PARP1- and immune-checkpoint inhibitors for the treatment of cancer patients overexpressing ubqln1 and / or ubqln4

Abstract

The present invention relates to an Poly(ADP-Ribose) Polymerase 1 (PARP-1) inhibitor and / or an immune checkpoint inhibitor for the treatment of cancer in a subject that overexpresses Ubiquilin 1 (UBQLN1) and / or Ubiquilin 4 (UBQLN4) as well as a method for determining whether a cancer subject is amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor based on the detection of the mRNA and / or protein expression level of UBQLN1 and / or UBQLN4 in a sample obtained from said cancer subject.

Description

[0001] PARP1- and immune-checkpoint inhibitors for the treatment of cancer patients overexpressing UBQLN1 and / or UBQLN4

[0002] The present invention relates to an Poly(ADP-Ribose) Polymerase 1 (PARP-1) inhibitor and / or an immune checkpoint inhibitor for the treatment of cancer in a subject that overexpresses Ubiquilin 1 (LIBQLN1) and / or Ubiquilin 4 (LIBQLN4) as well as a method for determining whether a cancer subject is amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor based on the detection of the mRNA and / or protein expression level of LIBQLN1 and / or LIBQLN4 in a sample obtained from said cancer subject.

[0003] 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.

[0004] In response to genotoxic stress, cells activate a complex signaling network, termed the DNA damage response (DDR). The main DNA double-strand break (DSB) repair pathways are non-homologous end joining (NHEJ) and homologous recombination (HR). Error-prone NHEJ operates throughout the cell cycle, whereas error-free HR is restricted to late S- and G2, when a repair DNA template is available. The balance between HR and NHEJ is essential for correct DSB repair, chemo- and radio-therapy sensitivity and cell survival.

[0005] HR-defective cancers are characterized by impaired DNA repair mechanisms and typically result from mutations or deficiencies in genes involved in the HR pathway, such as BRCA1 and BRCA2. Poly ADP-ribose Polymerase 1 (PARP1) inhibitors are a class of targeted therapies designed to exploit the vulnerabilities of HR-defective cancers. PARP1 is an enzyme involved in DNA repair, specifically in the Base Excision Repair (BER) pathway. In normal cells, when single-strand DNA breaks occur, PARP1 initiates the repair process. However, in HR-defective cancer cells, combining PARP1 inhibition with their already compromised DNA repair pathways creates a synthetic lethality. PARP1 inhibitors have shown clinical efficacy in treating HR-defective cancers, particularly those associated with BRCA1 and BRCA2 mutations. These inhibitors have gained approval for treating various malignancies, including ovarian, breast, and pancreatic cancers. Clinical trials and real-world evidence have demonstrated substantial benefits, including extended progression-free survival and improved overall survival rates. Genetic testing is performed to identify patients with HR pathway deficiencies, as it enables personalized treatment decisions and improves patient outcomes. However, only a small fraction of patients and only a limited number of cancers are HR-defective.

[0006] The precise biomarkers for a predicting either response or resistance to immune checkpoint inhibitors are unknown. Currently it is only known that a high tumor mutational burden, particularly in the context of MMR deficiency, and high level PD-L1 expression appear to be predictive of response.

[0007] In view of the above, there is an ongoing need in order to treat and identify those cancer patients that are amenable for the treatment with PARP1- and / or immune-checkpoint inhibitors. This need is addressed by the present invention.

[0008] Accordingly, the present invention relates in a first aspect to a Poly(ADP-Ribose) Polymerase 1 (PARP-1) inhibitor and / or an immune checkpoint inhibitor for the treatment of cancer in a subject that overexpresses Ubiquilin 1 (LIBQLN1) and / or Ubiquilin 4 (LIBQLN4).

[0009] Also described herein is a corresponding method of treatment. Hence, the present invention also relates to a method of treating a subject having cancer and overexpressing Ubiquilin 1 (UBQLN1) and / or Ubiquilin 4 (UBQLN4) by administering to the subject (an effective amount of) a PARP-1 inhibitor and / or an immune checkpoint inhibitor.

[0010] Poly(ADP-Ribose) Polymerase 1 (PARP-1) (ADPRT; EC 2.4.2.30) uses NAD as substrate to catalyze both the covalent transfer of ADP-ribose to a variety of nuclear protein acceptors and subsequently the transfer of an additional 60 to 80 ADP-ribose units to the initial moiety. Nuclear proteins that become predominantly poly(ADP-ribosyl)ated include nucleosomal core histones, histone H1 , HMG proteins, and topoisomerases I and II. ADP ribosyltransferase is required for cellular repair. Inhibitors of this enzyme potentiate the lethal effects of noxious agents. During repair, NAD+ is consumed and the NAD+ content of the cell decreases. Concomitantly, nuclear proteins are ADP-ribosylated. The enzyme is induced by singlestrand breaks in DNA, which serve as co-substrate for the reaction. The amino acid sequence of human PARP-1 is shown in SEQ ID NO: 1 and the nucleotide sequence of PARP-1 is shown in SEQ ID NO: 2.

[0011] Also described herein are for each of the recited sequences according to the SEQ ID NOs sequence which are with increasing preference at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5% and at least 99% identical thereto.

[0012] In accordance with the present invention, the term “percent (%) sequence identity” describes the number of matches (“hits”) of identical nucleotides / amino acids of two or more aligned nucleic acid or amino acid sequences as compared to the number of nucleotides or amino acid residues making up the overall length of the template nucleic acid or amino acid sequences. In other terms, using an alignment, for two or more sequences or subsequences the percentage of amino acid residues or nucleotides that are the same (e.g. 70%, 75%, 80%, 85%, 90% or 95% identity) may be determined, when the (sub)sequences are compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. This definition also applies to the complement of any sequence to be aligned.

[0013] Nucleotide and amino acid sequence analysis and alignment in connection with the present invention are preferably carried out using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Nucleic Acids Res. 25:3389-3402). BLAST can be used for nucleotide sequences (nucleotide BLAST) and amino acid sequences (protein BLAST). The skilled person is aware of additional suitable programs to align nucleic acid sequences.

[0014] Immune checkpoints engage when proteins on the surface of immune cells called T cells recognize and bind to partner proteins on other cells, such as some tumor cells. These proteins are called immune checkpoint proteins. When the checkpoint and partner proteins bind together, they send an “off” signal to the T cells. This can prevent the immune system from destroying the cancer. Immunotherapy drugs called immune checkpoint inhibitors work by blocking checkpoint proteins from binding with their partner proteins. This prevents the “off’ signal from being sent, allowing the T cells to kill cancer cells.

[0015] Ubiquilins contain an N-terminal ubiquitin-like domain and a C-terminal ubiquitin-associated domain. They physically associate with both proteasomes and ubiquitin ligases, and thus are thought to functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation.

[0016] Ubiquilin 1 (LIBQLN1) is associated with protein degradation and aggregation of misfolded proteins, and may be involved in neurodegenerative diseases. Ubiquilin-1 has been reported to act as a molecular chaperone for amyloid precursor protein (APP), a protein associated with Alzheimer's disease. It plays an important role in the regulation of different protein degradation mechanisms and pathways including ubiquitin-proteasome system (UPS), autophagy and endoplasmic reticulum-associated protein degradation (ERAD) pathway. Mediates the proteasomal targeting of misfolded or accumulated proteins for degradation by binding (via UBA domain) to their polyubiquitin chains and by interacting (via ubiquitin-like domain) with the subunits of the proteasome. UBQLN1 shares a high degree of similarity with related ubiquilins including UBQLN2 and UBQLN4.

[0017] The amino acid sequence of human UBQLN1 is shown in SEQ ID NO: 3 and the nucleotide sequences of UBQLN1 are shown in SEQ ID NOs 4 and 5 (noting that two splice variants are known; transcripts 1 and 2). The nucleotide sequences herein are cDNA sequences which means that in the corresponding mRNA sequences the nucleotide T is replaced by U.

[0018] Ubiquilin 4 (UBQLN4) regulates proteasomal protein degradation. It mediates the proteasomal targeting of misfolded, mislocalized or accumulated protein. It is a key regulator of DNA repair that represses homologous recombination repair: in response to DNA damage, recruited to sites of DNA damage following phosphorylation by ATM and acts by binding and removing ubiquitinated MRE11 from damaged chromatin, leading to MRE11 degradation by the proteasome. It acts as an adapter protein that recruits UBQLN1 to the autophagy machinery. Finally, UBQLN4 mediates the association of UBQLN1 with autophagosomes and the autophagy-related protein LC3 (MAP1LC3A / B / C) and may assist in the maturation of autophagosomes to autolysosomes by mediating autophagosome-lysosome fusion.

[0019] The amino acid sequence of human UBQLN4 is shown in SEQ ID NO: 6 and the nucleotide sequences of UBQLN4 are shown in SEQ ID NOs 7 and 8 (noting that two splice variants are known; transcripts 1 and 2).

[0020] The subject to be treated is a vertebrate, preferably a mammal and is most preferably human. The subject to be treated has cancer. Cancer is an abnormal malignant new 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 lung cancer, breast cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, bladder cancer, salivary gland cancer, pancreatic cancer, thyroid cancer, kidney 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 lung cancer is preferred.

[0021] With respect to preferred kinds of cancer reference is made to Figures 1 and 2. Here the overexpression of LIBQLN1 (Figure 2) and LIBQLN4 (Figure 1) has been tested in a number of different cancer types. The cancer overexpressing LIBQLN1 is preferably selected with increasing preference from the top 20, top 15 and top 10 kinds of cancer that show the highest LIBQLN1 expression levels in Figure 2. Similarly, the cancer overexpressing LIBQLN4 is preferably selected with increasing preference from the top 20, top 15 and top 10 kinds of cancer that show the highest LIBQLN1 expression levels in Figure 1.

[0022] The cancer is preferably a solid cancer. A solid cancer is an abnormal mass of tissue that usually does not contain cysts or liquid areas by contrast to a liquid tumor.

[0023] The subject to be treated overexpresses LIBQLN1 or LIBQLN4 and can also overexpress both.

[0024] A subject overexpressing LIBQLN1 or LIBQLN4 is preferably defined as a subject showing a greater than 1.25-fold upregulation of the mRNA and / or protein expression level of LIBQLN1 and / or LIBQLN4 as compared to the corresponding expression level in one or more healthy subjects. In this connection the greater than 1.25-fold upregulation is with increasing preference a greater than 1.5-fold, 1.75-fold, 2.0-fold, 2.25-fold, and 2.5-fold upregulation. The healthy subjects are with increasing preference at least 2, at least 5, at least 10, at least 20 and at least 50 healthy subjects in order to bias for potential expression level differences in healthy subjects.

[0025] Alternatively, or in addition a subject overexpressing LIBQLN1 or LIBQLN4 is preferably defined as a subject, wherein the mRNA and / or protein expression level of LIBQLN1 and / or LIBQLN4 is among the top 25% of corresponding expression levels as obtained from a plurality of cancer subjects. In this connection the top 25% are with increasing preference the top 20%, the top 15%, the top 10% and the top 5%. The plurality of cancer subjects are with increasing preference at least 2, at least 5, at least 10, at least 20 and at least 50 cancer subjects in order to bias for potential expression level differences in cancer subjects. The plurality of cancer subjects preferably have the same kind of cancer (e.g. lung cancer) as the subject to be treated. It is not required and even preferred that it is not known whether the plurality of cancer subjects are amenable for the treatment with an inhibitor of PARP-1 and / or an immune checkpoint protein.

[0026] Corresponding expression levels means, for example, that LIBQLN1 mRNA expression levels are compared with each other or LIBQLN4 protein expression levels are compared with each other and not, for example, that LIBQLN1 mRNA expression levels as compared with LIBQLN4 mRNA expression levels or LIBQLN1 protein expression levels with LIBQLN1 mRNA expression levels.

[0027] The term “treatment” as used herein is to be understood as encompassing curative treatment that defeats the cancer as well as treatment that prevents worsening of the cancer or that slows down the progression of the cancer, whereby a curative treatment is preferred.

[0028] The inhibitor of PARP-1 or an immune checkpoint protein may either be an inhibitor of the nucleic acid molecule encoding PARP-1 or an immune checkpoint, or an inhibitor PARP-1 or an immune checkpoint on the protein level. The inhibitor of PARP-1 or an immune checkpoint may interfere with the transcription of the genomic DNA encoding PARP-1 or an immune checkpoint protein in mRNA or with the translation of such mRNA into protein which is commonly referred to herein as the inhibition of the expression of PARP-1 or an immune checkpoint protein.

[0029] The inhibitor of PARP-1 or an immune checkpoint protein preferably specifically inhibits PARP-1 or an immune checkpoint protein. Specific inhibition designates that the inhibitor does not or essentially does inhibit other proteins or peptides than PARP-1 or the selected target immune checkpoint protein.

[0030] A compound inhibiting the expression of the PARP-1 or immune checkpoint nucleic acid molecule and / or protein is in accordance with the present invention (i) a compound lowering or preventing the transcription of the gene encoding the nucleic acid molecule and / or the protein, or (ii) is acompound lowering or preventing the translation of the mRNA encoding the protein. Compounds of (i) include compounds interfering with the transcriptional machinery and / or its interaction with the promoter of said gene and / or with expression control elements remote from the promoter such as enhancers. Compounds of (ii) include compounds interfering with the translational machinery. The compound inhibiting the expression of the nucleic acid molecule and / or the protein specifically inhibits the expression of the nucleic acid molecule and / or the protein, for example, by specifically interfering with the promoter region controlling the expression. Preferably, the transcription of the nucleic acid molecule and / or the protein or the translation of the protein is reduced by at least 50%, more preferred at least 75% such as at least 90% or 95%, even more preferred at least 98% and most preferred by about 100% (e.g., as compared to the same experimental set up in the absence of the compound).

[0031] A compound inhibiting the activity of the PARP-1 or immune checkpoint nucleic acid molecule and / or protein in accordance with the present invention causes said nucleic acid molecule and / or protein to perform its / their function with lowered efficiency. The compound inhibiting the activity of the nucleic acid molecule and / or the protein specifically inhibits the activity of said nucleic acid molecule and / or protein. Preferably, the activity of the nucleic acid molecule and / or the protein is reduced by at least 50%, more preferred at least 75% such as at least 90% or 95%, even more preferred at least 98%, and most preferably about 100% (e.g., as compared to the same experimental set up in the absence of the compound).

[0032] The activity of the PARP-1 nucleic acid molecule and / or the protein is in accordance with this invention preferably the PARP-1 enzymatic activity, noting that PARP-1 is a NAD+ ADP- ribosyltransferase (EC 2.4.2.30). The activity of the immune nucleic acid molecule and / or the protein is in accordance with this invention preferably their capability of binding with their partner proteins and / or their capability to send the above described “off” signal to T cells.

[0033] The efficiency of inhibition of an inhibitor can be quantified by methods comparing the level of activity in the presence of the inhibitor to that in the absence of the inhibitor. For example, the change in the amount of the nucleic acid molecule and / or the protein of the invention formed may be used in the measurement. The efficiency of several inhibitors may be determined simultaneously in high-throughput formats. High-throughput assays, independently of being biochemical, cellular or other assays, generally may be performed in wells of microtiter plates, wherein each plate may contain 96, 384 or 1536 wells. Handling of the plates, including incubation at temperatures other than ambient temperature, and bringing into contact of test compounds with the assay mixture is preferably effected by one or more computer-controlled robotic systems including pipetting devices. In case large libraries of test compounds are to be screened and / or screening is to be effected within a short time, mixtures of, for example 10, 20, 30, 40, 50 or 100 test compounds may be added to each well. In case a well exhibits the expected activity, said mixture of test compounds may be de- convoluted to identify the one or more test compounds in said mixture giving rise to said activity.

[0034] The compounds inhibiting the expression and / or the activity of the PARP-1 or immune checkpoint nucleic acid molecule and / or the protein of the invention may be formulated as vesicles, such as liposomes or exosomes. 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 to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, 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 the cellular niche. Exosomes are useful for diagnostic and therapeutic purposes, since they can be used as delivery vehicles, e.g. for contrast agents or drugs.

[0035] The compounds inhibiting the expression and / or the activity of the PARP-1 or immune checkpoint nucleic acid molecule and / or the protein can be administered to the subject at a suitable dose and / or a therapeutically effective amount. 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. 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”, Journal of the American statistical association, 97(457): 1-9.

[0036] The compounds inhibiting the expression and / or the activity of the PARP-1 or immune checkpoint nucleic acid molecule and / or the protein are preferably admixed with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, 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.

[0037] It was identified recently by the inventors that LIBQLN1 and LIBQLN4 promote the use of NHEJ, while curtailing HR. Both LIBQLN1 and LIBQLN4 belong to the LIBQLN family of proteins, UBQLN1-4. Their role as shuttles to direct cargo to the proteasome is mediated by binding of ubiquitylated proteins and simultaneous interaction with the proteasome. Specifically, LIBQLN1 and LIBQLN4 control the proteasome-dependent MRE11 turnover, a key nuclease needed to initiate DSB end resection.

[0038] The appended examples show that it was now surprisingly found by the inventors that LIBQLN1 and LIBQLN4 are frequently overexpressed in multiple cancers and associated with poor survival. Importantly, the overexpression of LIBQLN1 and LIBQLN4 is observed in tumors not formerly connected to HR-deficiency and thus extends the application of PARP1 inhibitors and immune check point inhibitors in these tumors and the examples show in a tumor mouse model that cancer subjects overexpressing overexpression LIBQLN1 and / or LIBQLN4 are amenable for the treatment with PARP1 inhibitors and / or immune check point inhibitors

[0039] Thus, the data in the application as field we propose that LIBQLN1 and / or LIBQLN4 serve as a biomarker to identify tumors that are amenable to the treatment with PARP-1 and / or immune-checkpoint inhibitors and that that those cancer patients showing PARP1 inhibitors and / or immune check point inhibitors are a subgroup of cancer subjects, wherein the treatment with PARP-1 and / or immune-checkpoint inhibitors will be exceptionally effective.

[0040] In accordance with a preferred embodiment the PARP-1 inhibitor and optionally the immune checkpoint inhibitor are used.

[0041] Hence, in accordance with this preferred embodiment the subject to be treated receives a PARP-1 inhibitor and may (optionally or preferably) also receive an immune checkpoint inhibitor in order to treat the cancer in the subject. Combination PARP inhibition with checkpoint blockade is an emerging area of investigation with potential synergy to produce robust responses with durability (Gupta et al., Breast Cancer Res Treat, 2023 Jan; 197(1):51-56).

[0042] In accordance with another preferred embodiment the subject overexpresses LIBQLN1 and optionally overexpresses LIBQLN4.

[0043] Hence, in accordance with this preferred embodiment the subject to be treated has to overexpress LIBQLN1 and may (optionally or preferably) also overexpress LIBQLN4.

[0044] In accordance with a further preferred embodiment the cancer is non-small cell lung cancer, preferably lung adenocarcinoma.

[0045] Non-small-cell lung cancer (NSCLC), or non-small-cell lung carcinoma, is any type of epithelial lung cancer other than small-cell lung cancer (SCLC). NSCLC accounts for about 85% of all lung cancers. The most common types of NSCLC are squamous-cell carcinoma, large-cell carcinoma, and adenocarcinoma, but several other types occur less frequently. Adenocarcinoma of the lung is currently the most common type of lung cancer in "never smokers" (lifelong nonsmokers). Adenocarcinomas account for about 40% of lung cancers.

[0046] As discussed herein, PARP-1 inhibitors are generally only or at least particularly suitable for the treatment of HR-defective cancers. In particular among NSCLC and adenocarcinoma of the lung, only a small fraction of patients and only a limited number of cancers are HR- defective.

[0047] In accordance with a preferred embodiment the immune checkpoint target is selected from PD-1 , PD-L1 and CTLA-4.

[0048] In other terms, in accordance with this preferred embodiment the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4. Examples of inhibitors of PD-1 , PD-L1 and CTLA-4 will be provided herein below.

[0049] PD-1 (programmed cell death protein 1 , also known as CD279 (cluster of differentiation 279)) is a protein on the surface of T and B cells that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells. PD-1 is an immune checkpoint and guards against autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T-cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells). PD-1 inhibitors activate the immune system to attack tumors and are used to treat certain types of cancer.

[0050] The amino acid sequence of human PD-1 is shown in SEQ ID NO: 9 and the nucleotide sequences of PD-1 is shown in SEQ ID NO: 10.

[0051] PD-L1 (programmed death-ligand 1 , also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) is a 40kDa type 1 transmembrane protein that functions as an immune checkpoint. It plays a major role in suppressing the adaptive arm of immune systems during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states such as hepatitis. Normally the adaptive immune system reacts to antigens that are associated with immune system activation by exogenous or endogenous danger signals. In turn, clonal expansion of antigen-specific CD8+ T cells and / or CD4+ helper cells is propagated. The binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM). This reduces the proliferation of antigen-specific T-cells in lymph nodes, while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells) - further mediated by a lower regulation of the gene Bcl-2.

[0052] The amino acid sequence of human PD-L1 is shown in SEQ ID NO: 11 and the nucleotide sequences of PD-L1 are shown in SEQ ID NOs 12 to 14 (noting that three splice variants are known).

[0053] CTLA-4 (cytotoxic T-lymphocyte-associated protein 4, also known as CD152 (cluster of differentiation 152)) is a protein receptor that functions as an immune checkpoint and downregulates immune responses. CTLA-4 is constitutively expressed in regulatory T cells but only upregulated in conventional T cells after activation - a phenomenon which is particularly notable in cancers. It acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells.

[0054] The amino acid sequence of human CTLA-4 is shown in SEQ ID NO: 15 and the nucleotide sequences of CCTLA-4 are shown in SEQ ID NOs 16 and 17 (noting that two splice variants are known). In accordance with another preferred embodiment the inhibitor(s) is / are (a) small molecule inhibitor(s), (a) nucleotide-based inhibitor(s) or (an) amino acid-based inhibitor(s).

[0055] The "small molecule" as used herein is preferably an organic molecule. Organic molecules relate or belong to the class of chemical compounds having a carbon basis, the carbon atoms linked together by carbon-carbon bonds. The original definition of the term organic related to the source of chemical compounds, with organic compounds being those carbon- containing compounds obtained from plant or animal or microbial sources, whereas inorganic compounds were obtained from mineral sources. Organic compounds can be natural or synthetic. The organic molecule is preferably an aromatic molecule and more preferably a heteroaromatic molecule. In organic chemistry, the term aromaticity is used to describe a cyclic (ring-shaped), planar (flat) molecule with a ring of resonance bonds that exhibits more stability than other geometric or connective arrangements with the same set of atoms. Aromatic molecules are very stable, and do not break apart easily to react with other substances. In a heteroaromatic molecule at least one of the atoms in the aromatic ring is an atom other than carbon, e.g. N, S, or O. For all above-described organic molecules the molecular weight is preferably in the range of 200 Da to 1500 Da and more preferably in the range of 300 Da to 1000 Da.

[0056] Alternatively, the "small molecule" in accordance with the present invention may be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds without carbon atoms (except carbon dioxide, carbon monoxide and carbonates). Preferably, the small molecule has a molecular weight of less than about 2000 Da, or less than about 1000 Da such as and most preferably less than about 500 Da. The size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry. The small molecules may be designed, for example, based on the crystal structure of the target molecule, where sites presumably responsible for the biological activity can be identified and verified in in vivo assays such as in vivo high-throughput screening (HTS) assays.

[0057] A nucleotide-based inhibitor is an inhibitor comprising or consisting of a nucleic acid molecule. The term “nucleic acid molecule” in accordance with the present invention includes DNA and RNA. In this regard, "DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone. DNA can have one strand of nucleotide bases, or two complimentary strands which may form a double helix structure. "RNA" (ribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (II), called nucleotide bases, that are linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and double-stranded hybrids 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 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. The nucleic acid molecule of the invention may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.

[0058] An amino acid-based inhibitor is an inhibitor comprising or consisting of an amino acid sequence, such a protein or a peptide. The term “protein” as used herein interchangeably with the term “polypeptide” describes linear molecular chains of amino acids, including single chain proteins or their fragments, containing at least 50 amino acids. The term “peptide” as used herein describes a group of molecules consisting of up to 49 amino acids, whereas the term “polypeptide” (also referred to as "protein") as used herein describes a group of molecules consisting of at least 50 amino acids. The term “peptide” as used herein describes a group of molecules consisting with increased preference of at least 15 amino acids, at least 20 amino acids at least 25 amino acids, and at least 40 amino acids. The group of peptides and polypeptides are referred to together by using the term "(poly)peptide". (Poly)peptides may further form oligomers consisting of at least two identical or different molecules. The corresponding higher order structures of such multimers are, correspondingly, termed homo- or heterodimers, homo- or heterotrimers etc.. Furthermore, peptidomimetics of such proteins / (poly)peptides where amino acid(s) and / or peptide bond(s) have been replaced by functional analogues are also encompassed by the invention. Such functional analogues include all known amino acids other than the 20 gene-encoded amino acids, such as selenocysteine. The terms “(poly)peptide” and “protein” also refer to naturally modified (poly)peptides and proteins where the modification is effected e.g. by glycosylation, acetylation, phosphorylation and similar modifications which are well known in the art.

[0059] In accordance with a more preferred embodiment the small molecule inhibitor(s) of PARP-1 is / are selected from Olaparib (Lynparza™; AZD2281), Niraparib, Rucaparib, Talazoparib, Veliparib, Pamiparib, Fuzuloparib, Senaparib, AZD-5305, AZD-9574, CVL-218, HWH-340, JPI-289, NMS-293, SC-10914, Stenoparib, TSL-1502, Venadaparib, HRS-1167, Nesuparib, and RP-12146; the small molecule inhibitor(s) of PD-1 is / are selected from CCX-559 and CS-17938; the small molecule inhibitor(s) of PD-L1 is / are selected from INCB-099280, INCB- 086550, WP-1066, ABSK-043, AN-4005, ASC-61 , BPI-371153, CCX-559, FH-2001 , IMMH- 010, INCB-099318, and Max-1 ; and the small molecule inhibitor of CTLA-4 is BPI-002.

[0060] Olaparib (Lynparza™; AZD2281) was approved in 2014 for use as a single agent PARP-1 inhibitor by the European Medicines Agency (EMA) in the European Union and by the Food and Drug Administration (FDA) in the United States. Its formula is C24H23FN4O3 and the Cas No. is 763113-22-0

[0061] Niraparib (Zejula) is also an approved PARP-1 inhibitor. Its formula is C19H20N4O and the CAS No. is 1038915-60-4.

[0062] Rucaparib (Rubraca) is a PARP inhibitor used as an anti-cancer agent. Its formula is C19H18FN3O and the CAS No. is 283173-50-2. Talazoparib (Talzenna) is a PARP inhibitor and approved for the treatment of breast cancer. Its formula is C19H14F2N6O and the CAS No. is 1207456-01-6. Veliparib (ABT-888) is an anti-cancer drug acting as a PARP inhibitor. Its formula is CI3HI6N4O and the CAS No. is 912444-00-9.

[0063] Pamiparib is also an approved PARP-1 inhibitor. Its formula is C16H15FN4O and the CAS No. is 1446261-44-4.

[0064] Fuzuloparib is also an approved PARP-1 inhibitor. Its formula is C22H16F4N6O2 and the CAS No. is 1358715-18-0.

[0065] Senaparib is a highly potent, selective and orally active PARP1 / 2 inhibitor. Its formula is C24H20F2N6O3 and the CAS No. is 1401682-78-7.

[0066] AZD-5305, also known as Saruparib, is another PARP-1 inhibitor, having CAS No. 2589531- 76-8.

[0067] AZD-9574 is another PARP-1 inhibitor, having CAS No. 2756333-39-6.

[0068] CVL-218, also known as Mefuparib, is a selective PARP-1 / 2 inhibitor. Its CAS No. is 1449746-00-2.

[0069] HWH-340is a PARP-1 and -2 inhibitor described in the US Patent No. 9,856,262.

[0070] JPI-289is another PARP-1 inhibitor, having CAS No. 1227156-72-0.

[0071] NMS-293 is another PARP-1 inhibitor, having CAS No. 1606995-47-4.

[0072] SC-10914 acts by inhibiting the enzymes poly (ADP-ribose) polymerase 1 and 2 (PARP-1 and PARP-2). Its CAS No. is 76985-08-5.

[0073] Stenoparib shows its therapeutic intervention by inhibiting nuclear enzymes poly (ADP- ribose) polymerase (PARP) 1 and 2 and tankyrase 1 and 2. Its CAS No. is 1140964-99-3.

[0074] TSL-1502is another PARP-1 inhibitor described in Wang L, Zhu X, Li L, et al. TSL-1502, a glucuronide prodrug of a poly (ADP-ribose) polymerase (PARP) inhibitor, exhibits potent antitumor activity in preclinical models. Am J Cancer Res. 2021 ;11(4): 1632-1645. Venadaparib, is a PARP-1 and -2 inhibitor having CAS No. 1681017-83-3.

[0075] Nesuparib acts by inhibiting poly (ADP-ribose) polymerase 1,2 and 3 (PARP) and tankyrase (TNKS). Its CAS No. is 2055357-64-5.

[0076] CCX-559 is a PD-1 and PD-L1 inhibitor described in Sullivan KMC, Vilalta M, Ertl LS, et al., PLoS One. 2023;18(6):e0286724.

[0077] CS-17938 acts by inhibiting the interaction of programmed cell death protein 1 (PD1) and programmed cell death 1 ligand 1 (PD L1) being produced by Shenzhen Chipscreen Biosciences Co., Ltd.

[0078] INCB-099280 exhibits therapeutic intervention by inhibiting programmed cell death 1 ligand 1 (PD L1), It is described by Prenen H, Lesimple T, Robert M, et al., Journal for ImmunoTherapy of Cancer 2022;10:doi: 10.1136 / jitc-2022-SITC2022.0734.

[0079] INCB-086550 exhibits therapeutic intervention by inhibiting programmed cell death 1 ligand 1 (PD L1). Its CAS No. is 2230911-59-6.

[0080] AN-4005 elicits anti-neoplastic by inhibiting PDL1 in a phase I clinical by Adlai Nortye Biopharma. Its IIIPAC name is ethyl 2-amino-4-methyl-thiazole-5-carboxylate.

[0081] IMMH-010 elicits anti-neoplastic efffcet in patients with advanced malignant solid tumors by inhibiting PDL1. Its IIIPAC name is 2-[4-(2-bromo-biphenyl-3-ylmethoxy)-5-chloro-2-(pyridin- 3-ylmethoxy)- benzylamino]-3-hydroxy-propionic acid isopropyl ester.

[0082] In accordance with a preferred embodiment the inhibitor(s) is / are (an) inhibitor(s) of the nucleic acid molecule encoding PARP-1 and / or the nucleic acid molecule encoding the immune check point target, and / or (an) inhibitor(s) of the activity of PARP-1 and / or the activity of the immune check point target.

[0083] As discussed above, the inhibitor of PARP-1 or an immune checkpoint may either be an inhibitor of the nucleic acid molecule encoding PARP-1 or an immune checkpoint or an inhibitor of the PARP-1 or an immune checkpoint protein. In the former case the inhibitor of PARP-1 or an immune checkpoint may interfere with the transcription of the genomic DNA encoding PARP-1 or an immune checkpoint protein in mRNA or with the translation of such mRNA into protein. In the latter case the inhibitor generally interferes with the activity of PARP-1 and / or the activity of the immune check point target at protein level. These activities of PARP-1 and of the immune check point target have been further descried herein above.

[0084] In accordance with a more preferred embodiment (i) the inhibitor(s) of the nucleic acid molecule encoding PARP-1 and / or the nucleic acid molecule encoding the immune check point target is / are selected from an aptamer, a siRNA, a shRNA, a miRNA, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas9-based construct, a CRISPR-Cpf1-based construct, a meganuclease, a zinc finger nuclease, and a transcription activator-like (TAL) effector (TALE) nuclease, and / or (ii) inhibitor(s) of the activity of PARP-1 and / or the activity of the immune check point target is / are selected from a small molecule, an antibody or antibody mimetic, and an aptamer, wherein the antibody mimetic is preferably selected from affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and probodies.

[0085] The term “antibody” as used in accordance with the present invention comprises, for example, polyclonal or monoclonal antibodies. Furthermore, also derivatives or fragments thereof, which still retain the binding specificity to the target, e.g. PARP-1 or an immune checkpoint protein, are comprised in the term "antibody". Antibody fragments or derivatives comprise, inter alia, Fab or Fab’ fragments, Fd, F(ab')2, Fv or scFv fragments, single domain VH or V-like domains, such as VhH or V-NAR-domains, as well as multimeric formats such as minibodies, diabodies, tribodies or triplebodies, tetrabodies or chemically conjugated Fab’- multimers (see, for example, Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 198; Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999; Altshuler EP, Serebryanaya DV, Katrukha AG. 2010, Biochemistry (Mose)., vol. 75(13), 1584; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 1126). The multimeric formats in particular comprise bispecific antibodies that can simultaneously bind to two different types of antigen. The first antigen can be found on the protein of the invention. The second antigen may, for example, be a tumor marker that is specifically expressed on cancer cells or a certain type of cancer cells. Non-limting examples of bispecific antibodies formats are Biclonics (bispecific, full length human IgG antibodies), DART (Dual-affinity Re-targeting Antibody) and BiTE (consisting of two single-chain variable fragments (scFvs) of different antibodies) molecules (Kontermann and Brinkmann (2015), Drug Discovery Today, 20(7): 838-847).

[0086] The term "antibody" also includes embodiments such as chimeric (human constant domain, non-human variable domain), single chain and humanised (human antibody with the exception of non-human CDRs) antibodies.

[0087] Various techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane (1988) and (1999) and Altshuler et al., 2010, loc. cit. Thus, polyclonal antibodies can be obtained from the blood of an animal following immunisation with an antigen in mixture with additives and adjuvants and monoclonal antibodies can be produced by any technique which provides antibodies produced by continuous cell line cultures. Examples for such techniques are described, e.g. in Harlow E and Lane D, Cold Spring Harbor Laboratory Press, 1988; Harlow E and Lane D, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999 and include the hybridoma technique originally described by Kohler and Milstein, 1975, the trioma technique, the human B-cell hybridoma technique (see e.g. Kozbor D, 1983, Immunology Today, vol.4, 7; Li J, et al. 2006, PNAS, vol. 103(10), 3557) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, Alan R. Liss, Inc, 77-96). Furthermore, recombinant antibodies may be obtained from monoclonal antibodies or can be prepared de novo using various display methods such as phage, ribosomal, mRNA, or cell display. A suitable system for the expression of the recombinant (humanised) antibodies may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., US patent 6,080,560; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 11265). Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies specific for an epitope of PARP-1 or an immune checkpoint protein. Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies.

[0088] As used herein, the term “antibody mimetics” refers to compounds which, like antibodies, can specifically bind antigens, such PARP-1 or an immune checkpoint protein in the present case, but which are not structurally related to antibodies. Antibody mimetics are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa. For example, an antibody mimetic may be selected from the group consisting of affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and prododies. These polypeptides are well known in the art and are described in further detail herein below.

[0089] The term “affibody”, as used herein, refers to a family of antibody mimetics which is derived from the Z-domain of staphylococcal protein A. Structurally, affibody molecules are based on a three-helix bundle domain which can also be incorporated into fusion proteins. In itself, an affibody has a molecular mass of around 6kDa and is stable at high temperatures and under acidic or alkaline conditions. Target specificity is obtained by randomisation of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. 899:103-26).

[0090] The term "adnectin" (also referred to as “monobody”), as used herein, relates to a molecule based on the 10th extracellular domain of human fibronectin III (10Fn3), which adopts an Ig- like p-sandwich fold of 94 residues with 2 to 3 exposed loops, but lacks the central disulphide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Adnectins with the desired target specificity, i.e. against PARP-1 or an immune checkpoint protein, can be genetically engineered by introducing modifications in specific loops of the protein.

[0091] The term "anticalin", as used herein, refers to an engineered protein derived from a lipocalin (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci U S A. 96(5): 1898- 903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Anticalins possess an eight-stranded p-barrel which forms a highly conserved core unit among the lipocalins and naturally forms binding sites for ligands by means of four structurally variable loops at the open end. Anticalins, although not homologous to the IgG superfamily, show features that so far have been considered typical for the binding sites of antibodies: (i) high structural plasticity as a consequence of sequence variation and (ii) elevated conformational flexibility, allowing induced fit to targets with differing shape.

[0092] As used herein, the term "DARPin" refers to a designed ankyrin repeat domain (166 residues), which provides a rigid interface arising from typically three repeated p-turns. DARPins usually carry three repeats corresponding to an artificial consensus sequence, wherein six positions per repeat are randomised. Consequently, DARPins lack structural flexibility (Gebauer and Skerra, 2009).

[0093] The term “avimer”, as used herein, refers to a class of antibody mimetics which consist of two or more peptide sequences of 30 to 35 amino acids each, which are derived from A- domains of various membrane receptors and which are connected by linker peptides. Binding of target molecules occurs via the A-domain and domains with the desired binding specificity, i.e. for PARP-1 or an immune checkpoint protein, can be selected, for example, by phage display techniques. The binding specificity of the different A-domains contained in an avimer may, but does not have to be identical (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).

[0094] A “nanofitin” (also known as affitin) is an antibody mimetic protein that is derived from the DNA binding protein Sac7d of Sulfolobus acidocaldarius. Nanofitins usually have a molecular weight of around 7kDa and are designed to specifically bind a target molecule, such as e.g. PARP-1 or an immune checkpoint protein, by randomising the amino acids on the binding surface (Mouratou B, Behar G, Paillard-Laurance L, Colinet S, Pecorari F., (2012) Methods Mol Biol.; 805:315-31).

[0095] The term “affilin”, as used herein, refers to antibody mimetics that are developed by using either gamma-B crystalline or ubiquitin as a scaffold and modifying amino-acids on the surface of these proteins by random mutagenesis. Selection of affilins with the desired target specificity, i.e. against PARP-1 or an immune checkpoint protein, is effected, for example, by phage display or ribosome display techniques. Depending on the scaffold, affilins have a molecular weight of approximately 10 or 20kDa. As used herein, the term affilin also refers to di- or multimerised forms of affilins (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).

[0096] A “Kunitz domain peptide” is derived from the Kunitz domain of a Kunitz-type protease inhibitor such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI). Kunitz domains have a molecular weight of approximately 6kDA and domains with the required target specificity, i.e. against PARP-1 or an immune checkpoint protein, can be selected by display techniques such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).

[0097] As used herein, the term "Fynomer®" refers to a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain. Fyn SH3-derived polypeptides are well-known in the art and have been described e.g. in Grabulovski et al. (2007) JBC, 282, p. 3196-3204, WO 2008 / 022759, Bertschinger et al (2007) Protein Eng Des Sei 20(2):57-68, Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255, or Schlatter et al. (2012), MAbs 4:4, 1-12).

[0098] The term “trispecific binding molecule” as used herein refers to a polypeptide molecule that possesses three binding domains and is thus capable of binding, preferably specifically binding to three different epitopes. At least one of these three epitopes is an epitope of the protein of the fourth aspect of the invention. The two other epitopes may also be epitopes of the protein of the fourth aspect of the invention or may be epitopes of one or two different antigens. The trispecific binding molecule is preferably a TriTac. A TriTac is a T-cell engager for solid tumors which comprised of three binding domains being designed to have an extended serum half-life and be about one-third the size of a monoclonal antibody.

[0099] As used herein, the term "probody" refers to a protease-activatable antibody prodrug. A probody consists of an authentic IgG heavy chain and a modified light chain. A masking peptide is fused to the light chain through a peptide linker that is cleavable by tumor-specific proteases. The masking peptide prevents the probody binding to healthy tissues, thereby minimizing toxic side effects.

[0100] Aptamers are nucleic acid molecules or peptide molecules that bind a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecule. These compound molecules have additional research, industrial and clinical applications (Osborne et. al. (1997), Current Opinion in Chemical Biology, 1:5-9; Stull & Szoka (1995), Pharmaceutical Research, 12, 4:465-483).

[0101] Nucleic acid aptamers are nucleic acid species that normally consist of (usually short) strands of oligonucleotides. Typically, they have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.

[0102] Peptide aptamers are usually peptides or proteins that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range). The variable peptide loop typically comprises 10 to 20 amino acids, and the scaffold may be any protein having good solubility properties. Currently, the bacterial protein Thioredoxin-A is the most commonly used scaffold protein, the variable peptide loop being inserted within the redoxactive site, which is a -Cys-Gly-Pro-Cys-loop (SEQ ID NO: 18) in the wild protein, the two cysteins lateral chains being able to form a disulfide bridge. Peptide aptamer selection can be made using different systems, but the most widely used is currently the yeast two-hybrid system.

[0103] Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival those of the commonly used biomolecules, in particular antibodies. In addition to their discriminatory recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamers' inherently low molecular weight. Unmodified aptamer applications currently focus on treating transient conditions such as blood clotting, or treating organs such as the eye where local delivery is possible. This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging. Several modifications, such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) linkage, fusion to albumin or other half-life extending proteins etc. are available to scientists such that the half-life of aptamers can be increased for several days or even weeks.

[0104] As discussed, the above-described small molecule, antibody or antibody mimetic and aptamer can specifically bind to the protein of the fourth aspect of the invention. This binding may block the immunosuppressive properties of the protein of the fourth aspect of the invention and preferably its capability to induce resistance to chemotherapy in cancer patients and / or to reduce progression free as well as overall survival in cancer patients. In this case the small molecule, antibody or antibody mimetic and aptamer are also referred to as blocking small molecule, antibody or antibody mimetic and aptamer. A blocking small molecule, antibody or antibody mimetic and aptamer blocks interactions of the protein of the fourth aspect of the invention with other cellular components, such as ligands and receptor which normally interact with the protein of the fourth aspect of the invention.

[0105] The small molecule, antibody or antibody mimetic and aptamer can also be generated in the format of drug-conjugates. In this case the small molecule, antibody or antibody mimetic and aptamer in itself may not have an inhibitory effect, but the inhibitory effect is only conferred by the drug. The small molecule, antibody or antibody mimetic and aptamer confer the sitespecificity binding of the drug to cells producing and / or binding to the protein of the invention. The drug is preferably capable to kill cells producing and / or binding to the protein of the invention. Hence, by combining the targeting capabilities of molecules binding to the protein of the invention with the cell-killing ability of the drug, the drug conjugates become inhibitors that allow for discrimination between healthy and diseased tissue and cells. Cleavable and non-cleavable linkers to design drug conjugates are known in the art. Non-limiting examples of drugs being capable of killing cells are cytostatic drugs and radioisotopes that deliver radiation directly to the cancer cells.

[0106] It is furthermore possible to confine the binding and / or inhibitory activity of the small molecule, antibody or antibody mimetic and aptamer to certain tissues or cell-types, in particular diseased tissues or cell-types. For instance, probodies may be designed. In a probody the small molecule, antibody or antibody mimetic or aptamer is bound to a masking peptide which limits or prevents binding to the protein of the invention and which masking peptide can be cleaved by a protease. Proteases are enzymes that digest proteins into smaller pieces by cleaving specific amino acid sequences known as substrates. In normal healthy tissue, protease activity is tightly controlled. In cancer cells, protease activity is upregulated. In healthy tissue or cells, where protease activity is regulated and minimal, the target-binding region of the probody remains masked and is thus unable to bind. On the other hand, in diseased tissue or cells, where protease activity is upregulated, the targetbinding region of the probody gets unmasked and is thus able to bind and / or inhibit.

[0107] In accordance with the present invention, the term "small interfering RNA (siRNA)", also known as short interfering RNA or silencing RNA, refers to a class of 18 to 30, preferably 19 to 25, most preferred 21 to 23 or even more preferably 21 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome. siRNAs naturally found in nature have a well-defined structure: a short double-strand of RNA (dsRNA) with 2-nt 3' overhangs on either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest. Essentially any gene for which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. The doublestranded RNA molecule or a metabolic processing product thereof is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and / or DNA methylation. Exogenously introduced siRNAs may be devoid of overhangs at their 3' and 5' ends, however, it is preferred that at least one RNA strand has a 5'- and / or 3'-overhang. Preferably, one end of the double-strand has a 3'-overhang from 1 to 5 nucleotides, more preferably from 1 to 3 nucleotides and most preferably 2 nucleotides. The other end may be blunt-ended or has up to 6 nucleotides 3'-overhang. In general, any RNA molecule suitable to act as siRNA is envisioned in the present invention. The most efficient silencing was so far obtained with siRNA duplexes composed of 21 -nt sense and 21 -nt antisense strands, paired in a manner to have a 2-nt 3'- overhang. The sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair (Elbashir et al. 2001). 2'-deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant. Delivery of siRNA may be accomplished using any of the methods known in the art, for example by combining the siRNA with saline and administering the combination intravenously or intranasally or by formulating siRNA in glucose (such as for example 5% glucose) or cationic lipids and polymers can be used for siRNA delivery in vivo through systemic routes either intravenously (IV) or intraperitoneally (IP) (Fougerolles et al. (2008), Current Opinion in Pharmacology, 8:280-285; Lu et al. (2008), Methods in Molecular Biology, vol. 437: Drug Delivery Systems - Chapter 3: Delivering Small Interfering RNA for Novel Therapeutics).

[0108] A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it. si / shRNAs to be used in the present invention are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer. Suppliers of RNA synthesis reagents are Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK). Most conveniently, siRNAs or shRNAs are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs. In general, the RNAs applicable in the present invention are conventionally synthesized and are readily provided in a quality suitable for RNAi. Further molecules effecting RNAi include, for example, microRNAs (miRNA). Said RNA species are single-stranded RNA molecules. Endogenously present miRNA molecules regulate gene expression by binding to a complementary mRNA transcript and triggering of the degradation of said mRNA transcript through a process similar to RNA interference. Accordingly, exogenous miRNA may be employed as an inhibitor of PARP-1 or an immune checkpoint protein after introduction into the respective cells.

[0109] A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyses a chemical reaction. Many natural ribozymes catalyse either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyse the aminotransferase activity of the ribosome. Non-limiting examples of well-characterised small self-cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro- selected lead-dependent ribozymes, whereas the group I intron is an example for larger ribozymes. The principle of catalytic self-cleavage has become well established in recent years. The hammerhead ribozymes are characterised best among the RNA molecules with ribozyme activity. Since it was shown that hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, it appears that catalytic antisense sequences for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site. The basic principle of constructing hammerhead ribozymes is as follows: A region of interest of the RNA, which contains the GUC (or CUC) triplet, is selected. Two oligonucleotide strands, each usually with 6 to 8 nucleotides, are taken and the catalytic hammerhead sequence is inserted between them. The best results are usually obtained with short ribozymes and target sequences.

[0110] A recent development, also useful in accordance with the present invention, is the combination of an aptamer, recognizing a small compound, with a hammerhead ribozyme. The conformational change induced in the aptamer upon binding the target molecule can regulate the catalytic function of the ribozyme.

[0111] The term “antisense nucleic acid molecule”, as used herein, refers to a nucleic acid which is complementary to a target nucleic acid. An antisense molecule in accordance with the invention is capable of interacting with the target nucleic acid, more specifically it is capable of hybridizing with the target nucleic acid. Due to the formation of the hybrid, transcription of the target gene(s) and / or translation of the target mRNA is reduced or blocked. Standard methods relating to antisense technology have been described (see, e.g., Melani et al., Cancer Res. (1991) 51:2897-2901).

[0112] CRISPR / Cas9, as well as CRISPR-Cpf1 , technologies are applicable in nearly all cells / model organisms and can be used for knockout mutations, chromosomal deletions, editing of DNA sequences and regulation of gene expression. The regulation of the gene expression can be manipulated by the use of a catalytically dead Cas9 enzyme (dCas9) that is conjugated with a transcriptional repressor to repress transcription a specific gene, here PARP-1 or an immune checkpoint protein. Similarly, catalytically inactive, "dead" Cpf1 nuclease (CRISPR from Prevotella and Francisella-1) can be fused to synthetic transcriptional repressors or activators to downregulate endogenous promoters, e.g. the promoter which controls PARP-1 or an immune checkpoint gene expression. Alternatively, the DNA-binding domain of zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) can be designed to specifically recognize the PARP-1 or an immune checkpoint protein gene or its promoter region or its 5'-UTR thereby inhibiting the expression of the PARP-1 or an immune checkpoint protein gene.

[0113] Such molecules, which reduce or abolish the expression of PARP-1 or an immune checkpoint protein or a regulatory molecule include, without being limiting, meganucleases, zinc finger nucleases and transcription activator-like (TAL) effector (TALE) nucleases. Such methods are described in Silva et al., Curr Gene Ther. 2011 ;11 (1):11-27; Miller et al., Nature biotechnology. 2011;29(2):143-148, and Klug, Annual review of biochemistry. 2010; 79:213- 231.

[0114] In accordance with a further more preferred embodiment the inhibitor(s) of the nucleic acid molecule encoding PARP-1 and / or the nucleic acid molecule encoding the immune check point target comprise(s) (a) a nucleic acid sequence which comprises or consists of a nucleic acid sequence being complementary to at least 12 continuous nucleotides of a nucleic acid sequence selected from SEQ ID NOs 2, 10, 12 to 14, 16 and 17, (b) a nucleic acid sequence which comprises or consists of a nucleic acid sequence which is at least 70% identical to the complementary strand of one or more nucleic acid sequences selected from SEQ ID NOs 2, 10, 12 to 14, 16 and 17, (c) a nucleic acid sequence which comprises or consists of a nucleic acid sequence according to (a) or (b), wherein the nucleic acid sequence is DNA or RNA, (d) an expression vector expressing the nucleic acid sequence as defined in any one of (a) to (c), preferably under the control of a lung-specific promoter, or (e) a host comprising the expression vector of (d). The nucleic acid sequences as defined in items (a) to (c) of this preferred embodiment comprise or consist of sequences being complementary to nucleotides of SEQ ID NOs 1, 10, 12 to 14, 15 and 17, noting that SEQ ID NO: 1 is the human PARP-1 cDNA, SEQ ID NO: is 10 the human PD-1 cDNA, SEQ ID NOs 12 to 14 are human PD-L1 cDNAs and SEQ ID NOs 16 and 17 are CTLA-4 cDNAs. Hence, the nucleic acid sequences as defined in items (a) to (c) comprise or are antisense nucleic acid sequences.

[0115] The nucleic acid sequence according to item (a) of this further preferred embodiment of the invention comprises or consists of a sequence which is with increasing preference complementary to at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides of a nucleotide sequence selected from SEQ ID NOs 2, 10, 12 to 14, 16 and 17. These at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, or at least 21 nucleotides are preferably a contiguous part of a nucleotide sequence selected from SEQ ID NOs 2, 10, 12 to 14, 16 and 17, i.e. the nucleotides are consecutive in the respective SEQ ID NO. The format of the nucleic acid sequence according to item (a) is not particularly limited as long as it comprises or consists of at least 12 continuous nucleotides being complementary to a nucleic acid sequence selected from SEQ ID NOs 2, 10, 12 to 14, 16 and 17. The nucleic acid sequence according to item (a) comprises or consists of an antisense oligonucleotide. Hence, the nucleic acid sequence according to item (a) reflects the above-mentioned basic principle of the antisense technology which is the use of an oligonucleotide for silencing a selected target RNA through the exquisite specificity of complementary-based pairing. Therefore, it is to be understood that the nucleic acid sequence according to item (a) is preferably in the format of an siRNA, shRNA or an antisense oligonucleotide as defined herein above. The antisense oligonucleotides are preferably LNA-GapmeRs, AntagomiRs, or antimiRs as defined herein above. LNA-GapmeRs are again preferred, particularly since they are used in the examples.

[0116] The nucleic acid sequence according to item (b) requiring at least 70% identity to the complementary strand of a nucleotide sequence selected from SEQ ID NOs 2, 10, 12 to 14, 16 and 17 is considerably longer than the nucleic acid sequence according to item (a) which comprises an antisense oligonucleotide and comprises at least 12 continuous nucleotides of a nucleotide sequence selected from SEQ ID NOs 2, 10, 12 to 14, 16 and 17. A nucleic acid sequence according to items (a) and (b) of the above preferred embodiment of the invention is capable of interacting with, more specifically hybridizing with the target nucleotide sequence. By formation of the hybrid the function of the target nucleotide sequence is reduced or blocked.

[0117] The sequence identity of the molecule according to item (b) in connection with a sequence selected from SEQ ID NOs 2, 10, 12 to 14, 16 and 17 is with increasing preference at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, at least 99% and 100%. The sequence identity in connection with each of SEQ ID NOs 2, 10, 12 to 14, 16 and 17 can be individually selected.

[0118] In the nucleic acid sequence according to item (c) the nucleotide sequences may be RNA or DNA. RNA or DNA optionally encompasses chemically modified RNA nucleotides or DNA nucleotides. As commonly known RNA comprises the nucleotide II while DNA comprises the nucleotide T.

[0119] In accordance with items (d) and (e) of the above preferred embodiment the inhibitor may also be an expression vector or host, respectively being capable of producing a nucleic acid sequence as defined in any one of items (a) to (c).

[0120] An expression vector may be a plasmid that is used to introduce a specific transcript into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes. The plasmid is in general engineered to contain regulatory sequences that act as enhancer and / or promoter regions and lead to efficient transcription of the transcript. In accordance with the present invention the expression vector preferably contains a lungspecific promoter. Using a lung-specific promoter ensures that the nucleic acid sequence is only expressed in the lug in a lung cancer subject and may avoid potential unwanted side effects by expression in other organs.

[0121] Non-limiting examples of expression vectors include prokaryotic plasmid vectors, such as the pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMCIneo (Stratagene), pXT1 (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 Intvitrogen). For the formulation of a pharmaceutical composition a suitable vector is selected in accordance with good manufacturing practice. Such vectors are known in the art, for example, from Ausubel et al, Hum Gene Ther. 2011 Apr; 22(4):489-97 or Allay et al., Hum Gene Ther. May 2011 ; 22(5): 595-604.

[0122] A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Moreover, elements such as origin of replication, drug resistance gene, regulators (as part of an inducible promoter) may also be included. The lac promoter is a typical inducible promoter, useful for prokaryotic cells, which can be induced using the lactose analogue isopropylthiol-b-D-galactoside ("IPTG"). For recombinant expression and secretion, the polynucleotide of interest may be ligated between e.g. the PelB leader signal, which directs the recombinant protein in the periplasm and the gene III in a phagemid called pHEN4 (described in Ghahroudi et al, 1997, FEBS Letters 414:521-526). Additional elements might include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from retroviruses, e.g., RSV, HTLVI, HIVI, and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter). Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Alternatively, the recombinant (poly)peptide can be expressed in stable cell lines that contain the gene construct integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells. The transfected nucleic acid can also be amplified to express large amounts of the encoded (poly)peptide. The DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al.1991 , Biochem J. 227:277 -279; Bebbington et al. 1992, Bio / Technology 70:169-175). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. As indicated above, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. For vector modification techniques, see Sambrook and Russel (2001), Molecular Cloning: A Laboratory Manual, 3 Vol. Generally, vectors can contain one or more origins of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes. Suitable origins of replication (ori) include, for example, the Col E1, the SV40 viral and the M 13 origins of replication.

[0123] The sequences 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 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 the transcription (e.g., translation initiation codon, promoters, enhancers, and / or insulators), 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, and / or naturally-associated or heterologous promoter regions. Preferably, the nucleotide sequence as defined in item (a) of the above preferred embodiment of the invention is operatively linked to such expression control sequences allowing expression in prokaryotic or eukaryotic cells.

[0124] The host may be a prokaryotic or eukaryotic cell. A suitable eukaryotic host may be a mammalian cell, an amphibian cell, a fish cell, an insect cell, a fungal cell or a plant cell. Representative examples of bacterial cells are E. coli, Streptomyces and Salmonella typhimurium cells; of fungal cells are yeast cells; and of insect cells are Drosophila S2 and Spodoptera Sf9 cells. It is preferred that the cell is a mammalian cell such as a human cell. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1 , Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells. The cell may be a part of a cell line, preferably a human cell line. Appropriate culture mediums and conditions for the above-described host cells are known in the art. The host is preferably a host cell and more preferably an isolated host cell. The host is also preferably a non-human host.

[0125] In accordance with another further more preferred embodiment (i) the inhibitor(s) is / are antibody / ies against (a) immune checkpoint target(s), wherein the antibody / ies against is / are preferably selected from one or more of an anti-PD-1 antibody, such as Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, Retifanlimab, Tislelizumab, APL-501 , Spartalizumab, Camrelizumab, Vopratelimab, AMP-514, MGA012, Acrixolimab, Toripalimab, Sintilimab, Penpulimab, Cadonilimab, Pucotenlimab, Camrelizumab, Prolgolimab, Serplulimab, Zimberelimab, Ivonescimab, Cetrelimab, Finotonlimab, IBI-318, MK-3475A, Nofazinlimab, QL-1604, Rulonilimab, Sasanlimab, or Tebotelimab;; an anti-PD-L1 antibody, such as Atezolimumab, Durvalumab, Avelumab, Cosibelimab, Envafolimab, Adebrelimab, Sugemalimab, APL-502, Socazolimab, Tagitanlimab, IBI-318, KN-035, KN-046, PM-8002, or SHR-1701 ; and an anti-CTLA-4 antibody, such as Ipilimumab, Tremelimumab, AGEN1884, Cadonilimab, Gotistobart, KN-046, ADG-126, BMS-986218, BMS-986249, BMS-986288, Botensilimab, Danvilostomig, GI-102, HL-06, JK-08, Lorigerlimab, Porustobart,

[0126] Quavonlimab, RP-2, Volrustomig, Vudalimab, XTX-101 , YH-001, or Zalifrelimab.

[0127] The above antibody / ies against (a) immune checkpoint target(s) are either approved antibody / ies being used in clinical practice for the treatment of cancer subjects or are currently in clinical trials, so that it can be expected that they might receive market approval as well.

[0128] In accordance with a preferred embodiment the inhibitor(s) is / are formulated as a pharmaceutical composition, preferably comprising a pharmaceutically acceptable excipient or diluent.

[0129] 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 pharmaceutical composition of the present invention may, optionally and additionally, comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are provided herein above. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will 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 should be in the range of 1 pg to 5 g units per day. However, a more preferred dosage might be in the range of 0.01 mg to 100 mg, even more preferably 0.01 mg to 50 mg and most preferably 0.01 mg to 10 mg per day. Furthermore, if for example said compound is an RNAi agent, 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 RNAi agent (e.g., about 4.4 x 1016 copies) 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 of RNAi agent 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.

[0130] In accordance with a preferred embodiment the subject that overexpresses LIBQLN1 and / or UE3QLN4 is characterized by a genomic aberration of the UE3QLN1 gene and / or the UE3QLN4 gene, wherein the genomic aberration is preferably a focal copy number gain or a gene amplification.

[0131] The cause of the overexpression of UE3QLN1 and / or UE3QLN4 in cancer subjects is generally a genomic aberration of the UE3QLN1 gene and / or the UE3QLN4 gene. In UE3QLN1 and UE3QLN4 and in particular in UE3QLN4 a focal copy number gain or a gene amplification are frequently observed as genomic aberration.

[0132] The present invention relate in a second aspect to a method for determining whether a cancer subject is amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor, wherein the method comprises: (a) detecting the mRNA and / or protein expression level of UE3QLN1 and / or UE3QLN4 in a sample obtained from said cancer subject, and (b) comparing the expression level of (a) with the corresponding expression level in a sample obtained from healthy subjects or a predetermined standard thereof, wherein a greater than 1.25-fold upregulation of the expression level of (a) is indicative for cancer patient being amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor, or (b’) comparing the expression level of (a) with the corresponding expression levels in a plurality of samples obtained from cancer subjects or a predetermined standard thereof, wherein an expression level of (a) falling into the top 25% of the expression levels of (b’) is indicative for cancer patient being amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor.

[0133] The definitions and preferred embodiments of the first aspect of the invention apply mutatis mutandis to the second aspect as far as they are applicable to the second aspect.

[0134] The method may further comprise (c) administering a PARP-1 inhibitor and / or an immune checkpoint inhibitor (in an effective amount) to the cancer subject, if the cancer subject is amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor.

[0135] The sample may be a body fluid of the subject or a tissue sample from an organ of the subject. Non-limiting examples of body fluids are whole blood, blood plasma, blood serum, urine, peritoneal fluid, and pleural fluid, liquor cerebrospinalis, tear fluid, or cells therefrom in solution. Non-limiting examples of tissue are colon, liver, breast, ovary, and testis. Tissue samples may be taken by aspiration or punctuation, excision or by any other surgical method leading to biopsy or resected cellular material. The sample may be a processed sample, e.g. a sample which has been frozen, fixed, embedded or the like. A preferred type of sample is a formaline fixed paraffin embedded (FFPE) sample. Preparation of FFPE samples are standard medical practice and these samples can be conserved for long periods of time. The sample obtained in (a) and the sample(s) in (b)((b’) are preferably the same kind of sample samples (for example, both blood or both tumor tissue).

[0136] In step (a) of the method the mRNA and / or protein expression level of LIBQLN1 and / or LIBQLN4 in a sample obtained from said cancer subject is / are determined and it is then determined by one of both of the alternative step (b) and (b’) whether the expression levels obtained in (a) indicate that the cancer subject is amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor.

[0137] In step (b) a comparison is made with the corresponding expression level in a sample obtained from healthy subjects. A healthy subject does not have cancer or any sign of proliferative disease in general. Since healthy subjects generally do not overexpress LIBQLN1 and / or LIBQLN4 the “sample obtained from healthy subjects” can be from only one subject. The healthy subjects are with increasing preference at least 2, at least 5, at least 10, at least 20 and at least 50 healthy subjects in order to bias for potential expression level differences in healthy subjects. The sample can be one mixed sample wherein the samples from the healthy subjects were mixed before the expression levels are determined or the sample can be parallel samples from individual healthy subjects wherein the expression levels are determined separately and then are averaged.

[0138] In step (b) a greater than 1.25-fold upregulation of the overexpression LIBQLN1 and / or UE3QLN4 indicates that a cancer subject is amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor. For example, here the cancer subject of a) may have 130% of the mRNA expression level of the healthy subjects (= 100%). The greater than 1.25-fold upregulation is with increasing preference a greater than 1.5-fold, 1.75-fold, 2.0-fold, 2.25-fold, and 2.5-fold upregulation.

[0139] In step (b’) a comparison is made with the corresponding expression levels in a plurality of samples obtained from other cancer subjects. As discussed above, these other cancer subjects may or may not overexpress LIBQLN1 and / or LIBQLN4. The plurality of cancer subjects are therefore with increasing preference at least 2, at least 5, at least 10, at least 20 and at least 50 cancer subjects in order to bias for potential expression level differences in healthy subjects. The plurality of cancer subjects preferably have the same kind of cancer (e.g. lung cancer) as the subject to be treated. It is not required and even preferred that it is not known whether the plurality of cancer subjects are amenable for the treatment with an inhibitor of PARP-1 or an immune checkpoint protein.

[0140] In step (b’) an expression level of (a) falling into the top 25% of the expression levels of (b’) indicates that the cancer subject is amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor. For example, here the cancer subject of a) may have an expression level that is higher than the expression level than in 3 / 4 of the other expression levels from other cancer subjects of (b’). In this connection the top 25% are with increasing preference the top 20%, the top 15%, the top 10% and the top 5%.

[0141] Predetermined standards in (b) and (b’) designate previously expression level(s). Such previously expression level(s) may be stored on paper, a computer or a storage medium (such as a cloud, CD-ROM, hard-disk or USB stick).

[0142] In accordance with a preferred embodiment the method is for determining whether a cancer subject is an amenable for the treatment with a PARP-1 inhibitor and optionally further for determining whether a cancer subject is an amenable for the treatment with an immune checkpoint inhibitor.

[0143] Hence, in accordance with this preferred embodiment it has be determined by the method whether the cancer subject is an amenable for the treatment with a PARP-1 inhibitor and it may (optionally or preferably) be further determined by the method whether the cancer subject is an amenable for the treatment with an immune checkpoint inhibitor.

[0144] In accordance with another preferred embodiment (a) the mRNA and / or protein expression level of LIBQLN1 is detected and optionally the mRNA and / or protein expression level of UE3QLN4 is detected.

[0145] Thus, in accordance with this preferred embodiment the mRNA and / or protein expression level of UE3QLN1 has to be detected and (optionally or preferably) also the mRNA and / or protein expression level of UE3QLN4 is detected.

[0146] In accordance with a further preferred embodiment the cancer subject is a lung cancer subject, preferably a lung adenocarcinoma subject.

[0147] As also discussed herein above, PARP-1 inhibitors are generally only or at least particularly suitable for the treatment of HR-defective cancers. In particular among NSCLC and adenocarcinoma of the lung, only a small fraction of patients and only a limited number of cancers are HR-defective and this small fraction can now be identified by the method of the second aspect of the invention.

[0148] In accordance with a preferred embodiment the detection of the mRNA expression level comprises (a) quantitative PCR, preferably quantitative real time PCR, or (b) a template / RNA amplification method followed by determining the expression level using a fluorescence- or luminescence-based quantification method.

[0149] In quantitative PCR (qPCR), the amount of amplified product is linked to fluorescence intensity using a fluorescent reporter molecule. The point at which the fluorescent signal is measured in order to calculate the initial template quantity can either be at the end of the reaction (endpoint semi-quantitative PCR) or while the amplification is still progressing (realtime qPCR). In endpoint semi-quantitative PCR, fluorescence data are collected after the amplification reaction has been completed, usually after 30-40 cycles, and this final fluorescence is used to back-calculate the amount of template present prior to PCR.

[0150] The more sensitive and reproducible method of real-time qPCR measures the fluorescence at each cycle as the amplification progresses. This allows quantification of the template to be based on the fluorescence signal during the exponential phase of amplification, before limiting reagents, accumulation of inhibitors, or inactivation of the polymerase have started to have an effect on the efficiency of amplification. Fluorescence readings at these earlier cycles of the reaction will measure the amplified template quantity where the reaction is much more reproducible from sample to sample than at the endpoint.

[0151] A non-limiting example of a template / RNA amplification method followed by determining the expression level of mRNAs using a fluorescence- or luminescence-based quantification method is a method combining transcription mediated amplification (TMA) and a hybridization protection assay (HPA). In more detail, such a method may comprise hybridizing one or more oligonucleotides ("capture oligonucleotides") that are complementary to the mRNA of interest. The hybridized target sequences are then captured onto magnetic microparticles that are separated from the sample in a magnetic field. Wash steps may be utilized to remove extraneous components. Target amplification typically occurs via TMA, which is a transcription-based nucleic acid amplification method that utilizes two enzymes, Moloney murine leukemia virus (MMLV) reverse transcriptase and T7 RNA polymerase. A unique set of primers is used for each target sequence. The reverse transcriptase is used to generate a DNA copy (containing a promoter sequence for T7 RNA polymerase) of the target sequence. T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy. Detection of mRNA expression level is achieved by HPA using single-stranded, chemiluminescent-labeled nucleic acid probes that are complementary to the one or more amplicon. Preferably, distinguishably labelled probes are used for each target amplicon. The labelled nucleic acid probes hybridize specifically to the amplicon. A “selection reagent” then differentiates between hybridized and unhybridized probes by inactivating the label on unhybridized probes. During the detection step, the chemiluminescent signal produced by the hybridized probe is measured in a luminometer and is reported as “Relative Light Units” (RLU), thereby quantifying the mRNA expression level.

[0152] In accordance with another preferred embodiment the detection of the protein expression level comprises immunohistochemistry via chromogenic, fluorescent detection or imaging mass cytometry (IMC).

[0153] Immunohistochemistry (IHC) is a technique that involves the labeling of cellular proteins in tissue samples with a specific primary antibody and visualization of the target with detection reagents. Using IHC, protein expression can be assessed via chromogenic or fluorescent detection. Both detection systems rely on antigen recognition mediated by a primary antibody. Chromogenic IHC utilizes enzyme-catalyzed deposition of chromogens at the site of the antigen, while fluorescent IHC employs fluorophores and direct or indirect immunofluorescence to help visualize antigens of interest.

[0154] Imaging Mass Cytometry (IMC) generates high-dimensional spatial data at subcellular resolution. IMC uses cytometry by time-of-flight (on which CyTOF systems are based) to overcome the multiplexing limitations of traditional immunohistochemistry (IHC) and immunofluorescence. By applying metal-tagged antibodies instead of fluorochromes, IMC has the unique capability to simultaneously stain, acquire and analyze 40-plus markers of interest (e.g. proteins of interest) on a tissue section without interference from autofl uorescent tissues or management of spectral overlap.

[0155] 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 from. 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.

[0156] 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.

[0157] The figures show.

[0158] Figure 1. UBQLN4 alteration frequency in a pan-cancer Analysis of whole genomes (ICGC / TCGA; Nature 2020). Colour legend: red = amplification; green = mutation; blue = deep deletion; grey = multiples alterations. Source: cbioportal.com

[0159] Figure 2. UBQLN1 alteration frequency in a pan-cancer Analysis of whole genomes and transcritomes (ICGC / TCGA; Nature 2020). Colour legend: red = amplification; green = mutation; blue = deep deletion; grey = multiples alterations; orange = mRNA High; light green = mRNA Low. Source: cbioportal.com

[0160] Figure 3. LIBQLN4 mRNA levels in tumor tissues (TCGA dataset). Statistical differences were calculated by the Mann- Whitney II test. Abbreviations: Breast Invasive Carcinoma (BRCA), Bladder Urothelial Carcinoma (BLCA), Esophageal Carcinoma (ESCA), Low Grade Glioma (LGG), Lung Squamous Cell Carcinoma (LUSC), Lung Adenocarcinoma (LUAD), Ovarian Serous Cystadenocarcinoma (OV), Rectal Adenocarcinoma (READ), Skin Cutaneous Melanoma (SKCM), Stomach Adenocarcinoma (STAD), Testicular Germ Cell Cancer (TGCT), Uterine Corpus Endometrial Carcinoma (UCEC) (see Kobayashi et al., Cells (2022))

[0161] Figure 4. Left Panel: Kaplan-Meier curves depicting overall survival of NSCLC patients with high and low mRNA expression levels of UBQLN1. Right Panel: Kaplan-Meier curves depicting overall survival of NSCLC patients with high and low mRNA expression levels of UBQLN4. Source: TCGA

[0162] Figure 5. Top panel: pCT images depicting tumor growth in K-RasLSL G12D / wt; p53m(KP) and KP+Rosa26LSL Ubqln1 / wt(KPU1) and KP+Rosa26LSL !Jbqln4 / wt(KPU4) mice after intratracheal instillation with Adeno-Cre virus at the indicated time points. Bottom panel: Kaplan-Meier curves depicting the overall survival of KP, KPU1 and KPU4 mice after intratracheal induction with Adeno-Cre virus. Data was analyzed via Mantel-Cox test. < 0.05, **p < 0.01, ***p < 0.001 Figure 6. Whole-Exome Sequencing analysis of tumors with indicated numbers extracted from KP, KPLI1 and KPLI4 animals. Left panel: single nucleotide variants (SNV); middle panel: Insertions; right panel: Deletions.

[0163] Figure 7. Whole-Exome Sequencing analysis of Microhomology and Deletion length of 5+ of tumors extracted from KP, KPLI1 and KPLI4 animals.

[0164] Figure 8. Left panel: IHC stainings of fixed murine tissue from KP, KPU1 and KPU4 lungs.

[0165] Right panel: tumor cells of the indicated genotypes were extracted, grown in vitro and subjected to 10 pM Olaparib for 7 days and confluency was measured. Error bars represent SD of three independent experiments. Data was analyzed via the t test with Welch’s correction not assuming equal variance. < 0.05, **p < 0.01, ***p < 0.001

[0166] Figure 9. Kaplan-Meier curves depicting the overall survival of KP, KPU1 and KPU4 mice after intratracheal induction with Adeno-Cre virus. Solid line indicates untreated animals. Dashed line indicates treated animals with PARP1 inhibitor olaparib. Data was analyzed via Mantel-Cox test. < 0.05, **p < 0.01 , ***p < 0.001

[0167] Figure 10. Top panel: Western Blot of indicated lung cancer cell lines. Bottom panel: indicated tumor cell lines were grouped into either high or low UBQLN1 and UBQLN4 expression and subjected to 10 pM olaparib for 7 days and confluency was measured. Error bars represent SD of three independent experiments. Data was analyzed via the t test with Welch’s correction not assuming equal variance. < 0.05, **p < 0.01 , ***p < 0.001

[0168] Figure 11. In silico neoantigen mutation count analysis of whole-exome-sequencing data of indicated animals.

[0169] Figure 12. Top panel: Kaplan-Meier curves depicting the overall survival of KP and KPU1 mice after intratracheal induction with Adeno-Cre virus. Solid line indicates untreated animals. Dashed line indicates treated animals with anti-PD-1 inhibitor RMP1-14. Data was analyzed via Mantel-Cox test and p values are indicated. Bottom panel: Kaplan-Meier curves depicting the overall survival of KP and KPU4 mice after intratracheal induction with Adeno- Cre virus. Solid line indicates untreated animals. Dashed line indicates treated animals with anti-PD-1 inhibitor RMP1-14. Data was analyzed via Mantel-Cox test and p values are indicated. Figure 13. ID signature mapping from whole-exome-sequencing data

[0170] Figure 14. A) Quantification of CD45 positive cells in tumor and representative images of CD45 staining on untreated and RMP1-14 treated KP, KPLI1, and KPLI4 tumors. B) Quantification of CD45 positive cells in tumor and representative images of CD3 staining on untreated and RMP1-14 treated KP, KPLI1 , and KPLI4 tumors.

[0171] The examples illustrate the invention

[0172] Example 1 - High expression of UBQLN1 and UBQLN4 is associated with reduced survival in lung adenocarcinoma patients

[0173] Utilizing publicly available TCGA datasets an amplification of UBQLN4 was identified as a common genetic alteration present in a multitude of various cancer entities (Figure 1)1. UBQLN1 showed lower levels of genetic alterations in this dataset, with cases of amplifications, deletions and other alterations (Figure 2). The inventors previously identified that UBQLN4 mRNA levels were significantly elevated in multiple cancer entities compared to healthy surrounding tissue (Figure 3)2. Utilizing TCGA gene expression data that high expression of UBQLN1 and UBQLN4 it is now identified is associated with reduced survival in lung adenocarcinoma patients (Figure 4)3.

[0174] Example 2 - Ubqln1 / 4-overexpressing autochthonous lung adenocarcinoma mouse models

[0175] Correlation of UBQLN4 expression to reduced survival is not limited to lung adenocarcinoma4. To further translate our findings, several autochthonous lung adenocarcinoma mouse models were successfully generated:

[0176] - KrasLSL G12D / wt;Tp53fl / fl(KP) (the KP model is expected to be HR- proficient),

[0177] - KP+Rosa26LSL Ubqln1 / wt(KPU1) and

[0178] - KP+Rosa26LSL Ubqln4 / wt(KPU4) mice.

[0179] Intratracheal AdenoCre application leads to tumor onset within 10 weeks in KP mice5. Expression of Ubqlnl or 4 results from a potent CAGS promoter, which was engineered into the Rosa26 locus. KPU1 and KPU4 mice show an earlier onset of lung tumors and a more aggressive behavior compared to KP controls (Figure 5). Strikingly, KPLI1 and KPLI4 animals have a significantly worse overall survival, just like the human counterparts (Figure 5).

[0180] An increased tumor mutational burden was further exposed indicated by increased single nucleotide variants (SNV) and number of Insertion and Deletions based on Ubqln1 / 4 overexpression of tumors extracted from KPLI1 and KPLI4 compared to KP animals (Figure 6). Moreover, increased Deletion length (5+) at microhomology regions indicative of HR- deficiency was identified (Figure 7)6.

[0181] Example 3 - PARP1 inhibitor treatment of Ubqln1 / 4-overexpressing autochthonous lung adenocarcinoma mice

[0182] AS HR-deficiency in cancer is known to be amenable to treatment with PARP1 inhibitors tumor cells of KP, KPLI1 and KPLI4 animals were isolated and an increased sensitivity to the PARP1 inhibitor olaparib in vitro was observed (Figure 8). To test whether Ubqln1 / 4- overexpressing lung adenocarcinoma may be sensitive to PARP1 inhibition in vivo, analogous to HR-defective BRCA 7-mutant breast and ovarian cancer, KP, KPU1 and KPU4 tumor bearing mice were generated and mice were treated with the PARP1 inhibitor olaparib (50 mg / kg, i.p. daily for 4 weeks). Tumor responses were assessed by pCT and recorded survival of these mice. Treatment with PARP1 inhibitor olaparib led to a significant survival benefit in KPLI1 and KPLI4 animals, in contrast, olaparib treated KP animals did not show a survival benefit (Figure 9). In order to translate these findings to humans, we assessed the expression levels of LIBQLN1 and LIBQLN4 in a human lung cancer cell line panel and identified a substantial amount of cell lines with high expression of LIBQLN1 and LIBQLN4 (Figure 10). We chose each 4 cell lines with either high or low UBQLN1 / 4 expression and verified that high expression of UBQLN1 / 4 conferred sensitivity to the PARP1 inhibitor olaparib in vitro, compared to no effect in UBQLN1 / 4 low expressing lung cancer cell lines (Figure 10). In line with an increased TMB in KPU1 and KPLI4 tumors, we further identified an increased neoantigen mutational count in tumors overexpressing Ubqlnl and Ubqln4 (Figure 11). These tumors (KP, KPU1 and KPLI4) were further analyzed for the occurrence of DNA insertions and deletions (ID) signatures in reference to the COSMIC database. Tumors of KPU1 and KPLI4 showed a specific prevalence of signatures ID8, which is proposed to be associated with repair of DNA double strand breaks by NHEJ and ID19, which is primarily composed of 5 base pair insertions (Figure 13). These signatures appear to be distinct for the KPU1 and KPLI4 genotype. Example 4 - Anti-PD-1 antibody treatment of Ubqln1 / 4-overexpressing autochthonous lung adenocarcinoma mice

[0183] Increased TMB in lung cancer is associated with increased sensitivity to immune checkpointblocking therapies7. Thus, in addition to PARP1 inhibition, we assessed the effectiveness of PD-1 blockade in our murine system. KP, KPLI1 and KPLI4 animals were treated with the anti-PD-1 antibody RMP1-14 (10 mg / kg; i.p. bi-weekly) after induction of lung tumors by adeno-cre. KPLI1 treated animals had a significantly improved overall survival compared to KP treated animals; whereas in KPLI4 animals a trend to better survival could be observed (Figure 12).

[0184] Example 5 - Overexpression of UBQLN1 and UBQLN4 improves attraction of immune effector cells killing tumor cells

[0185] Tumors of the respective genotype that were either untreated, or treated with PARPi or checkpoint-inhibitor were subjected to IHC to assess the tumor microenvironment. Following treatment with checkpoint inhibition KPLI1 and KPLI4 tumors showed an increased infiltration with immune cells (CD45+) and specifically CD3+ T cells (Figure 14). These data suggest that overexpression of LIBQLN1 and LIBQLN4 allow for the better attraction of immune effector cells killing tumor cells, which potentially explains the survival benefit observed in KPLI1 and KPLI4 tumor mice following checkpoint inhibition.

[0186] References Examples

[0187] 1. Cerami, E., Gao, J., Dogrusoz, II., Gross, B.E., Sumer, S.O., Aksoy, B.A., Jacobsen, A., Byrne, C.J., Heuer, M.L., Larsson, E., Antipin, Y., Reva, B., Goldberg, A.P., Sander, C., and Schultz, N. (2012). The eBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2, 401- 404. 10.1158 / 2159-8290.CD-12-0095.

[0188] 2. Kobayashi, Y., Bustos, M.A., Shoji, Y., Jachimowicz, R.D., Shiloh, Y., and Hoon, D.S.B. (2022). Genomic Amplification of LIBQLN4 Is a Prognostic and Treatment Resistance Factor. Cells 11. 10.3390 / cells11203311.

[0189] 3. Cancer Genome Atlas Research, N. (2014). Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543-550. 10.1038 / nature13385.

[0190] 4. Jachimowicz, R.D., Beleggia, F., Isensee, J., Velpula, B.B., Goergens, J., Bustos, M.A., Doll, M.A., Shenoy, A., Checa-Rodriguez, C., Wiederstein, J.L., Baranes- Bachar, K., Bartenhagen, C., Hertwig, F., Teper, N., Nishi, T., Schmitt, A., Distelmaier, F., Ludecke, H.J., Albrecht, B., Kruger, M., Schumacher, B., Geiger, T., Hoon, D.S.B. , Huertas, P., Fischer, M., Hucho, T., Peifer, M., Ziv, Y., Reinhardt, H.C., Wieczorek, D., and Shiloh, Y. (2019). LIBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors. Cell 176, 505-519 e522. 10.1016 / j. cell.2018.11.024.

[0191] 5. Schmitt, A., Knittel, G., Weicker, D., Yang, T.P., George, J., Nowak, M., Leeser, II., Buttner, R., Perner, S., Peifer, M., and Reinhardt, H.C. (2017). ATM Deficiency Is Associated with Sensitivity to PARP1- and ATR Inhibitors in Lung Adenocarcinoma. Cancer Res 77, 3040-3056. 10.1158 / 0008-5472.CAN-16-3398.

[0192] 6. Alexandrov, L.B., Kim, J., Haradhvala, N.J., Huang, M.N., Tian Ng, A.W., Wu, Y., Boot, A., Covington, K.R., Gordenin, D.A., Bergstrom, E.N., Islam, S.M.A., Lopez- Bigas, N., Klimczak, L.J., McPherson, J.R., Morganella, S., Sabarinathan, R., Wheeler, D.A., Mustonen, V., Group, P.M.S.W., Getz, G., Rozen, S.G., Stratton, M.R., and Consortium, P. (2020). The repertoire of mutational signatures in human cancer. Nature 578, 94-101. 10.1038 / s41586-020-1943-3.

[0193] 7. Ricciuti, B., Wang, X., Alessi, J.V., Rizvi, H., Mahadevan, N.R., Li, Y.Y., Polio, A., Lindsay, J., Umeton, R., Sinha, R., Vokes, N.I., Recondo, G., Lamberti, G., Lawrence, M., Vaz, V.R., Leonardi, G.C., Plodkowski, A. J., Gupta, H., Cherniack, A.D., Tolstorukov, M.Y., Sharma, B., Felt, K.D., Gainor, J.F., Ravi, A., Getz, G., Schalper, K.A., Henick, B., Forde, P., Anagnostou, V., Janne, P.A., Van Allen, E.M., Nishino, M., Sholl, L.M., Christiani, D.C., Lin, X., Rodig, S.J., Hellmann, M.D., and Awad, M.M. (2022). Association of High Tumor Mutation Burden in Non-Small Cell Lung Cancers With Increased Immune Infiltration and Improved Clinical Outcomes of PD-L1 Blockade Across PD-L1 Expression Levels. JAMA Oncol 8, 1160-1168. 10.1001 / jamaoncol.2022.1981.

Claims

CLAIMS1. A Poly(ADP-Ribose) Polymerase 1 (PARP-1) inhibitor and / or an immune checkpoint inhibitor for use in the treatment of cancer in a subject that overexpresses Ubiquilin 1 (UBQLN1) and / or Ubiquilin 4 (UBQLN4).

2. The PARP-1 inhibitor and / or immune checkpoint inhibitor for the use of claim 1, wherein the PARP-1 inhibitor and optionally the immune checkpoint inhibitor are used.

3. The PARP-1 inhibitor and / or immune checkpoint inhibitor for the use of claim 1 or 2, wherein the subject overexpresses UBQLN1 and optionally overexpresses UBQLN4.

4. The PARP-1 inhibitor and / or immune checkpoint inhibitor for the use of any one of claims 1 to 3, wherein the cancer is non-small cell lung cancer, preferably lung adenocarcinoma.

5. The PARP-1 inhibitor and / or immune checkpoint inhibitor for the use of any one of claims 1 to 3, wherein immune checkpoint target is selected from PD-1 , PD-L1 and CTLA-4.

6. The PARP-1 inhibitor and / or immune checkpoint inhibitor for the use of any one of claims 1 to 3, wherein the inhibitor(s) is / are (a) small molecule inhibitor(s), (a) nucleotide-based inhibitor(s) or (an) amino acid-based inhibitor(s).

7. The PARP-1 inhibitor and / or immune checkpoint inhibitor for the use of claim 6, wherein the small molecule inhibitor(s) ofPARP-1 is / are selected from Olaparib (Lynparza™; AZD2281), Niraparib, Rucaparib, Talazoparib, Veliparib, Pamiparib, Fuzuloparib, Senaparib, AZD-5305, AZD-9574, CVL-218, HTMC-0435, HWH-340, JPI-289, NMS-293, SC-10914, Stenoparib, TSL- 1502, Venadaparib, HRS-1167, Nesuparib, and RP-12146; the small molecule inhibitor(s) of PD-1 is / are selected from CCX-559 and CS-17938; the small molecule inhibitor(s) of PD-L1 is / are selected from INCB-099280, INCB- 86550, WP-1066, ABSK-043, AN-4005, ASC-61, BPI-371153, CCX-559, FH-2001 , IMMH-010, INCB-099318, Max-1, and AB-101 ; and the small molecule inhibitor of CTLA-4 is BPI-002.

8. The PARP-1 inhibitor and / or immune checkpoint inhibitor for the use of any one of claims 1 to 7, wherein the inhibitor(s) is / are(an) inhibitor(s) of the nucleic acid molecule encoding PARP-1 and / or the nucleic acid molecule encoding the immune checkpoint target, and / or(an) inhibitor(s) of the activity of PARP-1 and / or the activity of the immune checkpoint target.

9. The PARP-1 inhibitor and / or immune checkpoint inhibitor for the use of claim 8, wherein(i) the inhibitor(s) of the nucleic acid molecule encoding PARP-1 and / or the nucleic acid molecule encoding the immune checkpoint target is / are selected from an aptamer, a siRNA, a shRNA, a miRNA, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas9-based construct, a CRISPR-Cpf1 -based construct, a meganuclease, a zinc finger nuclease, and a transcription activator-like (TAL) effector (TALE) nuclease, and / or(ii) inhibitor(s) of the activity of PARP-1 and / or the activity of the immune check point target is / are selected from a small molecule, an antibody or antibody mimetic, and an aptamer, wherein the antibody mimetic is preferably selected from affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and probodies.

10. The PARP-1 inhibitor and / or immune checkpoint inhibitor for use of claim 8 or 9, wherein inhibitor(s) of the nucleic acid molecule encoding PARP-1 and / or the nucleic acid molecule encoding the immune check point target comprise(s)(a) a nucleic acid sequence which comprises or consists of a nucleic acid sequence being complementary to at least 12 continuous nucleotides of a nucleic acid sequence selected from SEQ ID NOs 3 to 6,(b) a nucleic acid sequence which comprises or consists of a nucleic acid sequence which is at least 70% identical to the complementary strand of one or more nucleic acid sequences selected from SEQ ID NOs 7 to 10,(c) a nucleic acid sequence which comprises or consists of a nucleic acid sequence according to (a) or (b), wherein the nucleic acid sequence is DNA or RNA,(d) an expression vector expressing the nucleic acid sequence as defined in any one of (a) to (c), preferably under the control of a lung-specific promoter, or(e) a host comprising the expression vector of (d).

11. The PARP-1 inhibitor and / or immune checkpoint inhibitor for the use of claim 9, wherein (i) the inhibitor(s) is / are antibody / ies against (a) immune checkpoint target(s), wherein the antibody / ies against is / are preferably selected from one or more of an anti-PD-1 antibody, such as Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, Retifanlimab, Tislelizumab, APL-501 , Spartalizumab, Camrelizumab, Vopratelimab, AMP-514, MGA012, Acrixolimab, Toripalimab, Sintilimab, Penpulimab, Cadonilimab, Pucotenlimab, Camrelizumab, Prolgolimab, Serplulimab, Zimberelimab, Ivonescimab, Cetrelimab, Finotonlimab, IBI-318, MK-3475A, Nofazinlimab, QL-1604, Rulonilimab, Sasanlimab, or Tebotelimab; an anti-PD-L1 antibody, such as Atezolimumab, Durvalumab, Avelumab, Cosibelimab, Envafolimab, Adebrelimab, Sugemalimab, APL-502, Socazolimab, Tagitanlimab, IBI- 318, KN-035, KN-046, PM-8002, or SHR-1701 ; and an anti-CTLA-4 antibody, such as Ipilimumab, Tremelimumab, AGEN1884, Cadonilimab, Gotistobart, KN-046, ADG-126, BMS-986218, BMS-986249, BMS- 986288, Botensilimab, Danvilostomig, GI-102, HL-06, JK-08, Lorigerlimab, Porustobart, Quavonlimab, RP-2, Volrustomig, Vudalimab, XTX-101 , YH-001 , or Zalifrelimab.

12. A method for determining whether a cancer subject is amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor, wherein the method comprises:(a) detecting the mRNA and / or protein expression level of LIBQLN1 and / or LIBQLN4 in a sample obtained from said cancer subject, and(b) comparing the expression level of (a) with the corresponding expression level in a sample obtained from healthy subjects or a predetermined standard thereof, wherein a greater than 1.25-fold upregulation of the expression level of (a) is indicative for cancer patient being amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor, or(b’) comparing the expression level of (a) with the corresponding expression levels in a plurality of samples obtained from cancer subjects or a predetermined standard thereof, wherein an expression level of (a) falling into the top 25% of the expression levels of (b’) is indicative for cancer patient being amenable for the treatment with a PARP-1 inhibitor and / or an immune checkpoint inhibitor13. The method of claim 12, wherein the method is for determining whether a cancer subject is an amenable for the treatment with a PARP-1 inhibitor and optionally further for determining whether a cancer subject is an amenable for the treatment with an immune checkpoint inhibitor.

14. The method of claim 12 or 13, wherein in (a) the mRNA and / or protein expression level of LIBQLN1 is detected and optionally the mRNA and / or protein expression level of LIBQLN4 is detected.

15. The method of any one of claims 12 to 14, wherein the cancer subject is a lung cancer subject, preferably a lung adenocarcinoma subject.

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