crystal structures of MGLs. Each subunit contains one molecule of both cofactor and norleucine. There are four monomers per asymmetric unit, named A, B, C and D. Catalytic dimers are constituted either of subunits from two different asymmetric units (A and A*, or B and B*), or from subunits from four asymmetric units (B and C#, or C’ and B”).

[0265] Active site with PLP and norleucine. The active site of TcMGL has been observed (data not shown). Residues surrounding the active site of the enzyme belong to two neighbouring subunits from the catalytic dimer. The residues that make direct hydrogen bonds with the PLP cofactor belong mainly to the same monomer (data not shown), namely Asp 187, Ser209, and Thr211. Molecule A also provides Tyrl 14 which ring makes a it stacking with that of PLP. In addition, two residues from the neighbouring monomer of the same catalytic dimer (molecule B, dark grey, left), namely Tyr59 and Arg61, interact with the phosphate group of PLP. All the above-mentioned residues have identical counterparts in PpMGL. Therefore, TcMGL and PpMGL crystal structures do not point to any active site residue directly interacting with PLP that could bring clues to explain the observed differences in PLP binding between the two enzymes.Crystal structure shows open and closed conformations of a subunit. Depending on subunits, the region containing residues Q350 to L374 (named from here “mobile domain”), has two possible conformations. In half of the subunits (monomers B, D on data not shown), this domain is close to the active site that contains the competitive inhibitor and the cofactor. In the other half (monomers A, C), it is more open, providing a larger space at the entrance of the active site. With regards to these open and closed conformations, the structure contains two types of tetramers. The catalytic dimer can be homogeneous, containing two open subunits, or two closed ones. Conversely, other tetramers contain heterogeneous catalytic dimers, which contain one open and one closed subunit (data not shown). Several intra-monomer interactions stabilize the closed conformation (data not shown). The side chain of Tyr360 from the mobile domain is hydrogen bonded to the main chain carbonyl of Leull3, and to the main-chain nitrogen of Thrll2. In addition, its ring makes a TI stacking with that of Phell8 (not shown). His357 from the mobile domain is H bonded to the main chain carbonyl atoms from Leull3 and Asnl61, and to the side chain oxygen atom of Thr356. Asnl61 makes H bonds with PLP, norleucine, and the side chain of Arg376 (data not shown). The two observed positions of the loop result in marked differences for the access to the active site (data not shown). In the closed conformation, the access channel is very narrow, whereas it forms a larger cleft in the open conformation.

[0266] The high stability of TcMGL in human plasma is greatly increased by PLP.

[0267] Mean baseline half-lives of TcMGL and PpMGL in plasma at 37°C without additional PLP (i.e., in samples containing approximately 0.05 pM PLP) were 9.2 h ± 0.8 hour and 6.7 ± I.9 hour, respectively. Pseudomonas putida MGL half-life increased slightly with increasing PLP concentration (Figure 3A): the highest mean half-life was 12.1 hours, i.e. 1.8-fold the value in the absence of supplemental PLP. Instead, mean TcMGL half-life in plasma increased gradually with increasing cofactor concentration, from 1.2- to 7.3-fold from baseline (i.e., from II.3 to 66.7 hours), in much greater magnitude than that achieved by PpMGL in same conditions. Normality test of transformed 6 / 2 did not exclude that data follows a normal distribution (Shapiro-Wilk Test, p = 0.143). Figure 3B shows a plot of the normalized halflives of TcMGL (black) and PpMGL (grey) according to supplemental PLP concentration. Two-way multivariate ANOVA of transformed data did not disclose any interaction between types of enzyme, and PLP concentration (p = 0.116). Statistical analysis demonstrated highly significant effect on plasma half-lives of both PLP concentration (p = 2.23 x 10'7) and type ofenzyme (i.e., PpMGL vs TcMGL) over the concentration range of supplemental PLP explored, ranging from none to 200 pM (p = 1.87 x 10'15).

[0268] From these findings, we concluded that plasma half-life changes with increasing concentration of cofactor are due to enzyme stabilization in active holo-form. Importantly, the gradual increase in TcMGL half-lives was observed with PLP concentrations from 2 to 20 pM, which can effectively be attained in mouse plasma in vivo following parenteral administration of pyridoxine (PN), an unphosphorylated B6 vitamer (data not shown).

[0269] TcMGL eliminates of L-methionine from human plasma

[0270] Initial methionine concentration measured in human plasma was 15.8 pM. L-Met elimination from plasma of normal subjects effectively occurred at all the concentrations of TcMGL tested, from 0.001 U / ml to 0.1 U / ml, and augmented with increasing amounts of enzyme (Figure 4). Elimination occurred rapidly; it continued between the first and eighth hour from start, indicating persisting activity of the enzyme during the time lapse.

[0271] EXAMPLE 2:

[0272] Material & Methods

[0273] Concentration-effect analysis including IC50 of TcMGL against the human colorectal HT29 and the human ovarian SKOV3 carcinoma cell lines was performed in cells growing in customized Dulbecco's modified Eagle medium (DMEM) without B6 vitamer, and in DMEM without B6 vitamer supplemented with five PLP concentrations ranging from 5 to 80 pM, added with 10 % fetal bovine serum and antibiotics. Cells were exposed for 72 h to TcMGL at nine increasing concentrations of enzyme ranging from 0.004 U / ml to 1.0 U / ml in 96-well microplates at 37 °C in an atmosphere with 5 % CO2. For accurate reproducibility, cell viability was assessed according to two different methods. In HT29 cells, it was determined using Crystal violet staining, by measuring the absorbance at 590 nm with a Tecan Infinite M200 Pro microplate reader. In SKOV3 cells, viability was assessed with resazurin by measurement of the fluorescence of newly formed resorufin at 544 nm / 590 nm (excitation / emission) using a Fluoroskan Ascent microplate reader. Experiments were performed in duplicate. Cell growth inhibition data were analyzed according to the median-effect principle for concentration-effect analysis. Dose-effect plots including 50 % inhibitory concentrations (IC50) were obtained with the CalcuSyn v2 software. Statistical analysis of cytotoxicity of the TcMGL against cancer cells growing in vitro in the presence of various concentrations of PLP used a model built withgeneralized linear model function in R including concentration of PLP and TcMGL as variables, and fraction of cells affected (fa) as outcome

[0274] Results

[0275] Cytotoxicity against cancer cells in vitro. Influence of PLP concentration on cell viability.

[0276] TcMGL exerted potent cytotoxicity against the human colorectal HT29 and ovarian SKOV3 carcinoma cell lines growing in cell culture medium. Under this condition which, contrary to plasma, does not restrain PLP availability, cytotoxicity significantly increased in both cell lines with rising concentrations of PLP (Figure 5). The striking influence of PLP concentration regarding both stability of the TcMGL in plasma, and potency of the cytotoxic effect against cancer cells in culture, depends on two intricately linked mechanisms. One is the need for high concentration of PLP according to the enzyme-ligand equilibrium equation, which postulates the requirement of great amounts of PLP, up to several folds the KD for binding of cofactor, to achieve high TcMGL active site occupation rates; the other is the unprecedented high intrinsic retention strength of PLP by TcMGL.

[0277] EXAMPLE 3:

[0278] Material & Methods

[0279] With the aim of preserving MGL function for long periods of time in vivo, we constructed a heterologous bioreactor by encapsulation of the TcMGL in human erythrocytes. These carrier cells internalize L-methionine, and their lifespan does not differ from, or is only slightly shorter than, that of intact cells, a characteristic that allows maintenance of therapeutic blood levels of loaded cells for prolonged periods of time. Moreover, carrier erythrocytes prevent loaded proteins from being released into the extracellular environment and are progressively degraded in the organism by the mononuclear phagocytic system without producing toxic substances.

[0280] Human blood was obtained from healthy adults and collected in heparin. Recombinant TcMGL was loaded into erythrocytes by sequential hypotonic dialysis, isotonic resealing, and reannealing. Erythrocytes were first washed twice at 4°C in Solution A (154 mM NaCl, 10 mM HEPES-NaOH, 5 mM glucose, 20 pM PLP; pH 7.4). Cells (800 pl of packed erythrocytes) were then suspended at 50% erythrocyte volume fraction in encapsulation solution consisting of Solution A supplemented with 200 pl TcMGL (8 mg / 97.6 Unit). Hypotonic hemolysis was performed by dialysis of 1 ml of cell suspension in a cellulose tube (14 kDa cutoff) versus 50ml of 60 mOsm hypotonic solution consisting in 10 mM NaHCCL, 10 mM NaH^PCU, 20 mM glucose, 2 mM ATP, 3 mM glutathione, 20 pM PLP; pH 7.4, for 90 minutes at 4°C. At the end of dialysis, osmolality of cell suspensions was approximately 90 mOsm. Erythrocyte resealing and reannealing were accomplished by incubating 10 volumes of cell suspension in one volume of hypertonic solution (approximately 3900 mOsm) of 100 mM inosine, 100 mM sodium pyruvate, 33 mM NaH2PO4, 1.66 M KC1, 190 mM NaCl, 10 mM glucose, 20 mM ATP, 4 mM MgCh, 20 pM PLP; pH 7.4 for 25 minutes at 37°C. Finally, after three washes in Solution A at 4°C, cells were suspended in this solution and used for experiments. Unloaded erythrocyte suspensions (i.e., cells subjected to the encapsulation procedure without enzyme) were used as control. Cells unloaded and loaded with TcMGL were incubated at 0.5% cell volume fraction in human plasma with additional L-m ethionine at 1 mM at 37°C in an atmosphere containing 5% CO2, not supplemented or supplemented with PLP at 20 pM. Potency and duration of L-methionine elimination in human plasma was assessed by measuring residual L-Met using LC-MS in cell supernatant at tO and after 3 hours, 24 hours, and 48 hours of incubation. Enzymatic activity was measured in loaded cells immediately after encapsulation ( / 0) and in samples obtained at various times after loading (t3h; / 24h; and / 48h) from cells incubated during 48 hours in human plasma. Assays for enzymatic activity in erythrocytes using the MBTH colorimetric assay were done in the absence and in the presence of supplementary PLP at 20 pM.

[0281] Results

[0282] Construction of a heterologous bioreactor with durable MGL activity by encapsulation of the TcMGL in human Erythrocytes.

[0283] Human erythrocytes encapsulated with TcMGL retain enzyme activity for long periods of time in vitro and form a bioreactor with sustained MGL function that strongly eliminates L-methionine from plasma in the presence and in the absence of supplementary cofactor (Figure 6).

[0284] We successfully built a bioreactor with durable catalytic MGL function (Figure 7) by encapsulation of the TcMGL into human erythrocytes. Under present experimental conditions, the intracellular erythrocyte environment does not prevent preservation of MGL activity. The intense and durable elimination of high concentrations of L-methionine from medium highlights the robustness of the bioreactor whose MGL function is retained in vitro. Plasma supplemented with 20 pl PLP had a slightly higher rate of L-methionine elimination than plasma without PLP. This is the consequence of the partial cofactor uptake by MGL-loadederythrocytes, resulting in higher saturation levels of the encapsulated enzyme. It suggests the possibility of modulating the bioreactor’s MGL activity in vivo by supplying cells with B6 vitamers.

[0285] Based on the naturally occurring rapid decline of PLP concentration in red blood cells, sustained MGL function within loaded erythrocytes in the long term is likely to result from the unprecedented high intrinsic retention strength of PLP by the MGL from Thermobrachium celere.

[0286] Loss of TcMGL activity within encapsulated RBCs in plasma ranged between 43% and 56% from baseline (tO) during the first 24 hours of incubation. Loss rate decreased to 3% to 19% during the next 24 hours of incubation (from the 24thto the 48thhour). Decrease in enzyme loss rate is likely to result in part from time-dependent erythrocyte permeability repair after sequential hypotonic dialysis, isotonic resealing, and reannealing procedures.

[0287] EXAMPLE 4:

[0288] Material & Methods

[0289] Groups of 3 to 4 BALB / c 6-week-old male and female mice caged separately and fed with standard diet and water ad libitum were subjected to four experimental administration schedules (Table 2). One control group including 7 animals (4 males; 3 females) received normal saline i.v., and i.p. at tQ and / 24h; one group of 8 animals (4 males; 4 females) received two consecutive injections of TcMGL at 10 U i.v. together with pyridoxine (PN) 300 mg / kg i.p. at Z0, and / 24h; one group of 8 animals (4 males; 4 females) received one single injection of TcMGL at 10 U i.v. and pyridoxine (PN) at 300 mg / kg i.p. at / 0; and one group of 8 animals (4 males; 4 females) received one single injection of TcMGL at 50 U i.v. with pyridoxine (PN) 300 mg / kg i.p. at tQ. All animals were left fasting for six hours before being euthanized and sampled at Z48h. The treatment was well tolerated; only minimal decrease in activity was observed in treated animals without any significant manifestation of pain or discomfort. Blood was collected from all animals and plasma was separated by centrifugation and frozen immediately at -80°C. Proteins were precipitated with sulfosalicylic acid and then derivatized with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC). Amino acids were separated on a gradient of acetonitrile and formic acid. Measurements of L-Met were performed using liquid chromatography (UPLC).

[0290]

[0291] Table 2, Plasma L-methionine concentration in mice treated with intravenous L-methionine y- lyase from Thermobrachium celere (TcMGL) accompanied by intraperitoneal pyridoxine (PN).

[0292] 1Groups 1 and 2 were injected daily during two consecutive days (tO and t24h), and groups 3 and 4 were injected only once at tO.2Groups of 3 or 4 six-week-old BALB / c males (M) and females (F) fed with standard diet and water ad libitum were caged and treated separately. Results among animals of different sex were similar; these are grouped in the Table.3Samples of plasma were obtained after sacrifice in the 48thhour after initiation of the experiment; i.e., animals were sampled (a) 24 hours after the last injection in Groups 1, and 2, and (b) 48 hours after the last injection in Groups 3, and 4.

[0293] Results

[0294] The methioninase from Thermobrachium celere (TcMGL) strongly depletes L- methionine (L-Met) from mouse plasma for long periods of time.

[0295] The TcMGL depleted L-Met from plasma to 2.17 pmol / 1 (4.1 % of mean control value) 24 hours after the second of two consecutive daily injections of the lowest dose (10 U / day). However, after 48 hours of a single injection at tQ of the same low dose (10 U), L-Met concentration recovered to 69 % of mean control value. TcMGL at the highest dose of 50 U administered in a single injection at tQ reduced L-Met plasma concentration to 6.61 pmol / 1 (12.4 % of mean control value) 48 hours after the injection. These results indicate that the enzyme remains active in holo-form in mouse plasma for periods of time between 24 hours and approximately 48 hours. Residual MGL activity in plasma, plasma pharmacokinetics of theTcMGL at various doses, and the influence of PN on enzyme activity will be assessed in further experiments.

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[0351] Ferchaud N., Kopecny D., Penna M., Boyer A., Hentati S., Pontoizeau C., Desterke C., Krystof V., Machover D., Briozzo P. The methioninase from the alkalithermophile Thermobrachium celere possesses suitable properties for treatment of cancer, Int. J. Biol. Macromol. 360(2): 147876, 2025

Claims

CLAIMS:

1. A polypeptide comprising an amino acid sequence as set forth by SEQ ID NO: 1 or a function-conservative variant thereof.

2. A nucleic acid encoding an amino acid sequence comprising SEQ ID NO: 1.

3. A vector comprising the nucleic acid according to claim 2.

4. A host cell comprising the vector according to claim 3.

5. A delivery system comprising the polypeptide according to claim 1.

6. The delivery system according to claim 5 is an erythrocyte, a nanoparticle, a liposome (for example a polymeric liposome or a lipidic liposome), a Virus-Like-Particle (VLP), a dendrimer, a micelle, a nanoemulsion, extracellular vesicle, nanosuspension or a bioreactor.

7. The delivery system according to claim 5 is an erythrocyte.

8. The delivery system according to claim 5 is a pegylated nanoparticle.

9. A drug conjugate comprising the polypeptide according to claim 1 linked to a heterologous moiety.

10. A polypeptide according to claim 1 or a drug conjugate according to claim 9 for use in a method for treating a cancer.

11. A method of treating a cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a polypeptide according to claim 1 or a drug conjugate according to claim 9.

12. The method according to claim 11, wherein the cancer is selected from the group consisting of but not limited to: neoplasm, malignant; carcinoma; carcinoma undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous feature; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmicastrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal sarcoma; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified nonHodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia, pleural cancer or colorectal cancer.

13. i) A polypeptide according to claim 1 or a drug conjugate according to claim 9 and ii) a classical treatment as a combined preparation for use by simultaneous, separate or sequential administration in the treatment of cancer and / or metastatic cancer in a subject in need thereof.

14. The combined preparation according to claim 13 wherein, the classical treatment refers to targeted therapy, radiation therapy, immunotherapy, cyclin-dependent kinase inhibitors (CDKi), hormonal therapy or chemotherapy.

15. The combined preparation according to the claim 13 wherein the classical treatment is fluoropyrimidines combined with folates and B6 vitamers in tandem.

16. A pharmaceutical composition comprising polypeptide according to claim 1 or a drug conjugate according to claim9, and a pharmaceutically acceptable carrier for use in the treatment of a cancer.

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