WO2025233381A1 - Enzymes that eliminate pyrimidine and/or purine nucleosides for treating cancer - Google Patents

Abstract

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.

Description

Enzymes that eliminate pyrimidine and/or purine nucleosides for treating cancer

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.

In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

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). Current anti-cancer therapies targeted at nucleotide metabolism make use (i) of small molecule inhibitors directed at intracellular enzymes that synthesize or convert nucleotides endogenously within a cancer cell or (ii) of nucleotide analogues that interfere with nucleotide metabolism (e.g., inhibitors of dihydrofolate reductase, Methotrexate; thymidylate synthase, 5-Fluorouracil; folate analogue, Pemetrexed, ribonucleotide reductase, Gemcitabine; etc.) (Mullen, N. J., et al. (2023). Nature Reviews Cancer 23, 275-294; Kaye, S. B. (1998). British Journal of Cancer 78, 1-7).

Inhibitors of other enzymes in nucleotide metabolism, such as dihydroorotate dehydrogenase (Leflunomide) and IMP dehydrogenase 1 and 2 (Mizoribine) were only approved for autoimmune diseases but have not yet shown clinical efficacy in cancer patients (Strand, V., et al. (1999). Archives of Internal Medicine 159, 2542-2550; Naffouje, R., et al. (2019). Cancers (Basel) 11). Overall, while targeting nucleotide metabolism has therapeutic success in certain cancer types, this is often linked with resistance and toxicity (Kaye, S. B. (1998). British Journal of Cancer 78, 1-7). Notably, the inhibition of internal nucleotide synthesis can be overcome by nucleosides (the unphosphorylated forms of nucleotides comprising an RNA base and a ribose sugar), or desoxynucleotides, that are taken up from the extracellular milieu (Lane, A. N., et al. (2015). Nucleic Acids Res 43, 2466-2485). This physiological mechanism can reduce the efficacy of the current anti-cancer approaches directed at intracellular biosynthetic/metabolic pathways (Halbrook, C. J., et al. (2019). Cell Metabolism 29, 1390-1399. e6).

Currently, it is not possible to target and eliminate these extracellular nucleosides, which are readily available to cancer cells even when their endogenous synthesis has been blocked, thus reducing the efficacy of anti-cancer therapy. 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.

Accordingly, the present invention relates in a first aspect 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.

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).

As used herein 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.

A purine (desoxy)nucleoside consists of a nucleobase and the sugar ribose or deoxyribose, wherein the nucleobase is a purine, preferably selected from adenine, guanine, hypoxanthine and xanthine. The purine nucleoside is preferably adenosine, deoxyadenosine, guanosine, deoxyguanosine, inosine or deoxyinosine, xanthosine or deoxyxanthosine.

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.

The term "nucleic acid molecule" in accordance with the present invention includes DNA, such as cDNA or double or single stranded genomic DNA and RNA. In this regard, "DNA" (deoxyribonucleic acid) 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) means any chain or sequence of the chemical building blocks of adenine (A), guanine (G), cytosine (C) and uracil (U), called nucleotide or nucleic acid bases, that are linked together on a ribose sugar backbone. 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.).

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. Further included are 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.

Also included are 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.

The term "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 (Novagen) and pCINeo (Promega). Examples for plasmid vectors suitable for Pichia pastoris comprise e.g. the plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen).

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) ensuring expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. 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. Acad. Sci. USA 98 (2001), 1471-1476) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Preferably, the polynucleotide encoding the 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. Furthermore, depending on the expression system used, 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.

Furthermore, it is preferred that the vector comprises a selectable marker. Examples of 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. Additionally, 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. Among this list of cancers, lung cancer (Examples 13 and 14) and 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.

As can be taken from the appended examples (Example 13 and Fig. 9) it was found that the administration of an enzyme that eliminates pyrimidine and/or purine nucleosides, i.e. a PASylated inosine uridine nucleotide hydrolase (lUNH)enzyme, to mice with lung cancer (orthotopic lung adenocarcinoma tumors) leads to significantly reduced tumor growth in several independent experiments. No adverse side effects were observed; for example, treatment did not affect mouse body weight.

The results of Example 13 were confirmed based on cancer cell lines in Example 14 and Fig. 10 (lung cancer and pancreatic cancer cells). The growth of the cancer cells was inhibited by the enzyme that eliminates pyrimidine and/or purine nucleosides extracellularly. An even stronger growth inhibition by the enzyme was found for cancer cells with impaired pyrimidine de novo synthesis; see Example 15 and Fig. 11. These data overall demonstrate that enzymes which eliminate pyrimidine and/or purine nucleosides are a novel treatment option for cancer.

As also mentioned above, the enzyme that eliminates pyrimidine and/or purine nucleosides in vivo according to the present invention cleaves and eliminates the nucleosides in vivo in the extracellular milieu, such as in the blood and in the interstitial space. This extracellular mode of action according to the present invention is distinct from the intracellular mode of action of uridine phosphorylase 1 (UPPl)-mediated conversion of uridine into uracil as described in Nwosu et al. (2023), Nature, 618(7963):151-158.

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.

In contrast, 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. When 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.

In accordance with a preferred embodiment the enzyme is a pyrimidine and/or purine hydrolase.

A hydrolase (EC 3.-.-.-) is an enzyme belonging to the class of enzymes that catalyze the cleavage of a chemical bond in the substrate under addition of water, e.g., esterases, glycosidases, lipases, nucleotidases, nucleosidases, peptidases, proteases and phosphatases. The hydrolase is preferably a glycosylase (EC 3.2.-.-) and more preferably a nucleosidases (EC 3.2.2.-).

A pyrimidine and/or purine hydrolase catalyzes (EC 3.2.2.-) the hydrolysis of the N-glycosidic bond in ribonucleosides. They are generally Ca²⁺-containing metalloenzymes (Singh et al. (2017), Protein Sci.; 26(5): 985-996).

In accordance with a more preferred embodiment 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).

An inosine-uridine preferring nucleoside hydrolase (IUNH) belonging to the class of purine nucleosidases is capable of catalyzing the conversion: purine/pyrimidine D-ribonucleoside + H2O => purine/pyrimidine nucleobase + D-ribose.

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.

In accordance with an even more preferred embodiment the inosine-uridine preferring nucleoside hydrolase (IUNH) has a kcat/Kivi value for uridine hydrolysis of 10⁴ M ¹ s ¹ or higher.

The kcat/K_M value (also called k_cat/K_M ratio) is a measure of the catalytic efficiency of an enzyme. The catalytic constant k_cat 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_cat/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_cat and a low value of K_M are expected for the best substrates. If k_cat/K_M approaches the diffusion limit (about 10⁸-10⁹ M^-1 s^-1), the enzyme cannot catalyze the reaction any better and is said to have reached "catalytic perfection". In accordance with a preferred embodiment (i) 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; (ii) 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: 3 or 4 and that encodes a polypeptide with IUNH activity; (ii-c) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or 2; (ii-d) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or 2 with a deletion, substitution, insertion, and/or addition of one or more amino acid(s) (such as with increasing preference 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or 1 and 1 amino acid(s)), and wherein said polypeptide comprises IUNH activity; and (ii-e) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence with at least 80%, preferably at least 90% and most preferably at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or 2, and wherein said polypeptide comprises IUNH activity; and/or (iii) the vector expresses the nucleotide sequence of SEQ ID NO: 3 or 4 or expresses a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

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.

Further embodiments of the present invention include IUNH enzymes from related parasite species of humans or mammals. Of note, 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. In preferred embodiments of the present invention, 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.

As defined herein, sequence identities of at least 80% are with increased preference at least 90% identical, and more preferred at least 95%. However, also envisaged by the invention 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%.

In accordance with another preferred embodiment the enzyme, nucleic acid molecule and/or vector is formulated into a pharmaceutical composition and said pharmaceutical composition is used for treating cancer.

Hence, the enzyme, nucleic acid molecule or vector of the invention can be formulated as a pharmaceutical composition.

In accordance with the present invention, 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. However, 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. Furthermore, if for example 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. More preferably, 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 may be administered, for example, orally, parenterally, such as subcutaneously, intravenously, intramuscularly, intraperitoneally, intrathecally, transdermally, transmucosally, subdurally, locally or topically via iontophoresis, sublingually, by inhalation spray, aerosol or rectally and the like in dosage unit formulations optionally comprising conventional pharmaceutically acceptable carriers or excipients. Intraperitoneal administration, in particular intraperitoneal injection, is preferred for preclinical studies because this route was used in the appended examples to treat cancer in mice.

The pharmaceutical composition preferably comprises in addition at least one pharmaceutically acceptable carrier, excipient or diluent. By "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.

Examples of suitable pharmaceutical carriers are well known in the art and 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.

In accordance with a more 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.

While pharmaceutical 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.

In accordance with a preferred embodiment 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. Apart from the nucleobase, 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.

If the growth of a cancer depends on extracellular uridine 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. This is in particular because the enzyme that eliminates pyrimidine and/or purine nucleosides in vivo according to the present invention cleaves and eliminates the nucleosides in vivo in the extracellular milieu, such as in the blood and in the interstitial space. This is also demonstrated in Example 14 for murine lung adenocarcinoma cells.

In accordance with a preferred embodiment the cancer is a lung cancer, wherein the lung cancer is preferably non-small cell lung cancer (NSCLC).

A significant reduction of tumor growth in a mouse model for lung cancer by an enzyme that eliminates pyrimidine and/or purine nucleosides is demonstrated herein below, in Example 13. Lung cancer is the leading cause of cancer-related deaths worldwide, accounting for the highest mortality rates among both men and women. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. It accounts for over 80% of lung cancer cases.

For these reasons the treatment of lung cancer, in particular non-small cell lung cancer (NSCLC), is preferred.

In accordance with another preferred embodiment the enzyme is used for treating cancer, preferably lung cancer, pancreatic cancer or breast cancer.

Among these three cancer types lung cancer is preferred in view of the Example 13.

As discussed herein above, for the treatment according to the invention the enzyme and/or the nucleic acid molecule encoding the enzyme and/or the vector expressing the nucleic acid molecule may be used. According to this preferred embodiment the enzyme is used and, thus, a protein is to be administered to the subject to be treated.

Also, in the appended examples the protein as such was used in cancerous mice and various cancer cell lines and is shown to inhibit tumor growth.

In accordance with a more preferred embodiment 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.

It is shown in the examples herein below that the enzyme of the invention can also be used and is stable enough for use in non-encapsulated form. This in particular holds true for the enzyme being in the form of a protein conjugate or a fusion protein as will be further detailed herein below. 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.

In accordance with another more preferred embodiment 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.

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.

In accordance with a preferred embodiment 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).

In case the enzyme is fused to a heterologous compound, the fusion partner is a proteinaceous compound (i.e. a compound having an amino acid sequence). In this case 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. In the case of a "fusion protein", 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.

In case the enzyme is conjugated to a heterologous compound, the conjugation partner is generally a non-proteinaceous compound. In this case 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.

The term "linker", as used in accordance with the present invention, preferably relates to peptide linkers, i.e. a sequence of amino acids, as well as to non-peptide linkers.

A peptide linker as envisaged by the present invention is a (poly)peptide linker of at least 1 amino acid in length. Preferably, the linker is 1 to 100 amino acids in length. More preferably, the linker is 5 to 50 amino acids in length and even more preferably, the linker is 10 to 20 amino acids in length. Preferably, the linker is a flexible linker using e.g. the amino acids glycine and/or serine. Preferably, the linker sequences are (Gly₄Ser)3, or (Gly₄Ser)2. The length and sequence of a suitable linker depends on the composition of the respective protein conjugate. Methods to test the suitability of different linkers are well known in the art and include e.g. the comparison of the enzymatic activity of the inosine-uridine preferring nucleoside hydrolases (IUNH). Preferably, if the fusion protein comprises a PAS biopolymer, which is structurally disordered, a linker is absent.

As is evident from the above, the linker may in certain embodiments, in particular for protein conjugates, be a non-peptide linker.

The term "non-peptide linker", as used in accordance with the present invention, refers to linkage groups having two or more reactive groups but excluding peptide linkers as defined above. For example, 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. Examples of 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). The reactive groups at both ends of the non-peptide linker may be the same or different. For example, the non-peptide linker may have a maleimide group at one end and an aldehyde group at the other end. Alternatively, 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. In this case, 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) is preferred and is illustrated in the appended examples. In a preferred embodiment, 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 (polySia) 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) 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. (2018) Bioconjug. Chem. 29(7):2232-2238.

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 (PEG) 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. In particular, 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.

In accordance with a more preferred embodiment 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 term "about" as used herein means with increasing preference ±20%, ±10% and ±5% or the respective value or number.

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. Among this list, ASPAAPAPASPAAPAPSAPA (SEQ. ID NO: 5) is most preferred, and this repeat unit is used for the PASylation tag PAS#l(200) as illustrated in the Example (10 repeats).

As used herein, 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. In particular, 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. Freeman and Company, New York; Creighton (1993) Proteins - Structures and Molecular Properties, 2nd ed., W. H. Freeman and Company, New York; Smith (1996) Fold. Des., l:R95-R106) and in the patent literature, e.g., WO 2011/144756 and WO 2008/155134.

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. The term "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. The term "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. For example, 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. With respect to "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).

In accordance with an even more preferred embodiment the polypeptide comprising proline and alanine and optionally serine consists of proline, alanine and serine. In accordance with a yet more preferred embodiment the proline residues constitute more than 4% and less than 40% of said polypeptide.

The 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.

Preferably, 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. More preferably, 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.

Also preferred herein is 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. As mentioned above, the serine residues comprise the remaining amount of said amino acid sequence. Accordingly, 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. For example, the term 10 % may also relate to 11 % or 12 % or to 9 % and 8 %, respectively.

In accordance with another even more preferred embodiment the polypeptide of proline and alanine and optionally serine consists of proline and alanine. In accordance with a yet more preferred embodiment the 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.

Preferably, 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.

Vice versa, 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. Also preferred herein is an 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. Even more preferably, 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.

Accordingly, 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. Alternatively, 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. For example, the term 10 % may also relate to 11 % or 12 % and to 9 % or 8 %, respectively.

In accordance with a further even more preferred embodiment 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_tot 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.

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. In this context the term repeat means that a nucleotide sequence occurs at least twice within the nucleotide sequence analyzed. When counting the frequencies 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.

Furthermore, each shorter repeat contained within a longer nucleotide sequence repeat is counted separately. For example, if the analyzed nucleotide sequence contains two GCACC nucleotide stretches (i.e., repeats), GCAC and 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. Of note, only repeats on the coding strand of the nucleic acid molecule are considered.

A person skilled in the art can identify nucleotide sequence repeats either manually or with the aid of generic software programs such as the Visual Gene Developer (Jung (2011) BMC Bioinformatics 12:340), available for download at http://www.visualgenedeveloper.net, or the Repfind tool (Betley (2002) Curr. Biol. 12:1756-1761), available at http://zlab.bu.edu/repfind. However, not every algorithm detects each kind of repeat, e.g., the result of the Visual Gene Developer does not include overlapping repeats. Thus, results of software tools have to be checked and, if necessary, manually corrected. Alternatively, the algorithm termed 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.

In accordance with a yet more preferred embodiment the Nucleotide Repeat Score (NRS) is lower than 5000, 2000, 1000, 750, 500, 200, 100, preferably lower than 50 and most preferably lower than 35.

In other words, 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.

Even more particularly preferred are 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.

In accordance with another yet more preferred embodiment the nucleotide sequence encoding the PAS polypeptide comprises said repeats, wherein said repeats have a maximum length n_max, wherein n_max is determined according to the formula: < 17 ₊ ^ N.2 , n L max 600 and wherein N_tot 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.

The term "maximum length" or "maximal length" or "n_max" 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. In other words, the term "maximum length" or "maximal length" or "n_max" 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.

As explained above, 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.

In accordance with an even more preferred embodiment the 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: ASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAP SAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPA APAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA. Also contemplated herein are 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%.

As shown in Fig 8 of the application the in vivo clearance of the enzyme of the invention with a PAS(#l)200 tag is surprisingly slow, with a half-live of more than 24h.

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 third aspect to a nucleic acid molecule encoding the fusion protein of the second aspect.

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 comprising the fusion protein of the second aspect, the nucleic acid molecule of the third aspect or the vector of the fourth aspect.

The definitions and preferred embodiments of the first aspect of the invention apply mutatis mutandis to the second to fifth aspect of the invention as far as being amenable for combination therewith.

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.

As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends on. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, 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, it is to be understood that 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, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1, a dependent claim 2 referring back to claim 1, and a dependent claim 3 referring back to both claims 2 and 1, it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to

3, it follows that the combination of the subject-matter of claims 4 and 1, of claims 4, 2 and 1, of claims

4, 3 and 1, as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The Figures show:

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. outside the expression cassette flanked by the Xba\ and Hindu 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 His6-CflUNH-PAS(#l)200 is identical except that CflUNH is encoded.

Fig. 3: Plasmid map of pASK75-T7RBS2-His₆-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. outside the expression cassette flanked by the Xba\ and H/ndlll 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 His6-PAS(#l)200-CflUNH is identical except that CflUNH is encoded.

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. This effect is clearly due to the Pro/Ala/Ser (PAS) biopolymer, which poorly binds SDS (Breibeck and Skerra (2018) Biopolymers 109:e23069), as the IUNH enzymes which are nor fused with the PAS polypeptide, having a calculated mass of approximately 35.6 kDa, exhibit normal electrophoretic mobility. Fig. 6: Analysis of the purified recombinant LmlUNH-PAS(#l)200, CflUNH-PAS(#l)200 and LmlUNH(C301G)-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 a subtractive cation exchange chromatography (CEX) followed by anion exchange (AEX) and sizeexclusion chromatography (SEC). 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. 5.

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. The data points were fitted to the equation vo = v_maxx[S]/(K_M+[S]) by non-linear regression, resulting in K_M = 1.47±0.22 mM and v_max = 0.31810.016 mAU/min. Using the total enzyme concentration [E_tot] = 0.369 pM, the turnover number was calculated as k_cat = v_max/[E_tot] = 81 s ¹.

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. Male C56BL/6N mice, 7-8 weeks old, were injected intraperitoneally (i.p.) with a dose of 5 mg of the test protein (enzyme) per kg body weight (b.w.). Blood samples were collected after 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h. Plasma samples were quantitatively assayed for the concentrations of Hisg- PAS#l(200)-CflUNH and LmlUNH(C301G)-PAS#l(200) using a sandwich ELISA. To estimate the plasma half-life, the resulting concentration values were plotted against time post i.p. injection and numerically fitted using Phoenix WinNonlin 6.3 software, assuming a first order invasion from peritoneum and first order-elimination via the kidneys in accordance with the Bateman function. As result, the elimination phase determined for His6-PAS#l(200)-CflUNH and LmlUNH(C301G)- PAS#l(200) is surprisingly slow, with half-lives of 25.816.3 h and 29.715.9 h, respectively, due to the fusion with a PAS biopolymer. 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. 3 days after the injection, 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. One group received treatment with His6-PAS#l(200)-CflUNH in PBS and the second group received vehicle (PBS). The data represent the mean of 7 individual animals per group (indicated by the open circles), error bars = S.E.M. (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. Treatment of mice was initialized 3 days after injection of HKP1 cells (and the initial luciferase measurement as shown in A). His6-PAS#l(200)-CflUNH and vehicle (PBS) were administered intraperitoneally every 3 days. A higher initial loading dose of His6-PAS#l(200)-CflUNH (7.9 mg/kg b.w.) was followed by a lower repeated maintenance dose (5 mg/kg b.w.). The signal is expressed as total emission ratio compared to signals from day 0 of treatment. Mean values of 7 individual animals are shown, error bars = S.E.M, **p<0.01 according to Sidak's multiple comparisons test. The results show that 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. Data are represented proportionally to 0 pg/ml His6-PAS#l(200)-CflUNH. Statistical comparison was performed against the value for 0 pg/ml His6-PAS#l(200)-CflUNH. Error bars represent S.E.M; *p<0.05,

**p<0.01, ***p<0.001 according to unpaired t-test. Fig. 11: In vitro effect of His6-PAS#l(200)-CflUNH treatment on HKP1 cancer cells with impaired pyrimidine de novo synthesis. (A) 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. (B) 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. (C) 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. 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. Mean values of 3 independent experiments are shown, error bars represent S.E.M, statistical comparison was performed against the value for 0 pg/ml Hisg-PAS#l(200)-CflUNH; *p<0.05, **p<0.01 according to 2-way ANOVA Tukey's multiple comparisons test.

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. Data are represented proportionally to 0 pg/ml LmlUNH(C301G)-PAS#l(200) and statistical comparison was performed against the value for 0 pg/ml LmlUNH(C301G)-PAS#l(200). Error bars represent S.E.M; *p<0.05 according to unpaired t-test.

The examples illustrate the invention.

Example 1: Gene cassettes for different IUNH enzymes

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. Four singular restrictions sites (Nde\, EcoQ109l, Sapl and H/ndlll) were incorporated into the synthetic gene to facilitate (a) insertion into the expression plasmid (see Example 2), using Nde\ and H/ndlll, and (b) fusing the enzymes with a PAS biopolymer at the genetic level either at its N-terminus, using EcoQ109l, or at its C-terminus, using Sap\. The corresponding gene cassettes (SEQ ID NOs 9 and 10) encoding Hisg-LmlUNH (SEQ ID NO: 11) and His₆-CflUNH (SEQ ID NO: 12), respectively, were obtained as DNA strings from Thermo Fisher Scientific (Munich, Germany). The Nde\ and H/ndlll restriction sites that were used for cloning can be seen in the nucleotide sequences of pASK75-T7RBS2-His₆-LmlUNH (SEQ ID NO: 14) and pASK75-T7RBS2-His₆-CflUNH (SEQ ID NO: 15).

Example 2: Construction of expression vectors for IUNH without the PAS biopolymer

For cloning of the synthetic gene fragments coding for the different enzymes from Example 1 a derivative of pASK75 (Skerra, A. (1994) Gene 151:131-135), pASK75-T7RBS2-MP-Sap/-Stop, which carries the T7 ribosomal binding site followed by a cloning region flanked by Nde\ and H/ndlll, including a Sap\ recognition site in reverse orientation (SEQ ID NO: 13), was employed. This vector was cut with Nde\ and H/ndlll and then ligated with the accordingly cut synthetic DNA fragment. Resulting plasmids were designated pASK75-T7RBS2-His₆-LmlUNH (SEQ ID NO: 14; Fig. 1) and pASK75-T7RBS2-His₆- CflUNH (SEQ ID NO: 15).

After transformation of E. coli XLl-Blue (Bullock (1987) Biotechniques 5:376-378), 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 using the Mix2Seq Kit (Eurofins Genomics, Ebersberg, Germany) with appropriate oligodeoxynucleotide primers (Eurofins Genomics) hybridizing on both sides.

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. coli XLl-Blue, 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, His₆-LmlUNH-PAS(#l)200 (SEQ ID NO: 16) was designated pASK75-T7RBS2-His₆-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-His₆-CflUNH-PAS(#l)200 (SEQ ID NO: 31) encoding His₆-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. coli XLl-Blue, 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 plasmids encoding the enzyme carrying a 200 residue PAS biopolymer, His6-PAS(#l)200-LmlUNH (SEQ ID NO: 18), was designated pASK75-T7RBS2-His₆-PAS(#l)200-LmlUNH (SEQ ID NO: 19; Fig. 3). 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 His₆-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 His₆-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-His₆-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.

Example 6: Substituting a critical Cys residue at position 301 in LmlUNH-PAS#l(200)

To replace Cys at position 301 (numbering according to the amino acid sequence of the mature protein; UniProt ID: P83851) in LmlUNH-PAS#l(200) by Gly, the QuikChange site-directed mutagenesis method was used (Papworth, C., Bauer, J. C., Braman, J. and Wright, D. A. (1996) Strategies 9(3): 3-4.). Therefore, a pair of mutagenic primers, SEQ ID NO: 27 and SEQ ID NO: 28, was used in a PCR reaction with pASK75-T7RBS2-LmlUNH-PAS(#l)200 as template. After digestion of the methylated template plasmid DNA with Dpn\, E. coli XLl-Blue was transformed with the reaction mixture and plated on LB/Amp agar.

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).

Example 7: Bacterial production and purification of IUNH enzymes

Hisg-LmlUNH (calculated mass: 35628 Da) and Hisg-CflUNH (calculated mass: 35644 Da) were produced at 30 °C in E. coli NEBExpress (New England Biolabs, Ipswich, MA) harboring the corresponding expression plasmids from Example 2 using shake flask cultures with 2 L LB medium (Sambrook et al., 1989) containing 100 mg/l ampicillin (LB/Amp). Induction of recombinant gene expression was performed by adding 200 pg/l anhydrotetracycline at OD550 = 0.6 and incubation was continued for 3 h (typically resulting in OD550 « 1.0 at harvest). Cells were harvested by centrifugation, resuspended in IMAC running buffer (50 mM NaPi, 500 mM NaCI pH 7.5) and disrupted using a French pressure cell (SLM Aminco, Urbana, IL). The total cell extract was cleared by centrifugation and dialyzed against IMAC running buffer. Then, each enzyme was purified by immobilized metal ion affinity chromatography (IMAC) using Ni²⁺-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 Na2HPO₄ 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.

SDS-PAGE was performed using a high molarity Tris buffer system (Fling and Gregerson (1986) Anal. Biochem. 155:83-88). Protein concentrations were determined according to the absorption at 280 nm using calculated extinction coefficients (Gasteiger, E., Gattiker, A., Hoogland, C. et al. (2003) Nucleic Acids Res. 31:3784-3788) of 21680 M ¹ cm'¹ both for Hisg-LmlUNH and Hisg-CflUNH as well as their respective N- and C-terminal PAS#l(200) fusion proteins (as PAS biopolymers do not contribute to UV absorption due to the lack of aromatic side chains).

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 (His₆-PAS(#l)200-LmlUNH and His₆-LmlUNH-PAS(#l)200: calculated mass of both proteins: 52146 Da; His₆-PAS(#l)200-CflUNH and His₆-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. Induction of recombinant gene expression was performed by adding 200 pg/l anhydrotetracycline at OD550 = 0.6 and incubation was continued for 3 h (typically resulting in OD550 « 1.0 at harvest). 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 Ni²⁺-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.

In this manner, homogeneous protein preparations were obtained for all four PASylated enzymes (Fig. 5) with yields of 1.2, 1.3, 1.0 and 0.9 mg L ¹ OD ¹ for His₆-PAS(#l)200-LmlUNH, Hisg-LmlUNH- PAS(#l)200, His₆-PAS(#l)200-CflUNH and His₆-CflUNH-PAS(#l)200, respectively.

Example 9: Bacterial production and purification of IUNH variants carrying a PAS#1 biopolymer at the C-terminus without a His₆-tag

LmlUNH-PAS(#l)200, CflUNH-PAS(#l)200 and LmlUNH(C301G)-PAS(#l)200 (calculated masses: 50899 Da, 50915 Da and 50853 Da, respectively), were produced at 30 °C in E. coli NEBExpress harboring the corresponding expression plasmids from Examples 5 and 6 using shake flask cultures with 2 L LB/Amp medium. Induction of recombinant gene expression was performed by adding 200 pg/l anhydrotetracycline at OD550 = 0.6 and incubation was continued for 3 h (typically resulting in OD550 « 1.0 at harvest). Cells were harvested by centrifugation, resuspended in IEX running buffer (20 mM Tris/HCI pH 8.0) and disrupted using a French pressure cell. The total cell extract was cleared by centrifugation and dialyzed against IEX running buffer. The IUNH variants were first purified by subtractive cation exchange chromatography (CEX) using a Resource S column (5 ml bed volume; Cytiva Europe). The flow through was then loaded on AEX column (Resource Q, 5 ml bed volume; Cytiva Europe) and elution was performed by applying an ascending NaCI concentration gradient, which was followed by gel filtration on a Superdex S200 HiLoad 16/60 column (Cytiva Europe) with PBS as running buffer.

For LmlUNH-PAS(#l)200, CflUNH-PAS(#l)200 and LmlUNH(C301G)-PAS(#l)200, homogeneous protein preparations were obtained (Fig. 6) with yields of 1.3, 1.3 and 1.4 mg L ¹ OD ¹, respectively.

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). To determine the kinetic constants, 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). The data points were fitted to the equation vo = v_maxx[S]/(K_M+[S]) by non-linear regression, resulting in K_M and Vmax values for the different tested lUNH/substrate combinations (exemplary shown for LmlUNH(C301G)-PAS(#l)200 in Fig. 7). The total enzyme concentration, [E_tot], in each reaction was used to calculate the turnover number, k_cat = v_max/E_tot. The ratio kcat/K_M defines the catalytic efficiency of an enzyme. The resulting kinetic parameters are summarized in the following Tables 1 and 2.

Table 1: IUNH activity parameters for uridine as substrate. Table 2: IUNH activity parameters for inosine as substrate.

Example 11: Determination of the PAS-IUNH pharmacokinetics in 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.

Example 12: Measurement of prolonged plasma half-life for PASylated IUNH enzymes

For the quantitative detection of IUNH enzymes in an ELISA, the wells of a 96-well microtitre plate (Maxisorb, NUNC, Denmark) 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. 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). After washing twice with PBS/T and twice with PBS, 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.

To estimate the plasma half-life of Hisg-PAS#l(200)-CflUNH and LmlUNH(C301G)-PAS#l(200) in mice, the concentration values, c(t), which had been determined for each time point in triplicate from these ELISA measurements were plotted against the time post injection, t. These data were numerically fitted using Phoenix WinNonlin 6.3 software (Certara, Princeton, NJ) assuming a first order invasion from peritoneum and first order-elimination via the kidneys as described by the Bateman function: wherein D is the applied dose in mg/kg b.w., V is the apparent volume of distribution in ml/kg b.w., kOl is the absorption rate and klO is the elimination rate in 1/h.

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

Orthotopic lung tumors were induced in 6-10-week-old male and female C56BL/6 N mice by tail vein injection of HKP1 cells (lxlO⁵ cells resuspended in 100 pl of PBS). 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. To this end, 10 pl of luciferin (15 mg/ml in PBS; Cat. No. 122799; PerkinElmer, Waltham, MA) per g animal weight was 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. After 5 min, mice were placed into the Lago X whole body spectral scanner (Spectral Instruments Imaging, Tucson, 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. The results show that His6-PAS#l(200)-CflUNH led to significantly reduced tumor growth over 11 days of His6-PAS#l(200)-CflUNH treatment as shown in Fig. 9B. This significant difference was observed in two independent experiments. Mouse weight was monitored throughout the treatment period and remained stable (Fig. 9C), indicating the absence of toxic side effects.

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; 2xl0³) and AsPCl human pancreatic adenocarcinoma cells (Sigma Aldrich, Burlington, MA; 5xl0³) were seeded in 96-well plates in RPMI medium. LLC1 Lewis lung carcinoma cells (ATCC, Manassas, VA; 5xl0³) were seeded in a 96-well plate in 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. 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. For quantification of cell growth, cells were stained with 0.05 % (w/v) crystal violet for 30 min at room temperature. The supernatant containing excess crystal violet was discarded, and cells were washed 3 times with PBS. Then, 100 pl of 1 % (w/v) SDS solution was added to each well, and the intensity of staining, corresponding to the number of cells in the well, was quantified by measuring the absorbance at 595 nm using an Infinite m200 spectrometer (Tecan, Mannedorf, Switzerland). Already 50 pg/ml of His6-PAS#l(200)-CflUNH significantly decreased the number of cells that had grown for all 3 tested cell lines compared to the condition in the absence of the enzyme (Fig. 10).

Example 15: Suppressive effect of His6-PAS#l(200)-CflUNH on the growth of HKP1 cancer cells deficient in pyrimidine synthesis

Pyrimidine synthesis deficient 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. Next, the effect of Hisg- PAS#l(200)-CflUNH treatment on the growth of the HKP1 DHODH KO cells in the presence of uridine was tested. To this end, 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. Cell growth was quantified using crystal violet as above. The data show that treatment with His6-PAS#l(200)-CflUNH at a concentration >0.5 pg/ml significantly affected the ability of HKP1 DHODH KO cells to proliferate (Fig 11C). This result demonstrates that His6-PAS#l(200)-CflUNH suppresses the growth-promoting effect of external uridine.

Example 16: Suppressive effect of LmlUNH(C301G)-PAS#l(200) on the growth of cancer cell lines

HKP1 murine lung adenocarcinoma cells (see Example 13 supra; 2xl0³) 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. After 48 h, cells were fixed for 30 min with 4 % (w/v) paraformaldehyde (Avantor, Gliwice, Poland) at room temperature and washed 3 times with PBS. For quantification of cell growth, cells were stained with crystal violet as described in Example 14 supra. The addition of 400 pg/ml of LmlUNH(C301G)-PAS#l(200) significantly decreased the number of cells compared to the condition in the absence of the enzyme (Fig. 12).

Claims

1. (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.

2. The enzyme, nucleic acid molecule and/or vector for use of claim 1, wherein the enzyme is a pyrimidine and/or purine hydrolase.

3. The enzyme, nucleic acid molecule and/or vector for use of claim 2, wherein the pyrimidine and/or purine hydrolase is an inosine-uridine preferring nucleoside hydrolase belonging to the class of purine nucleosidases (EC 3.2.2.1).

4. The enzyme, nucleic acid molecule and/or vector for use of claim 3, wherein the inosine-uridine preferring nucleoside hydrolase has a k_cat/l<M value for uridine hydrolysis of 10⁴ M ¹ s ¹ or higher.

5. The enzyme, nucleic acid molecule and/or vector for use of any one of claims 1 to 4, wherein

(i) 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;

(ii) the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 3 or 4 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto; and/or

(iii) the vector expresses the nucleotide sequence of SEQ ID NO: 3 or 4 or expresses a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

6. The enzyme, nucleic acid molecule and/or vector for use of any one of claims 1 to 5, wherein the enzyme, nucleic acid molecule and/or vector is the sole pharmaceutically active compound being used for treating cancer.

7. The enzyme, nucleic acid molecule and/or vector for use of any one of claims 1 to 6, wherein the cancer is a cancer whose growth depends on extracellular uridine.

8. The enzyme, nucleic acid molecule and/or vector for use of any one of claims 1 to 6, wherein the cancer is a lung cancer, wherein the lung cancer is preferably non-small cell lung cancer (NSCLC).

9. The enzyme, nucleic acid molecule and/or vector for use of any one of claims 1 to 8, wherein the enzyme is used for treating cancer.

10. The enzyme, nucleic acid molecule and/or vector for use of any one of claims 1 to 9, wherein 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 polyethylene glycol (PEG).

11. The enzyme, nucleic acid molecule and/or vector for use of claim 10, wherein the heterologous compound is a PAS biopolymer and 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 about 200, 400 or 600 amino acids, and wherein said polypeptide forms a random coil conformation.

12. The enzyme, nucleic acid molecule and/or vector for use of claim 11, wherein the polypeptide comprising proline and alanine and optionally serine consists of proline, alanine and serine.

13. The enzyme, nucleic acid molecule and/or vector for use of claim 11, wherein the polypeptide of proline and alanine and optionally serine consists of proline and alanine.

14. The enzyme, nucleic acid molecule and/or vector for use of any one of claims 11 to 13, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 9 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

15. 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 in any one of claims 10 to 14.