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WO2025233381A1 - Enzymes qui éliminent les nucléosides de pyrimidine et/ou de purine pour le traitement du cancer - Google Patents

Enzymes qui éliminent les nucléosides de pyrimidine et/ou de purine pour le traitement du cancer

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Publication number
WO2025233381A1
WO2025233381A1 PCT/EP2025/062447 EP2025062447W WO2025233381A1 WO 2025233381 A1 WO2025233381 A1 WO 2025233381A1 EP 2025062447 W EP2025062447 W EP 2025062447W WO 2025233381 A1 WO2025233381 A1 WO 2025233381A1
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WO
WIPO (PCT)
Prior art keywords
enzyme
nucleic acid
pas
acid molecule
vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/062447
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English (en)
Inventor
Katerina ROHLENOVÁ
Jakub ROHLENA
Arne Skerra
Martin Schlapschy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute Of Biotechnology Of Czech Academy Of Sciences
Technische Universitaet Muenchen In Vertretung Des Freistaates Bayern
Original Assignee
Institute Of Biotechnology Of Czech Academy Of Sciences
Technische Universitaet Muenchen In Vertretung Des Freistaates Bayern
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Filing date
Publication date
Application filed by Institute Of Biotechnology Of Czech Academy Of Sciences, Technische Universitaet Muenchen In Vertretung Des Freistaates Bayern filed Critical Institute Of Biotechnology Of Czech Academy Of Sciences
Publication of WO2025233381A1 publication Critical patent/WO2025233381A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2497—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing N- glycosyl compounds (3.2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/02—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2) hydrolysing N-glycosyl compounds (3.2.2)
    • C12Y302/02001—Purine nucleosidase (3.2.2.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to (a) an enzyme that eliminates pyrimidine and/or purine nucleosides in vivo, (b) a nucleic acid molecule encoding the enzyme of (a), and/or (c) a vector expressing the nucleic acid molecule of (b) for use in treating cancer.
  • Cancer cells need to proliferate in order to form tumors.
  • Proliferating cancer cells require nucleotides, the building blocks of nucleic acids, in particular DNA and RNA (Bajzikova, M., et al. (2019). Cell Metabolism 29, 399-416. elO; Sullivan, L.B., et al. (2015). Cell 162, 552-563).
  • the present invention relates to the therapeutic application of enzymes that cleave and eliminate nucleosides in vivo, in this way denying this source of nutrients to cancer cells. Importantly, this takes place in the extracellular milieu, such as in the blood and in the interstitial space. According to the best knowledge of the inventors the direct elimination of extracellular nucleotides in vivo by enzymes has not been considered before as treatment option for cancer.
  • the present invention also relates to the use of (a) an enzyme that eliminates pyrimidine and/or purine nucleosides in vivo, (b) a nucleic acid molecule encoding the enzyme of (a), and/or (c) a vector expressing the nucleic acid molecule of (b) for the manufacturing of a medicament for the treatment of cancer.
  • the present invention furthermore relates to a method of treating a subject having or being at risk of having cancer by administering to the subject a therapeutically effective amount of (a) an enzyme that eliminates pyrimidine and/or purine nucleosides in vivo, (b) a nucleic acid molecule encoding the enzyme of (a), and/or (c) a vector expressing the nucleic acid molecule of (b).
  • an enzyme is a biological catalyst that is a protein.
  • An enzyme accelerates a (bio)chemical reaction, usually in a living organism or cell.
  • the enzyme of the first aspect of the invention is capable of eliminating pyrimidine and/or purine nucleosides in vivo.
  • the elimination of pyrimidine and/or purine nucleosides means that pyrimidine and/or purine nucleosides are enzymatically converted by the enzyme, such that they are no longer usable; i.e. they can no longer be taken up by a cell and incorporated into and/or form nucleic acid molecules (RNA or DNA).
  • the "in vivo” requirement of the enzyme means that the enzyme, after it has been administered to the subject to be treated, is capable of eliminating pyrimidine and/or purine nucleosides within the body of the subject.
  • a pyrimidine (desoxy)nucleoside consists of a nucleobase and the sugar ribose or deoxyribose, wherein the nucleobase is a pyrimidine, preferably selected from uracil, thymine and cytosine.
  • the pyrimidine nucleoside is preferably uridine, thymidine, deoxythymidine, cytidine or deoxycytidine.
  • Nucleosides in contrast to nucleotides do not comprise a phosphate group.
  • a (desoxy)nucleoside consists of a nucleobase (also termed a nitrogenous base) and a five-carbon sugar (ribose or deoxyribose) whereas a (desoxy)nucleotide is composed of a nucleobase, a five-carbon sugar, and one (or more) phosphate groups.
  • the enzyme may, for example, be a hydrolase, phosphorylase or a reductase.
  • Hydrolases will be further described herein below, phosphorylases catalyze the cleavage of the N-glycosidic bond to the free nucleobase and pentose-l-phosphate, while reductases are enzymes that catalyze chemical reductions.
  • the subject to be treated is preferably a mammal, more preferably a primate and most preferably human.
  • nucleic acid molecule in accordance with the present invention includes DNA, such as cDNA or double or single stranded genomic DNA and RNA.
  • DNA deoxyribonucleic acid
  • DNA means any chain or sequence of the chemical building blocks of adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide or nucleic acid bases, that are linked together on a deoxyribose sugar backbone.
  • DNA can have one strand of (deoxy)nucleotides or two complimentary strands which may form a double helix structure.
  • RNA ribonucleic acid
  • A adenine
  • G guanine
  • C cytosine
  • U uracil
  • RNA typically has one strand of nucleotides, such as mRNA. Included are also single- and double-stranded hybrid molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA.
  • the nucleic acid molecule may also be modified by many means known in the art.
  • Non-limiting examples of such modifications include methylation, "caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • Nucleic acid molecules in the following also referred to as polynucleotides, may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators.
  • the polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.
  • nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers.
  • Such nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2'-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, Chem Biol 2001, 8: 1).
  • LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon.
  • nucleic acids containing modified bases for example thio-uracil, thio-guanine and fluoro-uracil.
  • a nucleic acid molecule typically carries genetic information, including the information used by cellular machinery to make proteins and/or polypeptides by way of transcription and/or translation.
  • the nucleic acid molecule of the invention may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- noncoding regions, and the like.
  • the nucleic acid molecule according to the invention encodes an enzyme that eliminates pyrimidine and/or purine nucleosides as described herein above.
  • the nucleic acid molecules as described herein above may be designed for direct introduction or for introduction via vesicles, such as liposomes into a cell.
  • Liposomes have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. Liposomal cell-type delivery systems have been used to effectively deliver nucleic acids, such as siRNA in vivo into cells (Zimmermann et al. (2006) Nature, 441:111-114). Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse with the cell membrane.
  • Non-cationic liposomes although not able to fuse as efficiently with the cell membrane, are phagocytosed by macrophages and other cells in vivo.
  • Exosomes are lipid packages which can carry a variety of different molecules including RNA (Alexander et al. (2015), Nat Commun; 6:7321). The exosomes including the molecules comprised therein can be taken up by recipient cells. Hence, exosomes are important mediators of intercellular communication and regulators of cellular function. Exosomes are useful for diagnostic and therapeutic purposes since they can be used as delivery vehicles, e.g. for contrast agents or drugs.
  • vector in accordance with the invention means preferably a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering which carries the nucleic acid molecule of the invention.
  • the nucleic acid molecule of the invention may, for example, be inserted into several commercially available vectors.
  • Non-limiting examples include prokaryotic plasmid vectors, such as of the pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2- dhfr, plZD35, pLXlN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (No
  • the nucleic acid molecules to be inserted into the vector can e.g. be synthesized by standard methods or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid encoding sequences can also be carried out using established methods.
  • Transcriptional regulatory elements parts of an expression cassette
  • These elements comprise regulatory sequences ensuring the initiation of transcription (e. g., promoters, such as naturally- associated or heterologous promoters and/or insulators; see above), translation initiation codon, ribosomal binding site (RBS), internal ribosomal entry sites (IRES) (Owens, Proc. Natl.
  • polypeptide/protein or fusion protein of the invention is operatively linked to such expression control sequences allowing expression in prokaryotes or eukaryotic cells.
  • the vector may further comprise nucleic acid sequences encoding secretion signals as further regulatory elements. Such sequences are well known to the person skilled in the art.
  • leader sequences capable of directing the expressed polypeptide to a cellular compartment or to the extracellular milieu may be added to the coding sequence of the polynucleotide of the invention.
  • Such leader sequences are well known in the art.
  • the vector comprises a selectable marker.
  • selectable markers include genes conferring resistance to neomycin, ampicillin, hygromycin, and kanamycin.
  • Specifically designed vectors allow the shuttling of DNA between different hosts, such as bacterial to fungal cells or bacterial to animal cells (e. g. the Gateway system available from Invitrogen).
  • An expression vector according to this invention is capable of directing the replication, and the expression, of the enzyme according to this invention.
  • baculoviral systems or systems based on vaccinia virus or Semliki Forest virus can be used as eukaryotic expression systems for the nucleic acid molecules of the invention.
  • Cancer is an abnormal malignant growth of tissue that possesses no physiological function and arises from uncontrolled, usually rapid, cellular proliferation.
  • the cancer is preferably selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulva cancer, bladder cancer, salivary gland cancer, pancreatic cancer, thyroid cancer, kidney cancer, lung cancer, cancer concerning the upper gastrointestinal tract, colon cancer, colorectal cancer, prostate cancer, squamous-cell carcinoma of the head and neck, cervical cancer, glioblastomas, malignant ascites, lymphomas and leukemias.
  • lung cancer Examples 13 and 14
  • pancreatic cancer Example 14 are preferred since these cancer types are illustrated by the appended examples.
  • the treatment of cancer comprises the curative and the disease preventive (i.e. prevention) treatment of cancer and is preferably the curative treatment; i.e. the treatment of a subject that has cancer.
  • the curative treatment may slow down or stop cancer progression or may even results in cancer remission in the subject.
  • the UPP1 enzyme is an intracellular component of a metabolic pathway that utilizes intracellular uridine inside the cancer cell for energy production. Hence, it is one of the routes to internally metabolize uridine taken up from the environment to support tumor growth.
  • the other major intracellular route of uridine metabolism is the interconversion into other pyrimidine nucleotides. Indeed, all these various pathways of uridine utilization are pro-tumorigenic.
  • the enzyme according to the present invention degrades uridine in the extracellular space before it can enter the cancer cell, denying it to the pro-tumorigenic intracellular uridine metabolism.
  • uridine is degraded extracellularly, the cancer cell cannot take up uracil, the product of hydrolysis of uridine, as uracil cannot be transported across the cell membrane.
  • the inability of extracellular uracil to rescue cancer cell proliferation is demonstrated in Example 15 and Fig 11.
  • the enzyme is a pyrimidine and/or purine hydrolase.
  • a pyrimidine and/or purine hydrolase catalyzes (EC 3.2.2.-) the hydrolysis of the N-glycosidic bond in ribonucleosides. They are generally Ca 2+ -containing metalloenzymes (Singh et al. (2017), Protein Sci.; 26(5): 985-996).
  • the pyrimidine and/or purine hydrolase is an inosine-uridine preferring nucleoside hydrolase (IUNH) belonging to the class of purine nucleosidases (EC 3.2.2.1).
  • IUNH inosine-uridine preferring nucleoside hydrolase
  • the enzyme may catalyze the hydrolysis of all of the commonly occurring purine and pyrimidine nucleosides into ribose and the associated base, but has a preference for inosine and, preferably, uridine as substrates.
  • the enzyme has been identified in protozoans and is important for these organisms, which are deficient in the de novo synthesis of purines, to salvage the purine nucleosides from the host.
  • the inosine-uridine preferring nucleoside hydrolase (IUNH) has a kcat/Kivi value for uridine hydrolysis of 10 4 M 1 s 1 or higher.
  • the kcat/K M value (also called k ca t/K M ratio) is a measure of the catalytic efficiency of an enzyme.
  • the catalytic constant k ca t is the turnover number and describes how many substrate molecules are transformed into products per unit time by a single enzyme.
  • the Michaelis constant K M describes the affinity of the substrate to the active site of the enzyme and can be determined from the substrate saturation in Michaelis/Menten kinetics, for example.
  • the k ca t/K M value indicates how effective the enzyme is on that particular substrate under physiological conditions. The higher this value the more specific the enzyme is for that substrate, in particular under conditions of non-saturating substrate concentration. This is because a high value of k ca t and a low value of K M are expected for the best substrates.
  • the enzyme comprises the amino acid sequence of SEQ ID NO: 1 or 2 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto;
  • the nucleic acid molecule comprises (ii-a) the nucleotide sequence of SEQ ID NO: 3, 4, 9 or 10 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto (wherein said polypeptide comprises or displays IUNH activity), (ii-b) a nucleic acid molecule that hybridizes (preferably under stringent conditions, such as 50% formamide and 45°C) with a nucleic acid molecule comprising or consisting of a nucleic acid sequence complementary to the nucleotide sequence of SEQ ID NO:
  • SEQ ID Nos 1 and 2 are the inosine-uridine preferring nucleoside hydrolases (IUNH) from Crithidia fasciculate and Leishmania major, respectively.
  • SEQ ID Nos 3 and 4 are the natural nucleic acid sequences that encode the amino acid sequences of SEQ ID Nos 1 and 2. Synthetic genes for these two enzymes with codon usage optimized for E. coll are shown in SEQ ID Nos 9 and 10, for example.
  • IUNH enzymes from related parasite species of humans or mammals.
  • these enzymes normally reside in the reducing cytoplasm and, as it often happens for cytoplasmic enzymes, they carry unpaired cysteine side chains. These free thiol groups are prone to oxidative modification in vitro and/or in vivo such as in blood or in extracellular fluids.
  • particularly reactive and/or exposed cysteine residues are replaced by more inert side chains, for example, the cysteine residue at position 301 in the IUNH from Leishmania major may be replaced by glycine.
  • Nucleotide and amino acid sequence analysis and alignment in connection with the present invention are preferably carried out using the NCBI BLAST algorithm (Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402).
  • BLAST can be used for nucleotide sequences (nucleotide BLAST) and for amino acid sequences (protein BLAST).
  • the skilled person is aware of additional suitable programs to align nucleic acid or protein sequences.
  • sequence identities of at least 80% are with increased preference at least 90% identical, and more preferred at least 95%.
  • the enzyme, nucleic acid molecule and/or vector is formulated into a pharmaceutical composition and said pharmaceutical composition is used for treating cancer.
  • the enzyme, nucleic acid molecule or vector of the invention can be formulated as a pharmaceutical composition.
  • the term "pharmaceutical composition” relates to a composition for administration to a patient, preferably a human patient.
  • the pharmaceutical composition of the invention comprises the compounds recited above. It may, optionally, comprise further molecules capable of altering the characteristics of the compounds of the invention thereby, for example, stabilizing, modulating and/or activating their function.
  • the composition may be in solid, liquid or gaseous form and may be, inter alia, in the form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s).
  • the dosage regimen of a pharmaceutical composition can be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The therapeutically effective amount for a given situation will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician. Generally, the regimen as a regular administration of the pharmaceutical composition, in particular biopharmaceuticals, should be in the range of 1 pg to 5 g units per day.
  • a more preferred dosage is in the range of 0.01 mg to 1000 mg, even more preferably 0.1 mg to 500 mg and most preferably 1 mg to 100 mg per day.
  • said compound comprises or is an nucleic acid molecule, such as an siRNA
  • the total pharmaceutically effective amount of pharmaceutical composition administered will typically be less than about 75 mg per kg of body weight, such as for example less than about 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg of body weight.
  • the amount will be less than 2000 nmol of nucleic acid molecule per kg of body weight, such as for example less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075 or 0.00015 nmol per kg of body weight.
  • the length of treatment needed to observe changes and the interval following treatment for responses to occur vary depending on the desired effect.
  • the particular amounts may be determined by conventional tests which are well known to the person skilled in the art.
  • the particular amounts may be determined by conventional tests which are well known to the person skilled in the art. Suitable tests are, for example, described in Tamhane and Logan (2002), “Multiple Test Procedures for Identifying the Minimum Effective and Maximum Safe Doses of a Drug", J. Am. Stat. Assoc. 97(457):1- 9.
  • the pharmaceutical composition preferably comprises in addition at least one pharmaceutically acceptable carrier, excipient or diluent.
  • pharmaceutically acceptable carrier, excipient or diluent a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type is meant (see also Handbook of Pharmaceutical Excipients 6ed. 2010, Published by the Pharmaceutical Press). Ways of administering inhibitors to humans are also described, for example, in De Fougerolles et al. (2008) Curr. Opin. Pharmacol. 8:280-285.
  • Suitable pharmaceutical carriers include phosphate buffered saline solutions, histidine or citrate buffers, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO etc. Compositions comprising such carriers can be formulated by well known conventional methods.
  • the enzyme (and/or the nucleic acid molecule encoding the enzyme and/or the vector expressing the nucleic acid molecule) is the only pharmaceutically active compound that is capable of treating cancer within the pharmaceutical composition.
  • compositions can generally comprise more than one active compound according to the above preferred embodiment the enzyme (and/or the nucleic acid molecule encoding the enzyme and/or the vector expressing the nucleic acid molecule) is the only pharmaceutically active compound that is capable of treating cancer within the pharmaceutical composition in the pharmaceutical composition of the invention. Hence, according to this more preferred embodiment any treatment success of cancer is caused by the enzyme (and/or the nucleic acid molecule encoding the enzyme and/or the vector expressing the nucleic acid molecule) of the invention.
  • the cancer is a cancer whose growth depends on extracellular uridine.
  • Extracellular uridine has been found to be a fuel for cancers, such as pancreatic cancer or breast cancer; see, for example, Nwosu et al. (2023) Nature 618:151-158 and Ma et al. (2016) Oncotarget 7(20): 29036-29050.
  • the uridine ribose ring fuels both energetic and anabolic metabolism in cancer cells.
  • Whether cancer growth depends on extracellular uridine can be determined by routine means, for example by the profiling of metabolite utilization in cancer cells; see Nwosu et al. (2023) Nature, 618:151-158.
  • this cancer is particularly amenable to treatment with an enzyme that eliminates pyrimidine and/or purine nucleosides in vivo, such as an inosine-uridine preferring nucleoside hydrolase (IUNH), this can be tested by measuring the growth of cancer cell lines.
  • an enzyme that eliminates pyrimidine and/or purine nucleosides in vivo such as an inosine-uridine preferring nucleoside hydrolase (IUNH)
  • IUNH inosine-uridine preferring nucleoside hydrolase
  • the cancer is a lung cancer, wherein the lung cancer is preferably non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • Lung cancer is the leading cause of cancer-related deaths worldwide, accounting for the highest mortality rates among both men and women.
  • NSCLC Non-small cell lung cancer
  • lung cancer in particular non-small cell lung cancer (NSCLC)
  • NSCLC non-small cell lung cancer
  • the enzyme is used for treating cancer, preferably lung cancer, pancreatic cancer or breast cancer.
  • Example 13 Among these three cancer types lung cancer is preferred in view of the Example 13.
  • the enzyme and/or the nucleic acid molecule encoding the enzyme and/or the vector expressing the nucleic acid molecule may be used.
  • the enzyme is used and, thus, a protein is to be administered to the subject to be treated.
  • the protein as such was used in cancerous mice and various cancer cell lines and is shown to inhibit tumor growth.
  • the enzyme is used in a non-encapsulated form.
  • Enzyme drug delivery can be achieved via various formulations. This includes encapsulated forms, in particular nanoparticles, including lipidic nanoparticles, polymer nanoparticles, and inorganic nanomaterials that protect the enzyme from degradation and/or prolong blood half-life of the enzyme.
  • nanoparticles including lipidic nanoparticles, polymer nanoparticles, and inorganic nanomaterials that protect the enzyme from degradation and/or prolong blood half-life of the enzyme.
  • a nonencapsulated form is any form wherein the enzyme after administration directly comes into contact with the body / body cells of the subject, and is thus not protected by a capsule or any other protective layer or shell.
  • the enzyme has a tetrameric quaternary structure.
  • a tetrameric quaternary structure is a protein structure with a quaternary structure comprising four subunits (tetrameric). Homotetramers have four identical subunits and heterotetramers are complexes of different subunits. It is believed that enzymes that eliminate pyrimidine and/or purine nucleosides in vivo, such as an inosine-uridine preferring nucleoside hydrolase (IUNH), in their active form have a tetrameric quaternary structure.
  • IUNH inosine-uridine preferring nucleoside hydrolase
  • the enzymes may from a tetrameric quaternary structure after administration in vivo or, in accordance with this more preferred embodiment, the enzyme may already be used / administered in the form of a tetrameric quaternary structure.
  • the enzyme is in the form of a protein conjugate or a fusion protein comprising the enzyme conjugated or fused to a heterologous compound, preferably conjugated or fused to a polymer, preferably a PAS biopolymer, polysialic acid, polysarcosine, hydroxyethyl starch (HES), or poly-ethylene glycol (PEG).
  • a polymer preferably a PAS biopolymer, polysialic acid, polysarcosine, hydroxyethyl starch (HES), or poly-ethylene glycol (PEG).
  • the fusion partner is a proteinaceous compound (i.e. a compound having an amino acid sequence).
  • the resulting compound can also be designated a "fusion protein".
  • a fusion protein may be encoded by a nucleic acid molecule and the nucleic acid molecule can be introduced into a vector as described herein above.
  • conjugation may be carried out by recombinant DNA technology using well established techniques. As a result, the conjugate is created as one continuous polypeptide chain through the joining of two or more genes that originally code for separate molecules. Translation of this fusion gene results in a fusion protein with functional properties derived from each of the original molecules. Suitable vectors are known in the art and have been described herein above. It will be appreciated that if the fusion protein of the invention is produced by recombinant DNA technology it may comprise a linker.
  • the conjugation partner is generally a non-proteinaceous compound.
  • the resulting compound can also be designated "protein conjugate”.
  • the conjugation partners can be linked by chemical methods, as e.g. described in (Hermanson, G.T. [2013] Bioconjugate Techniques, Academic Press, 3rd Ed), either by direct coupling of the molecules via functional or functionalized groups or by indirect coupling employing a linker.
  • the conjugation partner can be, for example, a polymer, preferably, a hydrophilic polymer like polysialic acid, polysarcosine, hydroxyethyl starch (HES), or poly-ethylene glycol (PEG), a lipid, a non-peptidic ligand, a small molecule drug, a toxic compound or diagnostically and therapeutically relevant radioactive moiety, including metal chelator, and fluorescent tracer.
  • a polymer preferably, a hydrophilic polymer like polysialic acid, polysarcosine, hydroxyethyl starch (HES), or poly-ethylene glycol (PEG), a lipid, a non-peptidic ligand, a small molecule drug, a toxic compound or diagnostically and therapeutically relevant radioactive moiety, including metal chelator, and fluorescent tracer.
  • linker preferably relates to peptide linkers, i.e. a sequence of amino acids, as well as to non-peptide linkers.
  • nucleoside hydrolases IUNH
  • a linker is absent.
  • non-peptide linker refers to linkage groups having two or more reactive groups but excluding peptide linkers as defined above.
  • the non-peptide linker may be a polymer having reactive groups at both ends, which individually bind to reactive groups of the molecules of the protein conjugate, for example, an amino terminus, a lysine residue, a histidine residue or a cysteine residue.
  • Suitable reactive groups of polymers include an aldehyde group, a propionic aldehyde group, a butyl aldehyde group, a maleimide group, a ketone group, a vinyl sulfone group, a thiol group, a hydrazide group, a carbonylimidazole group, an imidazolyl group, a nitrophenyl carbonate (NPC) group, a trysylate group, an isocyanate group, and succinimide derivatives.
  • succinimide derivatives include succinimidyl propionate (SPA), succinimidyl butanoic acid (SBA), succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA), succinimidyl succinate (SS), succinimidyl carbonate, and N-hydroxy succinimide (NHS).
  • SPA succinimidyl propionate
  • SBA succinimidyl butanoic acid
  • SCM succinimidyl carboxymethylate
  • SSA succinimidyl succinamide
  • SS succinimidyl succinate
  • NHS N-hydroxy succinimide
  • the reactive groups at both ends of the non-peptide linker may be the same or different.
  • the non-peptide linker may have a maleimide group at one end and an aldehyde group at the other end.
  • the polymer itself may already carry a reactive group that can bind to reactive groups of the inosine-uridine preferring nucleoside hydrolases (IUNH), for example, an amino terminus, a lysine residue, a histidine residue or a cysteine residue.
  • IUNH inosine-uridine preferring nucleoside hydrolases
  • the protein conjugate according to the invention may be devoid of a non-peptide linker.
  • the heterologous compound may, for example, modify or enhance the solubility of the resulting protein conjugate, to modify or enhance their stability, to prolong its half-life in vivo, or to facilitate the purification of the enzyme.
  • Purification can be simplified by conjugating the enzyme of the present invention with one or more peptide sequences that confer on the resulting protein conjugate an affinity to certain chromatography column materials.
  • Typical examples of such sequences are commonly known as fusion tags.
  • Fusion tags include, without being limiting, oligohistidine-tags, Strep-tag, glutathione S-transferase, maltose- binding protein or the albumin-binding domain of protein G.
  • the use of an oligohistidine-tags i.e. a 6xHis-tag
  • the fusion protein or protein conjugate of the invention may be devoid of such an affinity tag.
  • the heterologous compound is preferably a PAS biopolymer, polysialic acid, polysarcosine, hydroxyethyl starch (HES), or poly-ethylene glycol (PEG).
  • These heterologous compounds can be used in addition to the above-discussed purification tags, in particular by fusing or conjugating one heterologous compound to each end of the enzyme subunit or to a side chain of the enzyme or by fusing them in tandem to one end or a side chain of the enzyme.
  • PAS biopolymers are recombinant polypeptides comprising the small uncharged L-amino acids Pro, Ala and optionally Ser which resemble the widely used poly-ethylene glycol (PEG) in terms of pronounced hydrophilicity. Likewise, their random chain behavior in physiological solution results in a strongly expanded hydrodynamic volume. Hence, PAS biopolymers are ideal fusion partners for biopharmaceuticals to achieve prolonged half-life in vivo, see Binder & Skerra (2017) Curr. Opin. Colloid Interface Sci. 31, 10-17.
  • Polysialic acids are endogenous substances which are non-immunogenic and biodegradable. At the same time, polysialic acid modification of protein drugs can enhance the uptake by tumor cells, reduce the immunogenicity of the proteins and prolong the circulation of the modified protein drugs in the blood; see Zhang et al. (2014) Asian J. Pharma. Sci. 9(2):75-81.
  • Polysarcosine pSAR
  • pSAR is a polypeptoid based on the endogenous non-proteinogenic amino acid sarcosine (N-methylated glycine). pSAR combines excellent solubility in water, protease resistance, low cellular toxicity and has a non-immunogenic character. pSAR prolongs the circulation half-life of protein drugs in vivo; see Hu et al. (2016) Bioconjug. Chem. 29(7):2232-2238.
  • HES Hydroxyethyl starch
  • HES Hydroxyethyl starch
  • HES is a nonionic starch derivative commonly used for fluid resuscitation to replace intravascular volume.
  • HES is a clinical plasma volume expander, which may also offer potential as a drug carrier.
  • Various types of HES-based drug delivery systems have been described, including HES-protein drug conjugates but also HES-based nano-assemblies, HES-based nanocapsules and HES- based hydrogels; see Wang et al. (2021) RSC Adv. 11: 3226-3240.
  • Polyethylene glycol is a robust and strongly hydrophilic chemical polymer, available in different length distributions and, optionally, with various functionalized end groups (including NHS esters and maleimides), which has found wide application as reagent in biochemical and biophysical research, as ingredient in cosmetics and food products as well as in medical therapy.
  • PEG Polyethylene glycol
  • the chemical conjugation of recombinant proteins with PEG also known as PEGylation, has led to multiple clinically approved biopharmaceuticals, mainly with the goal of prolonging their intrinsically short circulation after parenteral administration; see Gao et al. (2023) PEGylated therapeutics in the clinic. Bioeng. Transl. Med. 9(l):el0600.
  • the heterologous compound is a PAS biopolymer, wherein each enzyme subunit is linked, preferably at its N-terminus or C-terminus to said PAS biopolymer, and wherein the PAS biopolymer is a polypeptide comprising proline and alanine and optionally serine, wherein the polypeptide comprises at least 100 amino acids and preferably (i.e. with increasing preference) about 200, 400 or 600 amino acids, and wherein said polypeptide forms a random coil conformation.
  • the PAS biopolymers or polypeptides comprise amino acid sequence repeats (i.e. amino acid sequence stretches which repeatedly occur within the polypeptide sequence).
  • Preferred examples of such repeats of the polypeptides comprising proline and alanine and optionally serine comprise or consist of an amino acid sequence selected of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 5); AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 6); and APSAAPSAAPSAAPSAAPSA (SEQ ID NO: 7), or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.
  • ASPAAPAPASPAAPAPSAPA SEQ. ID NO: 5
  • AAPAAPAPAAPAAPAPAAPAAPAAPA SEQ ID NO: 6
  • APSAAPSAAPSAAPSAAPSA SEQ ID NO: 7
  • ASPAAPAPASPAAPAPSAPA SEQ. ID NO: 5
  • this repeat unit is used for the PASylation tag PAS#l(200) as illustrated in the Example (10
  • the term "random coil” relates to any conformation of a polymeric molecule, including amino acid polymers, in particular polypeptides made of L-amino acids, in which the individual monomeric elements that form said polymeric structure are essentially randomly oriented towards the adjacent monomeric element or elements while still being chemically linked.
  • the encoded polypeptide or amino acid polymer adopting/having/forming the "random coil conformation” substantially lacks a defined secondary and tertiary structure.
  • the nature of the encoded polypeptide random coils and their methods of experimental identification are known to the person skilled in the art and have been described in the scientific literature (Cantor (1980) Biophysical Chemistry, 2nd ed., W. H.
  • the encoded random coil polypeptides of the present invention adopt/form a random coil conformation, for example, in aqueous solution and/or at physiological conditions.
  • physiological conditions is known in the art and relates to those conditions in which proteins usually adopt their native, folded conformation. More specifically, the term “physiological conditions” relates to the environmental biophysical parameters as they are typically valid for higher forms of life and, particularly, for mammals, most preferably human beings.
  • physiological conditions may relate to the biochemical and biophysical parameters as they are normally found in the body, in particular in body fluids of mammals and in particular in humans.
  • Said “physiological conditions” may relate to the corresponding parameters found in the healthy body as well as the parameters found under disease conditions or in human patients.
  • a sick mammal or human patient may have a higher, yet “physiological” body temperature (i.e., temperature condition) when said mammal or said human suffers from fever.
  • physiological conditions at which proteins adopt their native conformation/state, the most important parameters are temperature (37°C for the healthy human body), pH (7.35-7.45 for human blood), osmolality (280- 300 mmol/kg H2O), and, if necessary, general protein content (66-85 g/l serum).
  • polypeptide comprising proline and alanine and optionally serine consists of proline, alanine and serine.
  • proline residues constitute more than 4% and less than 40% of said polypeptide.
  • alanine and the serine residues constitute the remaining amount of said amino acid sequence.
  • Such polypeptides are also termed herein PAS-rich or proline/alanine/serine-rich polypeptides.
  • the encoded amino acid sequence comprises more than about 4 %, preferably more than about 6 %, more preferably more than about 10 %, more preferably more than about 15 %, more preferably more than about 20 %, more preferably more than about 22%, 23% or 24%, more preferably more than about 26%, 29%, or 30%, more preferably more than about 31 %, 32%, 33%, 34% or 35% and most preferably about 35 % proline residues.
  • the encoded amino acid sequence preferably comprises less than about 40 %, more preferably less than 39%, 38%, 37%, 36% proline residues, wherein the lower values are preferred.
  • the encoded amino acid sequence preferably comprises less than about 95 %, more preferably less than 90%, 86%, 84%, 82% or 80% alanine residues, wherein the lower values are preferred. More preferably, the encoded amino acid sequence comprises less than about 79%, 78%, 77%, 76% alanine residues, whereby lower values are preferred. More preferably, the encoded amino acid sequence comprises less than about 75%, 73%, 71%, or 70% alanine residues, whereby lower values are preferred. More preferably, the encoded amino acid sequence comprises less than about 69%, 67%, 66%, or 65% alanine residues, whereby lower values are preferred.
  • the encoded amino acid sequence comprises less than about 64%, 63%, 62%, or 60% alanine residues, whereby lower values are preferred. More preferably, the encoded amino acid sequence comprises less than about 59%, 57%, 56%, or 55% alanine residues, whereby lower values are preferred. More preferably, the encoded amino acid sequence comprises less than about 54%, 53%, or 51%, alanine residues, whereby lower values are preferred. Most preferably, the encoded amino acid sequence comprises about 50 % alanine residues.
  • an encoded amino acid sequence comprising more than about 10 %, preferably more than about 15%, 17%, 19%, or 20%, more preferably more than about 22%, 24%, or 25%, , more preferably more than about 27%, 29%, or 30%, more preferably more than about 32%, 34% or 35%, more preferably more than about 37%, 39%, or 40%, more preferably more than about 42%, 44% or 45%, more preferably more than about 46%, 47% or 49% alanine residues, wherein the higher values are preferred.
  • the serine residues comprise the remaining amount of said amino acid sequence.
  • the encoded random coil polypeptide may comprise an amino acid sequence consisting of about 35 % proline residues, about 50 % alanine and 15 % serine residues; see, for example, SEQ ID NO: 5.
  • Exemplary nucleotide sequences and the encoded polypeptides thereof can be found in Table 1.
  • the term "about X %" as used herein above is not limited to the concise number of the percentage, but also comprises values of 10 % to 20 % additional or 10 % to 20 % less residues.
  • the term 10 % may also relate to 11 % or 12 % or to 9 % and 8 %, respectively.
  • polypeptide of proline and alanine and optionally serine consists of proline and alanine.
  • proline residues constitute more than about 10 % and less than about 75 % of said polypeptide.
  • the alanine and the serine residues constitute the remaining amount of said amino acid sequence.
  • the alanine residues comprise the remaining at least 25 % to 90 % of said amino acid sequence.
  • Such polypeptides are also termed herein PA-rich or proline/alanine-rich polypeptides; see, for example, SEQ ID NO: 6.
  • the encoded amino acid sequence comprises more than about 10%, preferably more than about 12%, more preferably more than about 14%, 18%, 20%, more preferably more than about 22%, 23%, 24%, or 25%, more preferably more than about 27%, 29%, or 30%, and most preferably more than about 32%, 33%, or 34% proline residues.
  • the amino acid sequence preferably comprises less than about 75%, more preferably less than 70%, more preferably less than 65%, more preferably less than 60%, more preferably less than 55%, more preferably less than 50% proline residues, wherein the lower values are preferred. Even more preferably, the amino acid sequence comprises less than about 48%, 46%, 44%, 42% proline residues. More preferred are amino acid sequences comprising less than about 41%, 40%, 39% 38%, 37% or 36% proline residues, whereby lower values are preferred. Most preferably, the amino acid sequences comprise about 35% proline residues.
  • the amino acid sequence preferably comprises less than about 90 %, more preferably less than 88 %, 86 %, 84 %, 82 % or 80 % alanine residues, wherein the lower values are preferred. More preferably, the amino acid sequence comprises less than about 79 %, 78 %, 77 %, 76 % alanine residues, whereby lower values are preferred. More preferably, the amino acid sequence comprises less than about 74 %, 72 %, or 70 % alanine residues, whereby lower values are preferred. More preferably, the amino acid sequence comprises less than about 69 %, 67 %, or 66 % alanine residues, whereby lower values are preferred.
  • amino acid sequence comprising more than about 25%, preferably more than about 30%, more preferably more than about 35%, more preferably more than about 40%, more preferably more than about 45%, more preferably more than about 50%, more preferably more than about 52%, 54%, 56%, 58% or 59% alanine residues, wherein the higher values are preferred.
  • the amino acid sequence comprises more than about 60 %, 61 %, 62 %, 63 % or 64 % alanine residues. Most preferably, the amino acid sequence comprises about 65 % alanine residues.
  • the random coil polypeptide may comprise an amino acid sequence consisting of about 40% or 35 % or 30% proline residues and about 60% or 65% or 70%, respectively, alanine residues.
  • the random coil polypeptide may comprise an amino acid sequence consisting of about 35 % proline residues and about 65 % alanine residues.
  • the term "about X %" as used herein above is not limited to the concise number of the percentage, but also comprises values of 10 % to 20 % additional or 10 % to 20 % less residues.
  • the term 10 % may also relate to 11 % or 12 % and to 9 % or 8 %, respectively.
  • the PAS polypeptide is encoded by a nucleotide sequence having a length of at least 300 nucleotides, wherein said nucleotide sequence has a Nucleotide Repeat Score (NRS) lower than 10,000, wherein said Nucleotide Repeat Score (NRS) is determined according to the formula: wherein N to t is the length of said nucleotide sequence, n is the length of a repeat within said nucleotide sequence, and f i(n ) is the frequency of said repeat of length n, wherein if there is more than one repeat of length n, k(n) is the number of said different sequences of said repeat of length n, otherwise k(n) is 1 for said repeat of length n.
  • N to t is the length of said nucleotide sequence
  • n is the length of a repeat within said nucleotide sequence
  • f i(n ) is the frequency of said repeat of length n, wherein if there is more than one
  • the NRS is defined as the sum of the squared repeat length multiplied with the root of the respective overall frequency, divided through the total length of the analyzed nucleotide sequence.
  • the minimal repeat length considered for the calculation of NRS comprises 4 nucleotides, which includes all nucleotide sequences longer than one codon triplet, and it ranges up to Ntot 1 , that is the length of the longest nucleotide sequence repeat that can occur more than once in the analyzed nucleotide sequence.
  • repeat means that a nucleotide sequence occurs at least twice within the nucleotide sequence analyzed.
  • both nucleotide stretches with identical sequence that occur at least twice as well as different sequences of the same length which each also occur at least twice were considered. For example, if the overall frequency of a 14mer repeat is five, this can mean either that the same 14mer nucleotide stretch occurs 5 times, or one 14mer nucleotide sequence occurs twice and a different 14 nucleotide sequence occurs three times in the analyzed nucleotide sequence.
  • each shorter repeat contained within a longer nucleotide sequence repeat is counted separately.
  • GCACC nucleotide stretches i.e., repeats
  • CACC repeats are also counted individually, regardless if they occur within said GCACC nucleotide stretch or, possibly, in addition elsewhere within the analyzed nucleotide sequence.
  • only repeats on the coding strand of the nucleic acid molecule are considered.
  • NRS-Calculator described in Example 14 of WO 2017/109087 can be used to unambiguously identify nucleotide sequence repeats and to calculate the NRS automatically.
  • the Nucleotide Repeat Score is lower than 5000, 2000, 1000, 750, 500, 200, 100, preferably lower than 50 and most preferably lower than 35.
  • the nucleic acid molecule of the invention comprises a nucleotide sequence encoding a polypeptide consisting of proline, alanine and, optionally, serine, wherein said nucleotide sequence has a Nucleotide Repeat Score (NRS) lower than 5000, 2000, 1000, 750, 500, 200, 100, preferably lower than 50 and most preferably lower than 35.
  • NRS Nucleotide Repeat Score
  • nucleic acid molecules comprising a nucleotide sequence encoding a polypeptide consisting of proline, alanine and, optionally, serine, wherein said nucleotide sequence has a Nucleotide Repeat Score (NRS) of 34, 33, 32, 31, 30, 29, 28, 1 , 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 or an NRS lower than 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8.
  • NRS Nucleotide Repeat Score
  • the nucleotide sequence encoding the PAS polypeptide comprises said repeats, wherein said repeats have a maximum length n m ax, wherein n m ax is determined according to the formula: ⁇ 17 + ⁇ N.2 , n L max 600 and wherein N to t is the length of said nucleotide sequence, preferably wherein said repeats have a maximum length of about 14, 15, 16, or 17 nucleotides to about 21 nucleotides.
  • maximum length or “maximal length” or “n m ax” as used herein defines the number of nucleotides of the longest contiguous part/stretch/sequence of nucleotides that is present in at least two copies within said nucleotide sequence or nucleic acid molecule.
  • maximum length or “maximal length” or “n m ax” as used herein means that the nucleotide sequence of the nucleic acid molecule according to this invention has no repeats which are longer than this length.
  • the repeat analysis can be performed with any suitable tool such as the NRS analysis provided herein, manually or with the aid of generic software programs such as the dot plot analysis, for example using Visual Gene Developer (Jung (2011) loc. cit) or the Repfind tool (Betley (2002) loc. cit).
  • a dot plot is a visual representation of the similarities between two sequences, which may be different or the same.
  • polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.
  • SEQ ID NO: 8 is the sequence of PAS(#l)200 as used in the appended examples: ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPAP SAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPA APAPSAPAASPAAPAPASPA APAPSAPAASPAAPAPASPA APAPSAPAA.
  • sequences being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto. The at least 95% is with increasing preference at least 96%, at least 97%, at least 98% and at least 99%.
  • the present invention relates in a second aspect to a fusion protein comprising an enzyme that eliminates pyrimidine and/or purine nucleosides in vivo fused to a heterologous compound, wherein the enzyme comprises or consists of the amino acid sequence of SEQ ID NO: 1 or 2 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto; and wherein the heterologous compound is a PAS biopolymer as defined herein above in the context of the first aspect of the invention.
  • the present invention relates in a fourth aspect to a vector expressing the nucleotide sequence of the third aspect.
  • the present invention relates in a fifth aspect to a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein of the second aspect, the nucleic acid molecule of the third aspect or the vector of the fourth aspect.
  • sequence identities for instance, also in connection with the second to fifth aspect of the invention also envisaged are with increasing preference sequence identities of at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% with respect to SEQ. ID NOs 1 and 2.
  • Suitable vectors and details on pharmaceutical compositions are described hereinabove in connection with the first aspect of the invention and the same disclosure applies to the second to fifth aspect of the invention.
  • each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends on.
  • a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I
  • the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C,
  • Fig. 1 Plasmid map of pASK75-T7RBS2-HiS6-LmlUNH.
  • the structural gene for LmlUNH (comprising an N-terminal Hisg-tag and the LmlUNH) is under transcriptional control of the tetracycline promoter/operator (tet p/o ) and ends with the lipoprotein terminator (ti pp ).
  • the plasmid backbone i.e. outside the expression cassette flanked by the Xba ⁇ and Hind II I restriction sites, is identical with that of the generic cloning and expression vector pASK75 (Skerra (1994) Gene 151:131-135). Singular restriction sites are indicated.
  • the expression vector for Hisg-CflUNH is identical except that CflUNH is encoded.
  • Fig. 2 Plasmid map of pASK75-T7RBS2-HiS6-LmlUNH-PAS(#l)200.
  • the structural gene for LmlUNH- PAS(#l)200 (comprising an N-terminal Hisg-tag, the LmlUNH and the PAS#1 polypeptide with 200 residues, PAS(#l)200) is under transcriptional control of the tetracycline promoter/operator (tet p/o ) and ends with the lipoprotein terminator (ti PP ).
  • the plasmid backbone i.e.
  • Fig. 3 Plasmid map of pASK75-T7RBS2-His 6 -PAS(#l)200-LmlUNH.
  • the structural gene for PAS(#l)200- LmlUNH (comprising an N-terminal Hisg-tag, the PAS#1 polypeptide with 200 residues, PAS(#l)200), and LmlUNH) is under transcriptional control of the tetracycline promoter/operator (tet p/o ) and ends with the lipoprotein terminator (ti pp ).
  • the plasmid backbone i.e.
  • Fig. 4 Analysis of the purified recombinant Hisg-LmlUNH and Hisg-CflUNH proteins by SDS-PAGE, followed by staining with Coomassie brilliant blue R-250.
  • the recombinant proteins were produced in E. coli NEBExpress and purified by means of the Hisg-tag using immobilized metal-ion affinity chromatography (IMAC) followed by size exclusion chromatography (SEC).
  • the gel shows 4 pg protein samples each of Hisg-LmlUNH (lanes 1 and 3) and Hisg-CflUNH (lanes 2 and 4). Samples on the left side were reduced with 2-mercaptoethanol whereas corresponding samples on the right side were kept unreduced. Sizes of protein markers (kDa) - applied under reducing conditions - are indicated on the left. All proteins appear as single homogeneous bands with apparent molecular sizes of ca. 35 kDa.
  • Fig. 5 Analysis of the purified recombinant HiS6-PAS(#l)200-LmlUNH, HiS6-LmlUNH-PAS(#l)200, Hisg- PAS(#l)200-CflUNH and Hisg-CflUNH-PAS(#l)200 enzymes by SDS-PAGE, followed by staining with Coomassie brilliant blue R-250.
  • the recombinant proteins were produced in E. coli NEBExpress and purified by means of the Hisg-tag via IMAC followed by anion exchange chromatography (AEX).
  • the gel shows 4-8 pg protein samples each of Hisg-CflUNH-PAS(#l)200 (lane 1), Hisg-PAS(#l)200-CflUNH (lane 2), Hisg-LmlUNH-PAS(#l)200 (lane 3), and Hisg-PAS(#l)200-LmlUNH (lane 4). All samples were reduced with 2-mercaptoethanol. Sizes of protein markers (kDa) - applied under reducing conditions - are indicated on the left. All proteins appear as single homogeneous bands with apparent molecular sizes of around 80 kDa. These values are significantly larger than the calculated masses of approximately 52.1 kDa for all four PASylated IUNH enzymes.
  • coli NEBExpress and purified by means of a subtractive cation exchange chromatography (CEX) followed by anion exchange (AEX) and sizeexclusion chromatography (SEC).
  • CEX subtractive cation exchange chromatography
  • AEX anion exchange
  • SEC sizeexclusion chromatography
  • the gel shows 4 pg protein samples each of LmlUNH-PAS(#l)200 (lane 1), CflUNH-PAS(#l)200 (lane 2) and LmlUNH(C301G)-PAS(#l)200 (lane 3). All samples were reduced with 2-mercaptoethanol. Sizes of protein markers (kDa) - applied under reducing conditions - are indicated on the left. All proteins appear as single homogeneous bands with apparent molecular sizes of around 80 kDa. These values are significantly larger than the calculated masses of approximately 50.9 kDa for these PASylated IUNH enzymes, again due to the presence of the PAS biopolymer as described for
  • Fig. 7 Michaelis-Menten plot for the enzyme LmlUNH(C301G)-PAS(#l)200 with uridine as substrate.
  • Initial velocities (vo) for uridine hydrolysis with varying starting concentrations of the substrate (0.05, 0.1, 0.2, 0.3, 0.5, 1, 2.5, 5, 10 mM) were measured in 50 mM HEPES buffer at pH 7.4 °C and fitted by the Michaelis-Menten equation. Reactions were initiated by adding the purified enzyme solution (0.369 pM end concentration), and the decrease in absorbance at 280 nm was recorded at ambient temperature over 10 min. vo values were calculated from the initial slope of each reaction and plotted against the uridine substrate concentration [S] using KaleidaGraph.
  • Fig. 8 Analysis of the pharmacokinetics (PK) of His6-PAS#l(200)-CflUNH (filled triangles; solid line) and LmlUNH(C301G)-PAS#l(200) (filled circles; dashed line) in mice.
  • Fig. 9 In vivo efficacy of His6-PAS#l(200)-CflUNH treatment on murine orthotopic lung adenocarcinoma tumors.
  • A Orthotopic lung tumors were induced in male and female C56BL/6 N mice by a tail vein injection of HKP1 cells.
  • tumor size was measured by bioluminescence using a Lago X spectral instrument. The signal was expressed as total emission/s. After the measurement, mice were placed in a cage and observed until fully recovered. Based on this measurement, mice were distributed into 2 treatment groups with similar average tumor size.
  • B Analysis of the effect of His6-PAS#l(200)-CflUNH treatment on lung tumor progression. Tumor size was measured by bioluminescence imaging after 4 and 11 days of treatment.
  • His6-PAS#l(200)-CflUNH treatment led to significantly reduced tumor growth over the 11-day treatment period.
  • a significant effect of His6-PAS#l(200)-CflUNH was observed in two independent experiments.
  • (C) His6-PAS#l(200)-CflUNH treatment does not affect mouse body weight. Body weight of tumor-bearing mice (c.f. panels A and B) treated with His6-PAS#l(200)-CflUNH or vehicle (PBS) was monitored over a period of 12 days. No significant changes in body weight were observed for the Hisg- PAS#l(200)-CflUNH treated group compared to the vehicle-treated group.
  • Fig. 10 In vitro efficacy of His6-PAS#l(200)-CflUNH treatment on different cancer cell lines.
  • HKP1, LLC1, and AsPCl cells were treated with increasing concentrations of the purified His6-PAS#l(200)-CflUNH enzyme.
  • Cells were seeded in 96 well plates and treated with His6-PAS#l(200)-CflUNH at the following concentrations: 0, 5, 50, 100, 200, and 400 pg/ml. After 48 h (HKP1) or 72 h (LLC1 and AsPCl), cells were fixed and stained with crystal violet. After cell lysis, the intensity of staining was quantified and used as a measure of the number of cells. Mean values of 3 independent experiments are shown.
  • HKP1 WT and HKP1 DHODH knock-out (KO) cells were seeded in 96 well plates in the presence or absence of uridine for 72 h. Cells were fixed and stained with crystal violet to estimate the cell number. DHODH KO cells cannot grow without uridine supplementation. Mean values of 3 independent experiments are shown, error bars represent S.E.M; ***p ⁇ 0.001 according to 2-way ANOVA Sidak's multiple comparisons test.
  • HKP1 WT and HKP1 DHODH KO cells were seeded in 96 well plates in the presence of uridine (100 pM) or uracil (100 pM) for 72 h. Cells were fixed and stained with crystal violet to quantify the cell number. Uracil supplementation cannot rescue proliferation of DHODH KO cells. Mean values of 3 independent experiments are shown, error bars represent S.E.M; ****p ⁇ 0.0001 according to 2-way ANOVA Sidak's multiple comparisons test.
  • HKP1 WT and HKP1 DHODH KO cells were seeded in 96 well plates in the presence of uridine (204 pM) and treated with Hisg-PAS#l(200)-CflUNH at the following concentrations: 0, 0.01, 0.1, 0.2, 0.5, 1 and 2 pg/ml.
  • uridine 204 pM
  • Hisg-PAS#l(200)-CflUNH As a negative control, cells were grown in medium lacking uridine. After 72 h, cells were fixed and stained with crystal violet. The intensity of staining was quantified as a measure of the cell number.
  • Fig. 12 In vitro efficacy of LmlUNH(C301G)-PAS#l(200) treatment on the HKP1 cancer cell line.
  • HKP1 cells were treated with increasing concentrations of the purified LmlUNH(C301G)-PAS#l(200) enzyme.
  • Cells were seeded in 96 well plates with the addition of 204 pM uridine and treated with LmlUNH(C301G)-PAS#l(200) at the following concentrations: 0, 5, 50, 100, 200, and 400 pg/ml. After 48 h, cells were fixed and stained with crystal violet. After cell lysis, the intensity of staining was quantified and used as a measure of the number of cells. Mean values of 3 independent experiments are shown.
  • the synthetic gene cassettes encoding the Inosine-Uridine preferring Nucleoside Hydrolase (IUNH) enzymes from Leishmania major (Uniprot No. P83851) and Crithidia fasciculata (Uniprot No.Q27546) were designed by backtranslating the amino acid sequence using codons optimized for E. coli expression. To simplify purification, the codons for six consecutive His residues were inserted directly following the Met start codon.
  • IUNH Nucleoside Hydrolase
  • Nde ⁇ and H/ndlll restriction sites that were used for cloning can be seen in the nucleotide sequences of pASK75-T7RBS2-His 6 -LmlUNH (SEQ ID NO: 14) and pASK75-T7RBS2-His 6 -CflUNH (SEQ ID NO: 15).
  • Resulting plasmids were designated pASK75-T7RBS2-His 6 -LmlUNH (SEQ ID NO: 14; Fig. 1) and pASK75-T7RBS2-His 6 - CflUNH (SEQ ID NO: 15).
  • Example 3 Construction of expression vectors for IUNH enzymes with a C-terminal PAS biopolymer pASK75-T7RBS2-His6-LmlUNH was cut with Sap ⁇ , dephosphorylated with shrimp alkaline phosphatase (Thermo Fisher Scientific), and ligated with the 3-fold stoichiometric amount of a gene fragment encoding the 200 residue PAS(#1) biopolymer excised from the plasmid pXLl-PAS(#l)200 (XL-protein GmbH, Freising, Germany) via restriction digest with Sap ⁇ . After transformation of E.
  • plasmid DNA was prepared from individual colonies and the sequences of the cloned synthetic nucleic acids were confirmed by restriction analysis and automated double-stranded DNA sequencing as described in Example 2.
  • the plasmid encoding the enzyme fused with a C-terminal PAS biopolymer, His 6 -LmlUNH-PAS(#l)200 (SEQ ID NO: 16) was designated pASK75-T7RBS2-His 6 -LmlUNH-PAS(#l)200 (SEQ ID NO: 17; Fig. 2).
  • the plasmid encoding the enzyme from C.
  • fasciculate was constructed in the same manner using the corresponding plasmid pASK75-T7RBS2-His6-CflUNH from Example 2, resulting in pASK75-T7RBS2-His 6 -CflUNH-PAS(#l)200 (SEQ ID NO: 31) encoding His 6 -CflUNH-PAS(#l)200 (SEQ ID NO: 32).
  • Example 4 Construction of expression vectors for IUNH enzymes with an N-terminal PAS biopolymer pASK75-T7RBS2-HiS6-LmlUNH was cut with EcoQ109l, dephosphorylated using shrimp alkaline phosphatase, and ligated with the 3-fold stoichiometric amount of the gene fragment encoding the 200 residue PAS(#1) biopolymer excised from the plasmid pXLl-PAS(#l)200 via restriction digest with Sap ⁇ . After transformation of E.
  • plasmid DNA was prepared from individual colonies and the sequences of the cloned synthetic nucleic acids were confirmed by restriction analysis and automated double-stranded DNA sequencing as described in Example 2 (Eurofins Genomics).
  • the plasmid encoding the enzyme from C.
  • fasciculate was constructed in the same manner using the corresponding plasmid pASK75-T7RBS2-HiS6-CflUNH from Example 2, resulting in pASK75-T7RBS2-HiS6- PAS(#l)200-CflUNH (SEQ ID NO: 33) encoding His 6 -PAS(#l)200-CflUNH (SEQ ID NO: 34).
  • Example 5 Removal of the Hise-tag from the expression vectors for His6-LmlUNH-PAS(#l)200 and His 6 -CflUNH-PAS(#l)200
  • Two forward primers SEQ ID NO: 20 and SEQ ID NO: 21, were designed to anneal directly downstream of the nucleotide sequence encoding the Hisg-tag on the plasmids pASK75-T7RBS2-His6-LmlUNH (Seq ID NO: 14) and pASK75-HiS6-CflUNH (SEQ ID NO: 15), respectively. These plasmids were subjected to PCR using each of the forward primers together with a generic reverse primer, SEQ ID NO: 22.
  • the amplified DNA fragments were digested using Nde ⁇ and Nco ⁇ (an internal restriction site within each of the LmlUNH and CflUNH gene sequences), purified by agarose gel electrophoresis and combined in a ligation reaction with the correspondingly cut plasmids pASK75-T7RBS2-His6-LmlUNH-PAS(#l)200 and pASK75-T7RBS2-His 6 -CflUNH-PAS(#l)200.
  • the resulting plasmids were designated pASK75-T7RBS2- LmlUNH-PAS(#l)200 (SEQ ID NO: 23), encoding LmlUNH-PAS#l(200) (SEQ ID NO: 24), and pASK75- T7RBS2-CflUNH-PAS(#l)200 (SEQ ID NO: 25), encoding CflUNH-PAS#l(200) (SEQ ID NO: 26), respectively.
  • Plasmids were prepared from individual colonies and the sequences of the mutated nucleic acids were confirmed by automated double-stranded DNA sequencing as described in Example 2.
  • the plasmid coding for LmlUNH(C301G)-PAS#l(200) (SEQ ID NO: 29) was designated pASK75-T7RBS2- LmlUNH(C301G)-PAS#l(200) (SEQ ID NO: 30).
  • IMAC running buffer 50 mM NaPi, 500 mM NaCI pH 7.5
  • SLM Aminco, Urbana, IL French pressure cell
  • each enzyme was purified by immobilized metal ion affinity chromatography (IMAC) using Ni 2+ -charged NTA Sepharose FF (Cytiva Europe, Freiburg, Germany) and an imidazole concentration gradient, followed by gel filtration on a Superdex S200 HiLoad 16/60 column (Cytiva Europe) with PBS (115 mM NaCI, 4 mM KH2PO4, 16 mM Na2HPO 4 pH 7.4) as running buffer, yielding homogeneous protein preparations (Fig. 4) with 3.1 and 3.2 mg L 1 OD 1 for Hisg- LmlUNH and Hisg-CflUNH , respectively.
  • IMAC immobilized metal ion affinity chromatography
  • Example 8 Bacterial production and purification of IUNH enzymes carrying a PAS#1 biopolymer at the N- or C-terminus
  • N- or C -terminally PASylated IUNH (His 6 -PAS(#l)200-LmlUNH and His 6 -LmlUNH-PAS(#l)200: calculated mass of both proteins: 52146 Da; His 6 -PAS(#l)200-CflUNH and His 6 -CflUNH-PAS(#l)200: calculated mass of both proteins: 52162 Da), were produced at 30 °C in E. coli NEBExpress harboring the corresponding expression plasmids from Example 3 or 4 using shake flask cultures with 2 L LB/Amp medium.
  • Cells were harvested by centrifugation, resuspended in IMAC running buffer and disrupted using a French pressure cell. The total cell extract was cleared by centrifugation and dialyzed against IMAC running buffer.
  • Each PASylated enzyme was purified by IMAC using Ni 2+ -charged NTA Sepharose FF. Pure fractions were combined and dialyzed against 20 mM Tris/HCI pH 8.0.
  • Anion exchange chromatography (AEX) was subsequently performed on a Resource d column (Cytiva Europe) and the bound enzyme was eluted in an ascending NaCI concentration gradient.
  • Example 9 Bacterial production and purification of IUNH variants carrying a PAS#1 biopolymer at the C-terminus without a His 6 -tag
  • IUNH variants were first purified by subtractive cation exchange chromatography (CEX) using a Resource S column (5 ml bed volume; Cytiva Europe).
  • Example 10 Determination of the kinetic parameters of IUNH enzymes using a spectrophotometric enzyme activity test
  • the activity assay was based on the hydrolytic conversion of inosine into hypoxanthine or of uridine into uracil. Notably, both nucleosides inosine and uridine exhibit higher light absorption at 280 nm compared to their hydrolysis products (sugar and base).
  • initial velocities (vo) for the change in substrate concentration were measured and fitted by the Michaelis- Menten equation.
  • Reaction mixtures of 500 pl contained the substrates inosine or uridine at varying starting concentrations (0.05, 0.1, 0.2, 0.3, 0.5, 1, 2.5, 5, 10 mM) in 50 mM HEPES buffer at pH 7.4.
  • Reactions were initiated by adding 6 pl of the purified enzyme solution with appropriate concentrations (for example 1.56 mg/ml for LmlUNH(C301G)-PAS(#l)200), and the decrease in absorbance at 280 nm was recorded at ambient temperature over 10 min using a S-3100 UV/Vis spectrophotometer (Scinco, Seoul, Korea), vo values were calculated from the initial slope and plotted against the substrate concentration using KaleidaGraph software (Synergy Software, Reading, PA).
  • concentrations for example 1.56 mg/ml for LmlUNH(C301G)-PAS(#l)200
  • Table 1 IUNH activity parameters for uridine as substrate.
  • Table 2 IUNH activity parameters for inosine as substrate.
  • mice Male C56BL/6N mice, 7-8 weeks old, were injected intraperitoneally as follows:
  • the total volume of injected test compound was calculated according to the individual body weight on the day of administration (e.g., an animal with 20 g body weight (b.w.) received 100 pl of 1 mg/ml test compound). Blood sampling was performed as follows:
  • Blood samples (approximately 40 pl) were taken from the tail vein and kept on ice until centrifugation. Serum was separated by centrifugation for 20 min at 16000 g at 4°C and immediately frozen at minus 80°C. Mice were sacrificed by CO2 inhalation after the last blood sampling.
  • the wells of a 96-well microtitre plate were coated with 50 pl 20 pg/ml Avi-PAS Mab 2.1 antibody (XL-protein, Freising, Germany) in PBS for 2 h. After removal of the antibody solution the wells were blocked with 200 pl of 3 % (w/v) bovine serum albumin (BSA) in PBS/T (PBS supplemented with 0.1 % (v/v) Tween 20) for 1 h and washed three times with PBS/T.
  • BSA bovine serum albumin
  • mice The plasma samples from mice were applied in dilutions of 1:1000 or 1:2000 in PBS/T supplemented with 0.1 % (v/v) mouse plasma from an untreated animal and incubated for 1 h. The wells were then washed three times with PBS/T and incubated for 1 h with 50 pl of a 1:5000 diluted solution of Avi PAS Mab 1.1-alkaline phosphatase (AP) conjugate (0.8 mg/ml; XL-protein).
  • AP Avi PAS Mab 1.1-alkaline phosphatase
  • the chromogenic reaction was started by adding 50 pl of 0.5 mg/ml p-nitrophenyl phosphate in 100 mM Tris/HCI pH 8.8, 100 mM NaCI, 5 mM MgCL. After incubation for 10 min at 30 °C, the absorbance at 405 nm was measured using a BioTek Synergy 2 photometer (BioTek Instruments, Bad Friedrichshall, Germany).
  • Concentrations of Hisg- PAS#l(200)-CflUNH and LmlUNH(C301G)-PAS#l(200) in the initial plasma samples were quantified by comparison with standard curves, which were determined for dilution series of the corresponding purified recombinant proteins at defined concentrations in PBS/T containing 0.1 % (v/v) untreated mouse plasma, taking into consideration the applied dilution factor.
  • Fig. 8 depicts the kinetics of blood clearance in vivo.
  • the plasma half-life of His6-PAS#l(200)-CflUNH and LmlUNH(C301G)-PAS#l(200) was 25.8 ⁇ 6.3 h and 29.7 ⁇ 5.9 h, respectively.
  • These data show that the plasma half-life of CflUNH and LMIUNH(C301G) is significantly prolonged due to the fusion with the PAS biopolymers.
  • Example 13 Analysis of His6-PAS#l(200)-CflUNH treatment efficacy in vivo on murine orthotopic lung adenocarcinoma tumors
  • HKP1 cells a gift from Prof. Vivek Mittal, are a syngeneic mouse lung adenocarcinoma cell line able to form tumors in the lungs of immunocompetent mice (Choi, H., et al. Cell Reports 10:1187-1201).
  • HKP1 cells express luciferase, which allows the non-invasive monitoring of tumor growth. 3 days after injecting the cells, the tumor size was measured by bioluminescence imaging.
  • mice were administered intraperitoneally.
  • mice were anesthetized using isoflurane (Aerrane, Cat. No. 4DG9621, Baxter, Lessines, Belgium) inhalation at a concentration of 3.5-4.5 % (v/v) in air.
  • mice were placed into the Lago X whole body spectral scanner (Spectral Instruments Imaging, Arlington, AZ) and maintained on 2.5 % (v/v) isoflurane.
  • Luciferase signals from the HKPl-derived tumors, serving to estimate the tumor size were detected with the following settings: exposure time 300 s, binning middle (4), F stop 1.2 and using the total emission/s as readout. Based on these measurements, mice were distributed into 2 experimental groups with similar average tumor size (Fig. 9A). One group was injected intraperitoneally with PAS- IUNH in PBS (7 mice) and the other group was injected with vehicle (PBS) (7 mice). Treatment started 3 days after the injection of the HKP1 cells and continued as follows: To assess treatment efficiency, each tumor size was measured at days 4 and 11 after the start of the treatment.
  • Example 14 Suppressive effect of His6-PAS#l(200)-CflUNH on the growth of cancer cell lines.
  • HKP1 murine lung adenocarcinoma cells see Example 13 supra; 2xl0 3
  • AsPCl human pancreatic adenocarcinoma cells Sigma Aldrich, Burlington, MA; 5xl0 3
  • LLC1 Lewis lung carcinoma cells ATCC, Manassas, VA; 5xl0 3
  • DMEM medium DMEM medium.
  • Both, RPMI and DMEM media were supplemented with 10 % (v/v) dialyzed fetal bovine serum (FBS), 100 units/ml penicillin, 100 pg/ml streptomycin, 1 mM pyruvate, and 204 pM uridine.
  • FBS fetal bovine serum
  • Cells were treated with His6-PAS#l(200)-CflUNH at the following concentrations: 0, 5, 50, 100, 200, and 400 pg/ml.
  • After 48 h (HKP1) or 72 h (AsPCl and LLC1) cells were fixed for 30 min with 4 % (w/v) paraformaldehyde (Avantor, Gliwice, Poland) at room temperature and washed 3 times with PBS.
  • Example 15 Suppressive effect of His6-PAS#l(200)-CflUNH on the growth of HKP1 cancer cells deficient in pyrimidine synthesis
  • HKP1 DHODH knock-out (KO) cancer cells were produced by deleting the DHODH gene (NCBI Gene ID: 56749) in HKP1 wild-type (WT) cells (see Example 13 supra) using the CRISPR/Cas9 method according to a published protocol (Bajzikova et al (2019) Cell Metab. 29: 399- 416. elO.).
  • the dependency of the DHODH KO on uridine was tested by culturing these cells in RPMI medium supplemented with 10 % (v/v) dialyzed FBS, 100 units/ml penicillin, 100 pg/ml streptomycin, 1 mM pyruvate in the presence or absence of uridine (204 pM). Cell growth was quantified by staining with crystal violet as described in Example 14 supra. As evident from Fig. 11A and 11B, the HKP1 DHODH KO cells grew only when uridine was supplied in the culture medium, whereas supplementation with uracil (instead of uridine) did not rescue their growth.
  • both HKP1 WT and HKP1 DHODH KO cells were seeded in 96 well plates in RPMI medium supplemented with 10 % (v/v) dialyzed FBS, 100 units/ml penicillin, 100 pg/ml streptomycin, 1 mM pyruvate and 204 pM uridine.
  • the cells were treated with Hisg-PAS#l(200)- CflUNH at the following concentrations: 0, 0.01, 0.1, 0.2, 0.5, 1 and 2 pg/ml.
  • Example 16 Suppressive effect of LmlUNH(C301G)-PAS#l(200) on the growth of cancer cell lines
  • HKP1 murine lung adenocarcinoma cells were seeded in 96-well plates in RPMI medium supplemented with 10 % (v/v) dialyzed FBS, 100 units/ml penicillin, 100 pg/ml streptomycin, 1 mM pyruvate and 204 pM uridine.
  • Cells were treated with LmlUNH(C301G)- PAS#l(200) at the following concentrations: 0, 5, 50, 100, 200, and 400 pg/ml.

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Abstract

La présente invention concerne (a) une enzyme qui élimine les nucléosides de pyrimidine et/ou de purine in vivo, (b) une molécule d'acide nucléique codant pour l'enzyme de (a), et/ou (c) un vecteur exprimant la molécule d'acide nucléique de (b) pour une utilisation dans le traitement du cancer.
PCT/EP2025/062447 2024-05-07 2025-05-07 Enzymes qui éliminent les nucléosides de pyrimidine et/ou de purine pour le traitement du cancer Pending WO2025233381A1 (fr)

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WO2005108562A2 (fr) * 2004-05-10 2005-11-17 Fondazione Centro San Raffaele Del Monte Tabor Enzyme synthetique activant la fluoro-uridine dans les therapies anticancereuses
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