WO2021151974A1 - Interfering with mrna splicing to enhance response to checkpoint immunotherapies. - Google Patents

Abstract

The invention pertains to a compound that induces aberrant splicing in a tumor cell for use in the treatment of cancer, wherein in the treatment, the compound is combined with an immune checkpoint therapy. A preferred compound is a sulphonamide, preferable an aryl-sulphonamide. The invention further pertains to a diagnostic method to identifyi a subject that benefits from immune checkpoint therapy, comprising a step of detecting the presence of a mutated splicing factor in a tumor sample.

Description

Interfering with mRNA splicing to enhance response to checkpoint immunotherapies

Field of the invention

The present invention relates to the field of medicine, in particular to molecular oncology. More specifically, the invention concerns a compound that induces aberrant mRNA splicing in a tumor cell for use in the treatment of a cancer, wherein preferably the tumor cell is combined with an immune checkpoint therapy.

Background

Checkpoint immunotherapies (Cls, targeting molecules like CTLA4, PD1 and PDL1) can be effective for the treatment of cancers. Ample evidence exists to indicate that the mutation load of a tumor is a major factor in determining the effectiveness of these Cls against cancers. Tumors with a high mutation load, like melanoma and non-small cell lung cancer are among the most responsive tumors to Cls. Another cancer type with significant responses to Cl are the microsatellite instable (MSI) colon cancers (Le, D. T., et al, PD-1 Blockade in Tumors with Mismatch-Repair Deficiency, 2015, New England Journal of Medicine 372, 2509-2520).

More recently, the US FDA has approved Cls for all MSI tumors, irrespective of the tissue of origin of the tumor (Marcus, L, et al FDA Approval Summary: Pembrolizumab for the treatment of microsatellite instability-high solid tumors. Clin. Cancer Res. 2019; 25(13):3753-3758). Such somatic mutations are thought to result in non-self immunogenic antigens that can be recognized by cytotoxic T cells. Cls can enhance the responses to these non-self antigens, resulting in tumor shrinkage.

While point mutations that are present in lung cancer and melanoma can be potentially antigenic, the MSI tumors tend to generate “out of frame” mutant proteins due to slippage of di- to tri nucleotide repeats. Such out of frame proteins can have relatively long Carboxyl-terminal “nonsense” extensions encoded by another reading frame. Such frame shift peptides (FSPs) can be effective neo-antigens that can be presented by MHC class I antigens to T cells for tumor eradication.

Unfortunately, large subset of patients develop primary or secondary resistance upon immunotherapeutic treatment. Hence, there is still a need in the art for novel therapeutic approaches to and synergistic combinations for an effective anti-cancer treatment. In particular there is a need in the art for to increase to efficacy of checkpoint immunotherapy for the treatment of cancer. Summary of the invention

The invention may be summarized in the following embodiments:

Embodiment 1. A compound that induces aberrant mRNA splicing in a tumor cell for use in the treatment of a cancer, wherein in the treatment, the compound is combined with an immune checkpoint therapy.

Embodiment 2. A compound for a use according to embodiment 1 , wherein the aberrant mRNA splicing in the tumor cell alters the transcriptome profile of the tumor cell by increasing the level of aberrant mRNAs by at least one of: i) alternative 3’splice site usage; ii) alternative 5’splice site usage; iii) intron retention; iv) exon skipping; v) exon retention; and vi) presence of alternative exons, wherein preferably the transcriptome profile can be determined in an RNAseq experiment, preferably the RNAseq experiment of example 1 , and wherein the increase in the level of aberrant mRNAs is preferably at least a 2-fold increase as compared to the level of the same aberrant mRNAs in a control tumor cell not treated with the compound.

Embodiment 3. A compound for a use according to embodiment 1 or 2, wherein the compound is a small molecule.

Embodiment 4. A compound for a use according to any one of the preceding embodiments, wherein the compound is an aryl-sulfonamide, preferably selected from the group consisting of indisulam, NSC 719239 (E7820), NSC 339004 (CQS) and tasisulam.

Embodiment 5. A compound for a use according to embodiment 3 or 4, wherein the compound is indisulam or a functional equivalent thereof.

Embodiment 6. A compound for a use according to any one of the preceding embodiments, wherein the immune checkpoint therapy comprises the use of an immune checkpoint blocking agent that blocks at least one of PD-1 , PD-L1 and CTLA-4.

Embodiment 7. A compound for a use according to embodiment 6, wherein the immune checkpoint blocking agent is at least one of ipilimumab, nivolumab, pembrolizumab, antibody BGB-A31 and atezolizumab. Embodiment 8. A compound for a use according to any one of the preceding embodiments, wherein the compound is administered as a pretreatment of the immune checkpoint therapy, and wherein preferably treatment with the compound is continued during the immune checkpoint therapy.

Embodiment 9. A compound for a use according to any one of the preceding embodiments, wherein the compound is administered simultaneously, separately or sequentially with the immune checkpoint therapy.

Embodiment 10. A compound for a use according to any one of the preceding embodiments, wherein the compound is for a use in the prevention of resistance to immune checkpoint therapy or for a use in the treatment of a cancer comprising tumor cells that are resistant to immune checkpoint therapy.

Embodiment 11. A pharmaceutical composition comprising a compound as defined in any one of embodiments 1 - 5 and an immune checkpoint blocking agent as defined in embodiment 6 or 7, or a kit of parts comprising a pharmaceutical composition comprising a compound as defined in any one of embodiments 1 - 5 and a pharmaceutical composition comprising an immune checkpoint blocking agent as defined in embodiment 6 or 7, wherein optionally the kit of parts comprises a leaflet with instructions for use.

Embodiment 12. A pharmaceutical composition or a kit of parts according to embodiment 11 for use in the treatment of a cancer, wherein preferably the composition or the kit is used to prevent resistance to immune checkpoint therapy or to treat a cancer comprising tumor cells that are resistant to immune checkpoint therapy.

Embodiment 13. An agent for identifying a subject that benefits from immune checkpoint therapy, wherein the agent binds to a mutated splicing factor, or binds to a sequence encoding a mutated splicing factor.

Embodiment 14. An agent according to embodiment 13, wherein the agent is a nucleic acid molecule, an antibody or an antigen-binding fragment thereof.

Embodiment 15. A method for identifying a subject that benefits from immune checkpoint therapy, comprising a step of detecting the presence of a mutated splicing factor in a tumor sample, wherein preferably the method is an ex vivo method.

Embodiment 16. A method according to embodiment 15, wherein the mutated splicing factor is at least one of RBM39, SF3B1 , SRSF2 and U2AF1 . Embodiment 17. A method for treating a cancer in a subject comprising administering to the subject an effective amount of a compound that induces aberrant mRNA splicing as defined in any one of embodiments 1 - 5, wherein in the treatment, the compound is combined with an immune checkpoint therapy, wherein preferably the immune checkpoint therapy is a therapy as defined in embodiment 6 or 7.

Embodiment 18. The method of embodiment 17, wherein the method reduces the risk of developing resistance to the immune checkpoint therapy or wherein the cancer comprises tumor cells that are resistant to immune checkpoint therapy.

Definitions

Various terms relating to the methods, compositions, formulations, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.

“A,” “an,” and “the”: these singular form terms include plural referents unless the content clearly dictates otherwise. The indefinite article "a" or "an" thus usually means "at least one". Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

“About” and “approximately”: these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

“And/or”: The term “and/or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

As used herein, with "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ..., etc.

“Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

Exemplary: this terms means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein.

It is herein understood that the terms “RNA splicing”, “mRNA splicing” and “pre-mRNA splicing” can be used interchangeable herein and refer to the process of removing introns from the pre-mRNA and the joining of exons to obtain a mature mRNA molecule.

As used herein, with “transciptome” or “transcriptome profile” the sum of all messenger RNA molecules in one cell or population of cells, preferably identical cells, is intended.

As used herein "cancer" and "cancerous", refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer is also referred to as malignant neoplasm.

As used herein, "in combination with" is intended to refer to all forms of administration that provide a first drug together with a further (second, third) drug. The drugs may be administered simultaneous, separate or sequential and in any order. Drugs administered in combination have biological activity in the subject to which the drugs are delivered.

As used herein "simultaneous" administration refers to administration of more than one drug at the same time, but not necessarily via the same route of administration or in the form of one combined formulation. For example, one drug may be provided orally whereas the other drug may be provided intravenously. Separate includes the administration of the drugs in separate form and/or at separate moments in time, but again, not necessarily via the same route of administration. Sequentially indicates that the administration of a first drug is followed, immediately or in time, by the administration of the second drug.

A used herein "compositions", "products" or "combinations" useful in the methods of the present disclosure include those suitable for various routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral or mucosal application. The compositions, formulations, and products according to the disclosure invention normally comprise the drugs (alone or in combination) and one or more suitable pharmaceutically acceptable excipients. As used in the context of the invention, the terms "prevent", "preventing", and "prevention" refers to the prevention or reduction of the recurrence, onset, development or progression of a cancer, preferably a cancer as defined herein, or the prevention or reduction of the severity and/or duration of the cancer or one or more symptoms thereof.

As used in the context of the invention, the terms "therapies" and "therapy" can refer to any protocol(s), method(s) and/or agent(s), preferably as specified herein below, that can be used in the prevention, treatment, management or amelioration of cancer, preferably a cancer as defined herein below, or one or more symptoms thereof.

As used herein, the terms "treat", "treating" and "treatment" refer to the reduction or amelioration of the progression, severity, and/or duration of a cancer, preferably a cancer as defined herein below, and/or reduces or ameliorates one or more symptoms of the disease.

As used herein, the term "effective amount" refers to the amount of an agent, e.g., of a compound that induces aberrant mRNA splicing as defined herein, preferably together with an immune checkpoint therapy, which is sufficient to reduce the severity, and/or duration of a cancer, ameliorate one or more symptoms thereof, prevent the advancement of the cancer, or cause regression of the cancer, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of the cancer or one or more symptoms thereof. Preferably, the effective amount of the compound is an amount that enhances or improves the prophylactic and/or therapeutic effect(s) of another therapy, preferably enhances of improves the effects of an immune checkpoint therapy.

The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of a cancer varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. Thus, in connection with the administration of a drug which, in the context of the current disclosure, is "effective against" a cancer indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.

The term "agent" refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject. An agent can be a compound or a composition. An agent can e.g. be selected from the group consisting of: polynucleotides, polypeptides, small molecules, antibodies and functional fragments thereof.

The medical use herein described is formulated as a compound as defined herein for use as a medicament for treatment of the stated disease(s) preferably in combination with an immune checkpoint therapy, but could equally be formulated as a method of treatment of the stated disease(s) using a compound as defined herein preferably in combination with an immune checkpoint therapy, a compound as defined herein for use in the preparation of a medicament to treat the stated disease(s) preferably in combination with an immune checkpoint therapy, and use of a compound as defined herein for the treatment of the stated disease(s) by administering an effective amount, preferably in combination with an immune checkpoint therapy. Such medical uses are all envisaged by the present invention.

As used herein, the term "small molecule" can refer to compounds that are "natural productlike," but mostly will refer synthetic compounds. A small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 5000 Daltons (5 kD), preferably less than 3 kD, still more preferably less than 2 kD, and most preferably less than 1 kD. In some cases it is preferred that a small molecule have a molecular mass equal to or less than 700 Daltons.

The term “protein” or “polypeptide” refers to a molecule consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin. A “fragment” or “portion” of a protein may thus still be referred to as a “protein.” A protein as defined herein and as used in any method as defined herein may be an isolated protein. An “isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or animal host cell. Preferably, the protein comprises more than 50 amino acid residues.

The term “proteinaceous molecule" is herein understood as a molecule comprising a short chain of amino acid monomers linked by peptide (amide) bonds. The short chain of amino acid monomers comprise 2 or more amino acid residues. Preferably, the chain of amino acids has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 amino acid residues. Preferably, the there are no more than 100 amino acid residues. Preferably, there are no more than 50 amino acid residues in the proteinaceous molecule. Preferably, the proteinaceous molecule has about 2-100, 3-50, 4-40 or 5-30, or 6-20 amino acid residues. Preferably, the proteinaceous molecule has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acid residues. Optionally, the proteinaceous molecule comprises one or more additional organic moieties, such as, but not limited to a linking moiety to generate a cyclised proteinaceous molecule.

An “aptamer” preferably is a nucleic acid molecule having a particular nucleotide sequence. An aptamer can include any suitable number of nucleotides. An aptamer may comprise RNA or DNA, or comprises both ribonucleotide residues and deoxyribonucleotide residues. An aptamer may be single stranded, double stranded, or contain double stranded or triple stranded regions. In addition, an aptamer may comprise chemical modified residues, e.g. to improve its stability.

An aptamer will typically be between about 10 and about 300 nucleotides in length. More commonly, an aptamer will be between about 30 and about 100 nucleotides in length.

Aptamers to a given target (i.e. an aberrant splicing factor) include nucleic acids that may be identified from a candidate mixture of nucleic acids using a method comprising the steps of: (a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to other nucleic acids in the candidate mixture can be partitioned from the remainder of the candidate mixture; (b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to yield an enriched mixture of nucleic acids, whereby aptamers of the target molecule are identified. It is recognized that affinity interactions are a matter of degree; however, in this context, the “specific binding affinity” of an aptamer for its target means that the aptamer binds to its target generally with a much higher degree of affinity than it binds to other, non-target, components in a mixture or sample.

The term "antibody" is used in the broadest sense and specifically covers, e.g. monoclonal antibodies, including agonists and antagonist, neutralizing antibodies, full length or intact monoclonal antibodies, polyclonal antibodies, multivalent antibodies, single chain antibodies and functional fragments of antibodies, including Fab, Fab’, F(ab’)2 and Fv fragments, diabodies, triabodies, single domain antibodies (sdAbs), heavy-chain antibodies, nanobodies, as long as they exhibit the desired biological and/or immunological activity. An antibody can be human and/or humanized. "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.

An antibody "which binds" an antigen of interest, preferably the aberrant splicing factor, is one that binds said antigen with sufficient affinity such that the antibody is useful as a diagnostic marker.

"Antibody fragments" comprise a portion of an intact antibody, preferably at least the antigen binding and/or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; triabodies; linear antibodies (see U.S. Patent No. 5,641 ,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind the antigen.

The term “nanobody” is well-known in the art. A nanobody is an antibody fragment comprising or consisting of a VHH domain of a heavy chain only antibody. A preferred nanobody is derivable from the camelidae family, preferably derivable from a Llama.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, Ape etc.), and human constant region sequences.

Preferably, the antibody for binding to the diagnostic marker as defined herein does not significantly cross-react with other proteins. “Amino acid sequence”: This refers to the order of amino acid residues of, or within a protein. In other words, any order of amino acids in a protein may be referred to as amino acid sequence.

“Nucleotide sequence”: This refers to the order of nucleotides of, or within a nucleic acid. In other words, any order of nucleotides in a nucleic acid may be referred to as nucleotide sequence.

The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

The term “complementarity” is herein defined as the sequence identity of a nucleotide sequence to a fully complementary strand (e.g. the second, or reverse, strand). For example, a sequence that is 100% complementary (or fully complementary) is herein understood as having 100% sequence identity with the complementary strand and e.g. a sequence that is 80% complementary is herein understood as having 80% sequence identity to the (fully) complementary strand.

"Identity" and "similarity" can be readily calculated by known methods. “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blosum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred.

Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, word length = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

Nucleotide sequences of the invention may also be defined by their capability to hybridize with the specific nucleotide sequences disclosed herein or parts thereof, under moderate, or preferably under stringent hybridization conditions. Stringent hybridization conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridize at a temperature of about 65°C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at 65°C in a solution comprising about 0.1 M salt, or less, preferably 0.2 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is performed overnight, i.e. at least for 10 hours and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridization of sequences having about 90% or more sequence identity.

Moderate conditions are herein defined as conditions that allow a nucleic acid sequences of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridize at a temperature of about 45°C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is performed overnight, i.e. at least for 10 hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridization of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridization conditions in order to specifically identify sequences varying in identity between 50% and 90%. Detailed description of the invention

The inventors discovered that compounds inducing aberrant mRNA splicing can be effectively used for the treatment of a cancer. The induction of aberrant splicing in a tumor cell results in the expression of frame-shift proteins which will be presented by the MHC class I antigens to serve as effective neo-antigens for T cells fortumor eradication. The expression of these neoantigens will be particularly effective in combination with immune checkpoint therapy.

Compound In a first aspect, the invention therefore pertains to a compound that induces aberrant mRNA splicing in a tumor cell for use in the treatment of a cancer. Preferably in the treatment, the compound is combined with an immune checkpoint therapy.

The aberrant mRNA splicing can result in the expression of proteins that can serve as neoantigens. These proteins can be so-called “frame shift peptides” (FSPs), i.e. peptides that are expressed due to a shift in the reading frame of the aberrantly spliced mRNA molecule. Aberrant splicing may not only result in the expression of FSPs. As a non-limiting example, aberrant splicing may also result in the expression of proteins that comprise a section that is not normally present in the protein, e.g. due to the retention an in-frame intron, due to the presence of an alternative or additional in-frame exon and/or due to the absence of an in-frame exon. “Aberrant mRNA splicing” is understood herein as a splicing process that deviates or differs from a normal mRNA splicing process. Preferably, the aberrant splicing process deviates or differs from the normal splicing process in an otherwise identical cell that is not exposed to a compound as defined herein.

Aberrant splicing may comprise the use of an alternative 3’ acceptor splice site and/or may comprise the use of an alternative 5’ donor splice site by the splicing machinery. Alternatively or in addition, alternative splicing may comprise the skipping a 3’ acceptor splice site and/or may comprise the skipping a 5’ donor splice site by the splicing machinery, wherein the respective donor and/or acceptor splice site is normally used by the splicing machinery in an otherwise identical cell not having an aberrant RNA splicing machinery. Hence a compound for use in the treatment of a cancer a defined herein may induce aberrant mRNA splicing, wherein the aberrant mRNA splicing comprises at least one of the use of alternative 3’ acceptor splice site, the use of an alternative 5’ donor splice site, the skipping a 3’ acceptor splice site and the skipping a 5’ donor splice site. The production of these aberrant mRNA molecules may be increased at least about 2-, 10-, 50-, 100-, 500- or at least about 1000-fold as compared to an untreated control tumor cell. As a consequence of using an aberrant 5’ splice site and/or aberrant 3’ splice site, part of the pre-mRNA molecule that should normally be spliced out may be retained in the mRNA molecule and/or part of the RNA molecule that should normally be maintained is spliced out from the mRNA molecule. The result of an aberrant splicing is the presence of an aberrant mRNA molecule, e.g. encoding a frame shift protein. The compound-induced aberrant splicing typically does not only affect the splicing of a single or a few (specific) pre-mRNA molecule(s) but preferably more generally affects the splicing accuracy in a tumor cell. A compound for a use as defined herein preferably alters the transcriptome profile of a tumor cell by increasing the level of aberrant mRNAs by at least of an alternative 3’ splice site usage, an alternative 5’ splice site usage, intron retention, exon skipping, exon retention and the presence of an alternative exon. Preferably, the presence of an alternative exon is at least one of the presence of an alternative first exon, the presence of an alternative last exon, the presence of a mutually exclusive exon. The production of these aberrant mRNA molecules may be increased at least about 2-, 10-, 50-, 100-, 500- or at least about 1000-fold as compared to an untreated control tumor cell.

The transcriptome profile of the tumor cell can be determined by using any conventional means known to the skilled person, such a micro-array analysis and deep-sequencing of the mRNA molecules of a cell (RNAseq), including but not limited to single-cell sequencing. Preferably, the transcriptome profile of a tumor cell is determined using RNAseq. To determine the effect of a compound on the splicing accuracy of a tumor cell, i.e. whether the compound induces aberrant mRNA splicing, the transcriptome profile of a tumor cell treated with a compound can be compared with the transcriptome profile of an untreated control tumor cell. Preferably, the control tumor cell is identical to the tumor cell treated with a compound as defined herein, and is preferably cultured under identical conditions, with preferably the only exception that the control tumor cell is not treated with a compound as defined herein. Preferably, the genome of the untreated control tumor cell is substantially identical to the genome of the treated tumor cell.

RNA sequencing to determine the transcriptome profile can be performed using any conventional deep sequencing method known in the art, such as but not limited to Roche 454A and 454B sequencing, ILLUMINA™ SOLEXA™ sequencing, Applied Biosystems' SOLID™ sequencing, the Pacific Biosciences' SMRT™ sequencing, Pollonator Polony sequencing, Oxford Nanopore Technologies or the Complete Genomics sequencing.

Preferably, the compound increases the production of aberrant mRNA molecules in a cell due to the use of an alternative 3’ splice site by the splicing machinery. The production of these aberrant mRNA molecules may be increased at least about 2-, 10-, 50-, 100-, 500- or at least about 1000-fold as compared to an untreated control tumor cell.

Preferably, the compound increases the production of aberrant mRNA molecules in a cell due to the use of an alternative 5’ splice site by the splicing machinery. The production of these aberrant mRNA molecules may be increased at least about 2-, 10-, 50-, 100-, 500- or at least about 1000-fold as compared to an untreated control tumor cell.

Preferably, the compound increases the production of aberrant mRNA molecules in a cell due to an intron retention. The production of these aberrant mRNA molecules may be increased at least about 2-, 10-, 50-, 100-, 500- or at least about 1000-fold as compared to an untreated control tumor cell.

Preferably, the compound increases the production of aberrant mRNA molecules in a cell due to the skipping of an exon. The production of these aberrant mRNA molecules may be increased at least about 2-, 10-, 50-, 100-, 500- or at least about 1000-fold as compared to an untreated control tumor cell.

Preferably, the compound increases the production of aberrant mRNA molecules in a cell due to the erroneous retention of an exon. The production of these aberrant mRNA molecules may be increased at least about 2-, 10-, 50-, 100-, 500- or at least about 1000-fold as compared to an untreated control tumor cell.

Preferably, the compound increases the production of aberrant mRNA molecules in a cell due to the erroneous usage of alternative exons. Preferably, the erroneous usage an alternative exon is at least one of the presence of an alternative first exon, the presence of an alternative last exon, the presence of a mutually exclusive exon. The production of these aberrant mRNA molecules may be increased at least about 2-, 10-, 50-, 100-, 500- or at least about 1000-fold as compared to an untreated control tumor cell.

The compound for use in the invention induces aberrant mRNA splicing. Preferably, the compound is at least one of a small molecule, a peptide and a proteinaceous molecule. Preferably, the compound is a small molecule.

The compound for use in the invention preferably interferes with the splicing process in the tumor cell, e.g. by inhibiting one or more splicing factors. As a non-limiting example, the compound for use in the invention may induce degradation, such as proteasomal or lysosomal degradation, of one or more splicing factors. The degradation can be ubiquitin-induced proteasomal degradation.

The inhibited splicing factor may be a splicing activator or a splicing repressor. The splicing factor inhibited by a compound as defined herein may be an RNA-binding motif protein (RBM), preferably RBM39 (also annotated as CAPERa).

Preferably, the compound is a sulfonamide. The terms “sulfonamide”, “sulphonamide”, “sulfa drug” and “sulpha drug” may be used interchangeable herein and refers to a compound comprising a sulphonamide functional group. Preferably, the compound for use in the invention is an aryl- sulfonamide. Preferably, the compound for use in the invention is a so-called splicing inhibitor sulphonamide (SPLAM) as defined e.g. in Han et al ( Anticancer sufonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15, (2017), eaal3755). SPLAMs are compounds that target the splicing factor RBM39 for proteasomal degradation.

Preferred sulphonamides have general formula (I):

wherein X is a bond, C(=0), or-C(=0)-NH-;

Ar^N is a C5-12 aromatic hydrocarbon optionally comprising 1 , 2, or 3 heteroatoms and optionally substituted with 1 , 2, or 3 instances of R^N;

Ar³ is a C5-12 aromatic hydrocarbon optionally comprising 1 , 2, or 3 heteroatoms and optionally substituted with 1 , 2, or 3 instances of R^s; R^N is halogen, nitrile, Ci-3alkyl, Ci-3alkoxyl, C₂-3acyl, -0-C₂-3acyl, -NH-C₂-3acyl, amino, hydroxyl, sulphonamide, or guanidinyl, or two instances of R^N can together form a bridging moiety that can be -(CH₂)₂-5-, -0-(CH₂)I-4-, -NH-(CH₂)I-4-, -(CH₂)I-3-0-(CH₂)I-3-, or -(CH₂)I-3-NH-(CH₂)I-3- wherein the bridging moiety can be further unsaturated or halogenated; and

R^s is halogen, nitrile, Ci-3alkyl, Ci-3alkoxyl, C₂-3acyl, -0-C₂-3acyl, -NH-C₂-3acyl, amino, hydroxyl, sulphonamide, or guanidinyl, or two instances of R^s can together form a bridging moiety that can be -(CH₂)₂-5-, -0-(CH₂)I-4-, -NH-(CH₂)I-4-, -(CH₂)I-3-0-(CH₂)I-3-, or -(CH₂)I-3-NH-(CH₂)I-3- wherein the bridging moiety can be further unsaturated or halogenated.

When X is a bond, it can be considered to be absent. In this case Ar^N is directly attached to the nitrogen of the central sulphonamide, for example as in indisulam. When X is C(=0) an arylamide is formed, for example as in tasisulam. When X is -C(=0)-NH- it preferably forms a urea moiety, for example as in Ly295501. In preferred embodiments, X is a bond or C(=0), more preferably a bond. In preferred embodiments, X is a bond or -C(=0)-NH-. In preferred embodiments, X is -C(=0)-NH- or C(=0), more preferably C(=0). In other preferred embodiments, X is -C(=0)-NH-.

Ar^N is the aryl moiety that is on the nitrogen side of the central sulphonamide. Preferably it is a C5-10 aromatic hydrocarbon optionally comprising 1 , 2, or 3 heteroatoms and optionally substituted with 1 , 2, or 3 instances of R^N, more preferably it is C6-10. In preferred embodiments it is substituted with 0, 1 , or 2 instances of R^N, more preferably with 0 or 1 instances, most preferably with 1 . Preferably, when X is not a bond, Ar^N comprises an optionally substituted phenyl moiety. Preferably, when X is a bond, Ar^N comprises an optionally substituted indole or isoindole or quinoxaline moiety. Preferably, when at least 1 instance of R^N is present, it is para to the central sulphonamide. Preferably, when a second instance of R^N is present, it is meta to the central sulphonamide. In other embodiments, when a second instance of R^N is present, it is orto to the central sulphonamide.

Ar³ is the aryl moiety that is on the sulphur side of the central sulphonamide. Preferably it is a C5-10 aromatic hydrocarbon optionally comprising 1 , 2, or 3 heteroatoms and optionally substituted with 1 , 2, or 3 instances of R^N, more preferably it is C5-6. In preferred embodiments it is substituted with 0, 1 , or 2 instances of R^N, more preferably with 1 or 2 instances, most preferably with 1 . Preferably, when at least 1 instance of R^s is present, it is para to the central sulphonamide. Preferably, when a second instance of R^s is present, it is meta to the central sulphonamide. When 2 instances of R^s are present they preferably form a bridging moiety. Ar^ is preferably an optionally substituted phenyl orthiophenyl moiety.

R^N is halogen, nitrile, Ci-3alkyl, Ci-3alkoxyl, C₂-3acyl, -0-C₂-3acyl, -NH-C₂-3acyl, amino, hydroxyl, sulphonamide, or guanidinyl, or two instances of R^N can together form a bridging moiety that can be -(CH₂)₂-5-, -0-(CH₂)I-4-, -NH-(CH₂)I-4-, -(CH₂)I-3-0-(CH₂)I-3-, or -(CH₂)I-3-NH-(CH₂)I-3- wherein the bridging moiety can be further unsaturated or halogenated. Preferably, R^N is halogen, nitrile, Ci-2alkyl such as methyl, amino, or sulphonamide, or two instances of R^N can together form a bridging moiety. More preferably R^N is chlorine, methyl, or nitrile. Halogen in R^N is preferably chlorine, fluorine, bromine, or iodine, more preferably chlorine, fluorine, or bromine, even more preferably chlorine or bromine, most preferably chlorine. Bridging moieties formed by R^N are preferably -0-(CH2)I-4- such as -O-CH2-CH2-.

R^s is halogen, nitrile, Ci-3alkyl, Ci-3alkoxyl, C₂-3acyl, -0-C₂-3acyl, -NH-C₂-3acyl, amino, hydroxyl, sulphonamide, orguanidinyl, or two instances of R^s can together form a bridging moiety that can be -(CH₂)₂-5-, -0-(CH₂)I-4-, -NH-(CH₂)I-4-, -(CH₂)I-3-0-(CH₂)I-3-, or -(CH₂)I-3-NH-(CH₂)I-3- wherein the bridging moiety can be further unsaturated or halogenated. Preferably, R^s is halogen, nitrile, Ci-2alkyl such as methyl, amino, or sulphonamide, or two instances of R^s can together form a bridging moiety. More preferably R^s is sulphonamide, amino, halogen, nitrile, or comprised in a bridging moiety. Halogen in R^N is preferably chlorine, fluorine, bromine, or iodine, more preferably chlorine, fluorine, or bromine, even more preferably chlorine or bromine, most preferably chlorine. Bridging moieties formed by R^s are preferably -0-(CH2)I-4- such as -O-CH2-CH2-.

Preferred moieties for Ar³ or Ar^N are shown below, with * indicating where the moiety is attached to the central sulphonamide, and with reference names below each moeity.

In preferred embodiments, Ar^ is selected from Ar1-Ar12, more preferably from Ar1-Ar11 , even more preferably from Ar6-Ar11 , most preferably from Ar7-Ar10. It is most preferably Ar7. In preferred embodiments, Ar^N is selected from Ar1-Ar12, more preferably from Ar1-Ar11 , even more preferably from Ar1-Ar6, most preferably from Ar1-Ar4. It is most preferably Ar1.

In preferred embodiments, when X is a bond, Ar^N is Ar1 , Ar2, or Ar4. In preferred embodiments, when X is C(=0), Ar^N is Ar3. In preferred embodiments, when X is -C(=0)-NH-, Ar^N is Ar4 or Ar5. Preferred sulphonamides of formula (I) have the following substituents:

Preferably, when X is as listed for an entry in the table above, Ar³ is as listed as for that same entry in the table above. Preferably, when X is as listed for an entry in the table above, Ar^N is as listed as for that same entry in the table above, most preferably both Ar³ and Ar^N are as listed for that same entry in the table above. Preferably, when Ar³ is as listed for an entry in the table above, X is as listed as for that same entry in the table above. Preferably, when Ar³ is as listed for an entry in the table above, Ar^N is as listed as for that same entry in the table above, most preferably both X and Ar^N are as listed for that same entry in the table above. Preferably, when Ar^N is as listed for an entry in the table above, X is as listed as for that same entry in the table above. Preferably, when Ar^N is as listed for an entry in the table above, Ar³ is as listed as for that same entry in the table above, most preferably both X and Ar³ are as listed for that same entry in the table above.

A preferred compound is a sufonamide selected from the group consisting of E7070 (indisulam, A/-(3-chloro-1 H-indol-7-yl)-4-sulfamoylbenzenesulfonamide), CQS (4-Amino-N-(5- chloro-2-quinoxalinyl)benzenesulfonamide), tasisulam (N-(2,4-Dichlorobenzoyl)-5- bromothiophene-2-sulfonamide), E7820 (3-cyano-N-(3-cyano-4-methyl-1 H-indol-7-yl)- benzenesulfonamide), LY186641 (N-(4-Chlorophenylaminocarbonyl)indane-5-sulfonamide), LY295501 (N-(5-(2,3-Dihydrobenzofuryl)sulfonyl)-N'-(3,4-dichlorophenyl)urea) and LY-ASAP (N- (2,4-dichlorobenzoyl)-4-chlorophenyl-sulfonamide), e.g. as described in US2009047278. Preferably, the compound is a sulfonamide selected from the group consisting of indisulam, CQS, E7820 and tasisulam. Preferably, the compound is indisulam, or a functional equivalent thereof.

Indisulam (E7070), CQS and tasisulam has been shown previously to interfere with mRNA splicing by binding both to mRNA splicing factor RBM39 and the ubiquitin ligase component DCAF15, causing degradation of RBM39 (Han T et al, supra). In addition, Uehara et al, ( Selective degradation of splicing factor CAPERa by anticancer sulphonamides (2017), Nat Chem Biol.;13(6):675-680) showed that the sulfonamides E7820 (NSC 719239), indisulam and CQS (NSC 339004, chloroquinoxaline sulphonamide) induce degradation of the U2AF-related splicing factor coactivator of activating protein-1 and estrogen receptors (CAPERa) by ubiquitination.

The terms “indisulam” and “E7070” can be used interchangeable herein. Indisulam has PubChem CID no. 216468 and may also be annotated with the lUPAC name “4-N-(3-chloro-1 H- indol-7-yl) benzene- 1 ,4-disulfonamide”. The terms “CQS”, “NSC 339004” and “chloroquinoxaline sulphonamide” can be used interchangeably herein

The terms “E7820” and “NSC 719239” can be used interchangeable herein.

The terms “tasisulam” and “LY573636” can be used interchangeably herein.

The compound may be a pharmaceutically acceptable salt thereof, or a solvate thereof. Pharmaceutically acceptable salts are those salts that are suitable to be administered as drugs or pharmaceuticals to humans and/or animals.

The sulfonamide compound for use in the invention may form a a pharmaceutically acceptable salt with an acid or a base. The sulfonamide compound of the invention also comprises these pharmacologically acceptable salts. Examples of salts formed with acids include inorganic acid salts such as hydrochloride salts, hydrobromide salts, sulfate salts and phosphate salts, and salts formed with organic acids such as formic acid, acetic acid, lactic acid, succinic acid, fumaric acid, maleic acid, citric acid, tartaric acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and trifluoroacetic acid. Examples of salts formed with bases include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, arginine and lysine (organic amine salts), and ammonium salts.

The sulfonamide compound may be in anhydride form, and may form a solvate such as a hydrate. The solvate may be either a hydrate or a nonhydrate, preferably a hydrate. The solvent used may be water, alcohol (e.g., methanol, ethanol or n-propanol), dimethylformamide or the like.

If solvates and/or enantiomers of these compounds exist, the sulfonamide compound of the invention comprises these solvates and/or enantiomers. The sulfonamide compound of the invention may also comprise a sulfonamide compound that undergoes metabolism such as oxidation, reduction, hydrolysis and conjugation e.g. in vivo. Moreover, the sulfonamide compound of the invention also comprises compounds that generate a sulfonamide compound by undergoing metabolism such as oxidation, reduction and hydrolysis e.g. in vivo.

Checkpoint therapy

In a preferred embodiment, the compound as defined herein is combined with an immune checkpoint therapy. The term "immune checkpoint" refers to a group of molecules on the cell surface of CD4⁺ and/or CD8⁺ T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response. Immune checkpoint proteins are well-known in the art and include, without limitation, CTLA-4, PD-1 , VISTA, B7-H2, B7- H3, PD-L1 , B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1 , TIM-3, TIM-4, LAG-3, GITR, 4- IBB, OX-40, BTLA, SIRP, CD47, CD48, 2B4 (CD244), B7.1 , B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, IDO, CD39, CD73 and A2aR (see, for example, WO 2012/177624). The term further encompasses biologically active protein fragments, as well as nucleic acids encoding full-length immune checkpoint proteins and biologically active protein fragments thereof. In some embodiment, the term further encompasses any fragment according to homology descriptions provided herein. Immune checkpoints and their sequences are well-known in the art and representative embodiments are described below. For example, the term "PD-1" refers to a member of the immunoglobulin gene superfamily that functions as a coinhibitory receptor having PD-L1 and PD- L2 as known ligands. PD-1 was previously identified using a subtraction cloning based approach to select for genes upregulated during TCR-induced activated T cell death. PD-1 is a member of the CD28/CTLA-4 family of molecules based on its ability to bind to PD-L1 . Like CTLA-4, PD-1 is rapidly induced on the surface of T- cells in response to anti-CD3 (Agata et al. 25 (1996) Int. Immunol. 8:765). In contrast to CTLA-4, however, PD-1 is also induced on the surface of B-cells (in response to anti-lgM). PD-1 is also expressed on a subset of thymocytes and myeloid cells (Agata et al. (1996) supra; Nishimura et al. (1996) Int. Immunol. 8:773).

The nucleic acid and amino acid sequences of a representative human PD-1 biomarker is available to the public at the GenBank database under NM 005018.2 and NP_005009.2 (see also Ishida et al. (1992) 20 EMBO J 11 :3887; Shinohara et al. (1994) Genomics 23:704; U.S. 5,698,520). PD-1 has an extracellular region containing immunoglobulin superfamily domain, a transmembrane domain, and an intracellular region including an immunoreceptor tyrosine-based inhibitory motif (ITIM) (Ishida et al. (1992) EMBO J. 11 :3887; Shinohara et al. (1994) Genomics 23:704; and U.S. Patent 5,698,520) and an immunoreceptor tyrosine-based switch motif (ITSM). These features also define a larger family of polypeptides, called the immunoinhibitory receptors, which also includes gp49B, PIR-B, and the killer inhibitory receptors (KIRs) (Vivier and Daeron (1997) Immunol. Today 18:286). It is often assumed that the tyrosyl phosphorylated ITIM and ITSM motif of these receptors interacts with SH2-domain containing phosphatases, which leads to inhibitory signals. A subset of these immunoinhibitory receptors bind to MHC polypeptides, for example the KIRs, and CTLA4 binds to B7-1 and B7-2. It has been proposed that there is a phylogenetic relationship between the MHC and B7 genes (Henry et al. (1999) Immunol. Today 20(6):285-8).

PD-1 polypeptides are inhibitory receptors capable of transmitting an inhibitory signal to an immune cell to thereby inhibit immune cell effector function, or are capable of promoting costimulation {e.g., by competitive inhibition) of immune cells, e.g., when present in soluble, monomeric form. Preferred PD-1 family members share sequence identity with PD-1 and bind to one or more B7 family members, e.g., B7-1 , B7-2, PD-1 ligand, and/or other polypeptides on antigen presenting cells.

The term "PD-1 activity," includes the ability of a PD-1 polypeptide to modulate an inhibitory signal in an activated immune cell, e.g., by engaging a natural PD-1 ligand on an antigen presenting cell. Modulation of an inhibitory signal in an immune cell results in modulation of proliferation of, and/or cytokine secretion by, an immune cell. Thus, the term "PD-1 activity" includes the ability of a PD-1 polypeptide to bind its natural ligand(s), the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate the immune response.

The term "PD-1 ligand" refers to binding partners of the PD-1 receptor and includes both PD- L1 (Freeman et al. (2000) J Exp. Med. 192:1027-1034) and PD-L2 (Latchman et al. (2001) Nat. Immunol. 2:261). At least two types of human PD-1 ligand polypeptides exist. PD-1 ligand proteins comprise a signal sequence, and an IgV domain, an IgC domain, a transmembrane domain, and a short cytoplasmic tail. Both PD-L1 (See Freeman et al. (2000) for sequence data) and PD-L2 (See Latchman et al. (2001) Nat. Immunol. 2:261 for sequence data) are members of the B7 family of polypeptides. Both PD-L1 and PD-L2 are expressed in placenta, spleen, lymph nodes, thymus, and heart. Only PD-L2 is expressed in pancreas, lung and liver, while only PD-L1 is expressed in fetal liver. Both PD-1 ligands are upregulated on activated monocytes and dendritic cells, although PD- L1 expression is broader. For example, PD-L1 is known to be constitutively expressed and upregulated to higher levels on murine hematopoietic cells (e.g., T cells, B cells, macrophages, dendritic cells (DCs), and bone marrow-derived mast cells) and non- hematopoietic cells (e.g., endothelial, epithelial, and muscle cells), whereas PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow-derived mast cells (see Butte et al. (2007) Immunity 27: 111).

PD-1 ligands comprise a family of polypeptides having certain conserved structural and functional features. The term "family" when used to refer to proteins or nucleic acid molecules, is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology, as defined herein. Such family members can be naturally or non- naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of nonhuman origin. Members of a family may also have common functional characteristics. PD-1 ligands are members of the B7 family of polypeptides. The term "B7 family" or "B7 polypeptides" as used herein includes costimulatory polypeptides that share sequence homology with B7 polypeptides, e.g., with B7-1 , B7-2, B7h (Swallow et al. (1999) Immunity 11 :423), and/or PD-1 ligands (e.g., PD- L1 or PD-L2). For example, human B7-1 and B7-2 share approximately 26% amino acid sequence identity when compared using the BLAST program at NCBI with the default parameters (Blosum62 matrix with gap penalties set at existence 11 and extension 1 (See the NCBI website). The term B7 family also includes variants of these polypeptides which are capable of modulating immune cell function. The B7 family of molecules share a number of conserved regions, including signal domains, IgV domains and the IgC domains. IgV domains and the IgC domains are art-recognized Ig superfamily member domains. These domains correspond to structural units that have distinct folding patterns called Ig folds. Ig folds are comprised of a sandwich of two b sheets, each consisting of anti-parallel b strands of 5-10 amino acids with a conserved disulfide bond between the two sheets in most, but not all, IgC domains of Ig, TCR, and MHC molecules share the same types of sequence patterns and are called the Cl -set within the Ig superfamily. Other IgC domains fall within other sets. IgV domains also share sequence patterns and are called V set domains. IgV domains are longer than IgC domains and contain an additional pair of b strands.

Preferred B7 polypeptides are capable of providing costimulatory or inhibitory signals to immune cells to thereby promote or inhibit immune cell responses. For example, B7 family members that bind to costimulatory receptors increase T cell activation and proliferation, while B7 family members that bind to inhibitory receptors reduce costimulation. Moreover, the same B7 family member may increase or decrease T cell costimulation. For example, when bound to a costimulatory receptor, PD-1 ligand can induce costimulation of immune cells or can inhibit immune cell costimulation, e.g., when present in soluble form. When bound to an inhibitory receptor, PD-1 ligand polypeptides can transmit an inhibitory signal to an immune cell. Preferred B7 family members include B7-1 , B7-2, B7h, PD-L1 or PD-L2 and soluble fragments or derivatives thereof. In one embodiment, B7 family members bind to one or more receptors on an immune cell, e.g., CTLA4, CD28, ICOS, PD-1 and/or other receptors, and, depending on the receptor, have the ability to transmit an inhibitory signal or a costimulatory signal to an immune cell, preferably a T cell.

Modulation of a costimulatory signal results in modulation of effector function of an immune cell. Thus, the term "PD-1 ligand activity" includes the ability of a PD-1 ligand polypeptide to bind its natural receptor(s) (e.g. PD-1 or B7-1), the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate the immune response.

The term "PD-L1" refers to a specific PD-1 ligand. Two forms of human PD-L1 molecules have been identified. One form is a naturally occurring PD-L1 soluble polypeptide, i.e., having a short hydrophilic domain and no transmembrane domain, and is referred to herein as PD-L1 S (SEQ ID NO: 4 in WO2018/14837, incorporated herein by reference). The second form is a cell- associated polypeptide, i.e., having a transmembrane and cytoplasmic domain, referred to herein as PD-L1 M (SEQ ID NO: 6 in WO2018/14837, incorporated herein by reference). The nucleic acid and amino acid sequences of representative human PD-L1 biomarkers regarding PD-L1 M are also available to the public at the GenBank database under NM_014143.3 and NP 054862.1 .

The term "PD-L2" refers to another specific PD-1 ligand. PD-L2 is a B7 family member expressed on various APCs, including dendritic cells, macrophages and bone- marrow derived mast cells (Zhong et al. (2007) Eur. J. Immunol. 37:2405). APC-expressed PD-L2 is able to both inhibit T cell activation through ligation of PD-1 and costimulate T cell activation, through a PD-1 independent mechanism (Shin et al. (2005) J. Exp. Med. 201 : 1531 ). In addition, ligation of dendritic cell-expressed PD-L2 results in enhanced dendritic cell cytokine expression and survival (Radhakrishnan et al. (2003) J. Immunol. 37:1827; Nguyen et al. (2002) J. Exp. Med. 196:1393). The nucleic acid and amino acid sequences of representative human PD-L2 biomarkers are well- known in the art and are also available to the public at the GenBank database under NM_025239.3 and NP_079515.2. PD-L2 proteins are characterized by common structural elements. In some embodiments, PD-L2 proteins include at least one or more of the following domains: a signal peptide domain, a transmembrane domain, an IgV domain, an IgC domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

The term "PD-L2 activity," "biological activity of PD-L2," or "functional activity of PD-L2," refers to an activity exerted by a PD-L2 protein, polypeptide or nucleic acid molecule on a PD-L2 - responsive cell or tissue, or on a PD- L2 polypeptide binding partner, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, a PD-L2 activity is a direct activity, such as an association with a PD-L2 binding partner. As used herein, a "target molecule" or "binding partner" is a molecule with which a PD-L2 polypeptide binds or interacts in nature, such that PD- L2-mediated function is achieved. In an exemplary embodiment, a PD-L2 target molecule is the receptor RGMb. Alternatively, a PD-L2 activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the PD- L2 polypeptide with its natural binding partner (i.e., physiologically relevant interacting macromolecule involved in an immune function or other biologically relevant function), e.g., RGMb. The biological activities of PD-L2 are described herein. For example, the PD-L2 polypeptides of the present invention can have one or more of the following activities: 1) bind to and/or modulate the activity of the receptor RGMb, PD-1 , or other PD-L2 natural binding partners, 2) modulate intra-or intercellular signaling, 3) modulate activation of immune cells, e.g. , T lymphocytes, and 4) modulate the immune response of an organism, e.g., a mouse or human organism.

An "immune checkpoint therapy" refers to the use of agents that inhibit immune checkpoint nucleic acids and/or proteins. Inhibition of one or more immune checkpoints can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer. Exemplary agents useful for inhibiting immune checkpoints include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint nucleic acids, or fragments thereof. Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint proteins block the interaction between the proteins and its natural receptor(s); a nonactivating form of one or more immune checkpoint proteins (e.g. , a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint nucleic acid transcription or translation; and the like. Such agents can directly block the interaction between the one or more immune checkpoints and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response. Alternatively, agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response. For example, a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand. In one embodiment, anti-PD-1 antibodies, anti-PD-L1 antibodies, and/or anti-PD-L2 antibodies, either alone or in combination, are used to inhibit immune checkpoints. These embodiments are also applicable to specific therapy against particular immune checkpoints, such as the PD-1 pathway (e.g., anti-PD-1 pathway therapy, otherwise known as PD-1 pathway inhibitor therapy).

In a preferred embodiment, the immune checkpoint therapy that is combined with the use of the compound as defined herein in the treatment of the cancer in accordance to the invention, preferably, comprises the use (or administration) of at least one of an immune checkpoint blocking agent that blocks at least one of PD-1 , PD-L1 , PD-L2 and CTLA-4. Preferably, the immune checkpoint blocking agent is at least one of ipilimumab (anti-CTLA-4), nivolumab, pembrolizumab, antibody BGB-A31 (anti-PD-1) and atezolizumab (anti-PD-L1).

Cancer types

In a preferred embodiment, a compound as defined herein attracts inflammatory cells to tumors in vivo. Without wishing to be bound by any theory, the aberrant splicing induced by a compound as defined herein results in the expression of neo-antigens, such as Frame Shift Proteins. These neo-antigens can boost the recruitment of CD8+ T cells, which can increase the response to an immune checkpoint therapy. In an embodiment, the tumor is a tumor wherein mRNA splicing interference results in the attraction of T-cells, preferably CD8+ T cells to the tumor cell.

The term “cancer” as used herein includes, but is not limited to, a proliferative disease such as a cancer or malignancy, including e.g. atypical and/or non-classical cancers. The term “cancer” may further include precancerous conditions such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia.

In one embodiment, the compound as defined herein is for use in the treatment of a hematological cancer. Preferably the treatment is combined with an immune checkpoint therapy. Preferably, the hematologic cancer is chosen from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non- Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia.

In one embodiment, the compound as defined herein is for use in the treatment of a solid cancer. Preferably the treatment is combined with an immune checkpoint therapy. Preferably, the solid cancer is selected from the group consisting of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

The cancer may be sensitive or resistant to immunotherapy. An immunotherapy may include at least one of a adoptive cell transfer (ACT) and immune checkpoint therapy. In an embodiment, the tumor is a so-called “cold tumor”, i.e. a tumor comprising cells that are resistant or substantially resistant to immunotherapy. The tumor may have become a cold tumor due to a previous immunotherapy treatment, such as, but not limited to an immune checkpoint therapy. In an embodiment, the compound for use in the invention, preferably together with an immune checkpoint therapy, may be a second line treatment for cancer, e.g. because the tumor has become resistant to an immunotherapy.

A compound as defined herein may therefore be for use in the treatment of a cancer comprising tumor cells that are resistant to immune checkpoint therapy, wherein the compound is preferably used in combination with an immune checkpoint therapy. The immune checkpoint therapy that resulted in the resistance of the tumor cell may be the same immune checkpoint therapy subsequently used in combination with a compound as defined herein for the treatment of a cancer. Alternatively, the immune checkpoint therapy that resulted in the resistance of the tumor cell may be a different immune checkpoint therapy that is subsequently used in combination with a compound as defined herein for the treatment of a cancer.

The compound as defined herein may be for use in the treatment of a cancer, wherein the compound is preferably used in combination with an immune checkpoint therapy, and wherein the cancer comprises one or more tumor cells that are sensitive to immunotherapy, preferably are sensitive to immune checkpoint therapy. The compound for use as defined herein may augment the effectiveness of the immune checkpoint therapy. In addition or alternatively, the compound for use as defined herein may prevent resistance to immune checkpoint therapy. The compound as defined herein, preferably in combination with an immune checkpoint therapy, may be used in a first line of anti-cancer treatment.

Compositions

In an aspect, the invention pertains to a composition comprising a compound as defined herein, optionally together with an immune checkpoint blocking agent. The composition may be suitable for use in cell culture, preferably animal cell culture, more preferably mammalian cell culture. The composition preferably is a pharmaceutical composition.

A composition may comprise one type of compound as defined herein, or a combination of compounds as defined herein, e.g. a combination of indisulam and another sulphonamide compound. A composition may comprise at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of compounds that induce aberrant mRNA splicing in a tumor cell.

A composition may further comprise an immune checkpoint blocking agent. In an embodiment, the composition may comprise indisulam and at least one of PD-1 , PD-L1 and CTLA- 4. A composition of the invention, preferably for use in the treatment of a cancer, may be a composition comprising one or more compounds as defined herein, i.e. a compound that induces aberrant mRNA splicing in a tumor cell. The composition may additionally comprise a compound for immune checkpoint therapy, preferably an immune checkpoint blocking agent. Alternatively, the immune checkpoint blocking agent may be present in a separate composition.

Hence a composition as further described herein preferably comprises:

- one or more compounds as defined herein, wherein the one or more compounds induce aberrant RNA splicing in a tumor cell;

- one or more immune checkpoint blocking agents as defined herein; or

- a combination of one or more compounds as defined herein and one or more immune checkpoint blocking agent as defined herein.

When referring to “a composition” the compositions indicated above are intended, except if it is clear from its context that a specific composition or compositions are intended.

The composition or compositions are preferably for a use in the treatment of a cancer, preferably a cancer as defined herein. The composition or compositions may be used to prevent resistance to immune checkpoint therapy or for the treatment of a cancer comprising tumor cells that are resistant to immune checkpoint therapy.

The composition may be administered in combination with further pharmaceutical agents and/or can be combined with a physiologically acceptable carrier. In particular the composition can be formulated as pharmaceutical composition by formulation with additives such as pharmaceutically or physiologically acceptable excipients, carriers, and vehicles.

Suitable pharmaceutically or physiologically acceptable excipients, carriers and vehicles can include processing agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-P- cyclodextrin, polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof. Other suitable pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences, " Mack Pub. Co., New Jersey (1991), and "Remington: The Science and Practice of Pharmacy, " Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003), 21^st edition (2005) and 22^nd edition (2012), incorporated herein by reference.

Pharmaceutical compositions as described herein for use according to the invention may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion. In a preferred embodiment, a composition as defined herein is administered in a solid form or in a liquid form.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms the composition, preferably a composition comprising a compound as described herein, may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water or saline. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.

Liquid carriers are typically used in preparing solutions, suspensions, and emulsions. In a preferred embodiment, liquid carriers / liquid dosage forms contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvents), pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof.

In a preferred embodiment a composition as described herein is admixed with an aqueous solution prior to administration. The aqueous solution should be suitable for administration and such aqueous solutions are well known in the art. It is further known in the art that the suitability of an aqueous solution for administration may be dependent on the route of administration.

In a preferred embodiment, the aqueous solution is an isotonic aqueous solution. The isotonic aqueous solution preferably is almost (or completely) isotonic to blood plasma. In an even more preferred embodiment, the isotonic aqueous solution is saline.

The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, flavorants and the like. Preferred flavorants are sweeteners, such as monosaccharides and / or disaccharides. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like.

For parenteral administration, the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like. Compositions for use in the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.

Time-release, sustained release or controlled release delivery systems may be used for administration of one or more of the compositions as described herein, such as a diffusion controlled matrix system or an erodible system, as described for example in: Lee, "Diffusion-Controlled Matrix Systems", pp. 155-198 and Ron and Langer, "Erodible Systems", pp. 199-224, in "Treatise on Controlled Drug Delivery", A. Kydonieus Ed. , Marcel Dekker, Inc. , New York 1992. The matrix may be, for example, a biodegradable material that can degrade spontaneously in situ and in vivo for, example, by hydrolysis or enzymatic cleavage, e.g. , by proteases. The delivery system may be, for example, a naturally occurring or synthetic polymer or copolymer, for example in the form of a hydrogel. Exemplary polymers with cleavable linkages include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly(phosphoesters), polyamides, polyurethanes, poly(imidocarbonates) and poly(phosphazenes). A composition as defined herein can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound, immune checkpoint blocking agent or a combination of a compound and an immune checkpoint blocking agent as defined herein, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N. Y., p. 33 et seq (1976).

A pharmaceutical composition as defined herein, i.e. comprising a compound, an immune checkpoint blocking agent or a combination of a compound and an immune checkpoint blocking agent as defined herein, can comprise a unit dose formulation, where the unit dose is a dose sufficient to have a therapeutic or suppressive effect of a disorder or condition as defined herein, and/or an amount effective to reduce, or knock out, the expression of a membrane-bound protein.

The unit dose may be sufficient as a single dose to have a therapeutic effect of a cancer, preferably of a cancer as defined herein. Alternatively, the unit dose may be a dose administered periodically in a course of treatment of a cancer, preferably a cancer as defined herein. During the course of the treatment, the concentration of the subject compositions may be monitored to insure that the desired level is maintained.

While the compound for use as described herein can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents used in the treatment or suppression of a cancer. In particular, the compound as defined herein can be administered in combination with an immune checkpoint blocking agent. Further representative agents useful in combination with the compound, and optionally the immune checkpoint blocking agent, forthe treatment of cancer include, but are not limited to, Coenzyme Q, vitamin E, idebenone, MitoQ, EPI-743, vitamin K and analogues thereof, naphtoquinones and derivatives thereof, other vitamins, and antioxidant compounds.

When further active agents are used in combination with a compound as defined herein and optionally an immune checkpoint blocking agent, the further active agents may generally be employed in therapeutic amounts as indicated in the Physicians' Desk Reference (PDR) 53rd Edition (1999), which is incorporated herein by reference, or such therapeutically useful amounts as would be known to one of ordinary skill in the art. The ccompound and/or immune checkpoint blocking agent as defined herein can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the compound as defined herein and/or immune checkpoint blocking agent in the compositions of the invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. When administered in combination with further active agents, the active agents can be formulated as separate compositions that are given at the same time or different times, or the active agents can be given as a single composition. A composition as described herein, can be prepared as a medicinal or cosmetic preparation or in various other media, such as foods for humans or animals, including medical foods and dietary supplements.

A "medical food" is a product that is intended forthe specific dietary management of a disease or condition for which distinctive nutritional requirements exist. By way of example, but not limitation, medical foods may include vitamin and mineral formulations fed through a feeding tube (referred to as enteral administration).

A "dietary supplement" shall mean a product that is intended to supplement the human diet and is typically provided in the form of a pill, capsule, and tablet or like formulation. By way of example, but not limitation, a dietary supplement may include one or more of the following ingredients: vitamins, minerals, herbs, botanicals; amino acids, dietary substances intended to supplement the diet by increasing total dietary intake, and concentrates, metabolites, constituents, extracts or combinations of any of the foregoing. Dietary supplements may also be incorporated into food, including, but not limited to, food bars, beverages, powders, cereals, cooked foods, food additives and candies.

The subject composition thus may be compounded with other physiologically acceptable materials which can be ingested including, but not limited to, foods. In addition or alternatively, the compositions for use as described herein may be administered orally in combination with (the separate) administration of food.

Modes of administration

As indicated above, unless it is clear from its context that it a specific composition is intended, a composition as specified herein comprises:

- one or more compounds as defined herein, wherein the one or more compounds induce aberrant RNA splicing in a tumor cell;

- one or more immune checkpoint blocking agents as defined herein; or

- a combination of one or more compounds as defined herein and one or more immune checkpoint blocking agents as defined herein.

A composition as defined herein may be administered enterally, orally, parenterally, sublingually, by inhalation (e. g. as mists or sprays), rectally, or topically, preferably in dosage unit formulations containing conventional nontoxic pharmaceutically or physiologically acceptable carriers, adjuvants, and vehicles as desired. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal (e. g. via nasal mucosa), subdural, rectal, gastrointestinal, and the like, and directly to a specific or affected organ or tissue, e.g. a cancerous tissue. For delivery to the central nervous system, spinal and epidural administration, or administration to cerebral ventricles, can be used. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.

The compound as defined herein and/or the checkpoint blocking agent as defined herein can be mixed with pharmaceutically acceptable carriers, adjuvants, and vehicles appropriate for the desired route of administration. The compound as defined herein and/or the checkpoint blocking agent as defined herein may be administered by supplementation via gastric or percutaneous tubes.

In a preferred embodiment the invention pertains to a compound as defined herein above, optionally in combination with a checkpoint immune therapy, for use in treating, preventing, or suppressing symptoms associated with a cancer by administration of an effective total daily dose for a specified period of time.

The dosage form for oral administration can be a solid oral dosage form. The class of solid oral dosage forms consists primarily of tablets and capsules, although other forms are known in the art and can be equally suitable. When used as a solid oral dosage form, a composition as defined herein may e.g. be administered in the form of an immediate release tablet (or a capsule and the like) or a sustained release tablet (or a capsule and the like). Any suitable immediate release or sustained release solid dosage forms can be used in the context of the invention as will be evident for the skilled person.

A composition as described herein, for a use as described herein, can be administered in solid form, in liquid form, in aerosol form, or in the form of tablets, pills, powder mixtures, capsules, granules, injectables, creams, solutions, suppositories, enemas, colonic irrigations, emulsions, dispersions, food premixes, and in other suitable forms. The composition can also be administered in liposome formulations. The compound and/or the immune checkpoint blocking agent can be administered as prodrugs, where the prodrug undergoes transformation in the treated subject to a form which is therapeutically effective. Additional methods of administration are known in the art.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in propylene glycol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Suppositories for rectal administration of the compound and/or immune checkpoint blocking agent as defined herein can be prepared by mixing with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols that are solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum and release the compound and/or immune checkpoint blocking agent.

Preferably a composition comprising a compound as defined herein, preferably a sulphonamide as defined herein, is administered orally or parenterally. Preferably a composition comprising an immune checkpoint blocking agent, optionally in combination with a compound as defined herein, is administered parenterally.

In a preferred embodiment the compound as defined herein is used in combination with an immune checkpoint therapy.

In an embodiment, the compound that induces aberrrant mRNA splicing in a tumor cell is administered prior to the immune checkpoint therapy. The compound may be administered as a pretreatment of the immune checkpoint therapy. The compound may induce aberrant mRNA splicing, which can result in the expression of neo-antigens. These neoantigens can be presented by MHC classs I antigens to T cells for tumor eradication. Subsequent to the administration of a compound as defined herein, the subject may receive an immune checkpoint therapy. During the immune checkpoint therapy, treatment with the compound may be continued. Treatment with the compound may be continued during the full time period of the immune checkpoint therapy or may be continued during part of the immune checkpoint therapy. The compound can be administered simultaneuously, separately or sequentially with the immune checkpoint therapy. Expression of the neoantigens may increase the repsonse to immune checkpoint therapy strategies.

Alternatively, the treatment with a compound that induces aberrant mRNA splicing is in combination with an immune checkpoint therapy, without pretreatment with the compound. The compound can be administed simultaneuously, separately or sequentially with the immune checkpoint therapy.

A subject may receive several rounds of treatment. During each round of treatment, the compound may be administered prior to the immune checkpoint therapy and optionally treatment with the compound may be continued during the immune checkpoint therapy, i.e. is in combination with an immune checkpoint therapy.

Alternatively during each round of treatment, treatment with the compound may be in combination with the immune checkpoint therapy, without pretreatment.

Alternatively the subject may receive several rounds of treatment, wherein in one or more rounds the compound may be administered as a pretreatment and optionally the treatment may be continued during (part of) the immune checkpoint therapy and and in one or more rounds the treatment with the compound may be in combination with the immune checkpoint therapy, without a pretreatment.

Kit of parts

One or more of the pharmaceutical compositions as defined herein may be comprised in a kit of parts. In an aspect, the invention relates to a kit of parts comprising one or more compositions, preferably pharmaceutical compositions, as defined herein.

The kit of parts is preferably for a use in the treatment of a cancer, preferably a cancer as defined herein. The kit of parts may be used to prevent resistance to immune checkpoint therapy or may be used for the treatment of a cancer comprising tumor cells that are resistant to immune checkpoint therapy. Preferably the kit of part comprises at least one of:

- A vial comprising a composition, preferably a pharmaceutical composition, wherein the composition comprises one or more compounds as defined herein, wherein the one or more compounds induce aberrant RNA splicing in a tumor cell;

- A vial comprising a composition, preferably a pharmaceutical composition, wherein the composition comprises one or more immune checkpoint blocking agents as defined herein; and

- a vial comprising a composition, preferably a pharmaceutical composition, wherein the composition comprises a combination of one or more compounds as defined herein and one or more immune checkpoint blocking agents as defined herein.

Preferably the kit of part comprises:

- A vial comprising a composition, preferably a pharmaceutical composition, wherein the composition comprises one or more compounds as defined herein, wherein the one or more compounds induce aberrant RNA splicing in a tumor cell; and

- A vial comprising a composition, preferably a pharmaceutical composition, wherein the composition comprises one or more immune checkpoint blocking agents as defined herein; Preferably, a kit of parts as defined herein is for a use in the treatment of a cancer, preferably a cancer as defined herein.

Optionally, the kit of parts further comprises a leaflet. The leaflet may comprise instructions for use. In addition or alternatively, the leaflet may be at least one of a patient information leaflet and a Summary of Product Characteristics (an SmPC).

Diagnostic method

The presence of aberrant splicing in a tumor cell can be indicative of the responsiveness of a tumor cell to immune checkpoint therapy. In an aspect, the invention therefore pertains to a (diagnostic) method for identifying a subject that benefits from immune checkpoint therapy, comprising a step of detecting the presence of a diagnostic marker in a tumor sample. The term “diagnostic marker”, “marker for diagnosing” and “marker for diagnosis”, as used herein, is intended to indicate a biological parameter capable of (aiding in) identification of a tumor cell that will be responsive to an immune checkpoint therapy. The diagnostic marker as used herein may be indicative of an aberrant mRNA splicing in the tumor cell. The diagnostic marker can be a mutated splicing factor, or a genomic, cDNA, mRNA or pre-mRNA sequence encoding a mutated splicing factor. The mutated splicing factor may be at least one of RBM39, SF3B1 , SRSF2 and U2AF1 . Alternatively or in addition, the diagnostic marker may be the presence of an aberrant splicing pattern of one or more RNA molecules. The diagnostic marker may be a transcriptome profile that differs from a reference transcriptome profile.

A method as disclosed herein can be a method for identifying a subject that benefits from immune checkpoint therapy, comprising a step of detecting the presence of a mutated splicing factor in a tumor sample. The method may be an in vivo method. Preferably the method is an ex vivo method, preferably an in vitro method. The method can comprise a step of obtaining a biopsy from the tumor tissue and detecting a diagnostic marker in, preferably detecting the presence of a mutated splicing factor, ex vivo.

The mutated splicing factor is preferably at least one of RBM39, SF3B1 , SRSF2 and U2AF1 . The mutated splicing factor can be a splicing factor as described in Garraway LA and Lander ES ( Lessons from the cancer genome, Cell (2013), 153(1 ):17-37), Yoshida K and Ogawa S. ( Splicing factor mutations and cancer, Wiley Interdiscip Rev RNA. 2014 ;5(4):445-59) and/or Yoshida et al ( Frequent pathway mutations of splicing machinery in myelodysplasia, Nature. (2011);478(7367):64-9).

An agent binding a diagnostic marker as described herein can be selected from the group consisting of a nucleic acid, a small molecule, a proteinaceous molecule, an aptamer, an antibody or an antigen-binding fragment thereof. Such agents may further comprise a detectable signal or label, such as a radioisotope, a fluorescent molecule or biotin.

A nucleic acid molecule for detecting a biomarker as described herein may selectively hybridize to a genomic, cDNA, mRNA and/or pre-mRNA sequence encoding a biomarker as described herein. Preferably a nucleic acid molecule for detecting a biomarker as described herein may selectively hybridize to a genomic, cDNA, mRNA and/or pre-mRNA sequence encoding a mutated splice factor as described herein, wherein preferably the mutated splice factor is selected from the group consisting of RBM39, SF3B1 , SRSF2 and U2AF1 .

An antibody or antigen-binding fragment thereof for detecting a biomarker as described herein may selectively bind to a biomarker as described herein. Preferably an antibody or antigenbinding fragment thereof may bind to a mutated splice factor as described herein, wherein preferably the mutated splice factor is selected from the group consisting of RBM39, SF3B1 , SRSF2 and U2AF1 .

The step of detecting a diagnostic marker can be performed using any conventional method known in the art. As non-limiting examples, the diagnostic marker can be detected using an agent capable of binding a diagnostic marker as described herein, or by using deep-sequencing technologies e.g. to detect one or more aberrantly spliced mRNA molecules.

Non-limiting examples for detecting the biomarker as defined herein include, but are not limited to, (quantitative) PCR, enzyme-linked immunosorbent assay (ELISA), gel electrophoresis, surface plasmon resonance (SPR), Mass-sensing BioCD protein array, surface enhanced Raman spectroscopy (SERS), colorimetric assay, electrochemical assay, and fluorescence methods.

In an aspect, the invention pertains to an agent for identifying a subject that benefits from immune checkpoint therapy. Preferably, the agent can detect a biomarker as described herein. Preferably, the agent is an agent as described herein above. Preferably, the agent binds to a mutated splicing factor, or binds to a sequence encoding the mutated splicing factor. Preferably, the agent binds specifically to a mutated splicing factor or binds (or hybridizes) specifically to a sequence encoding the mutated splicing factor. Preferably, the mutated splicing factor is at least one of RBM39, SF3B1 , SRSF2 and U2AF1 . The invention further pertains to a kit comprising an agent as defined herein and to the use of an agent as defined herein to identify a subject that benefits from immune checkpoint therapy.

The present invention has been described above with reference to a number of exemplary embodiments as shown in the drawings. Modifications and alternative implementations of some parts or elements are possible, and are included in the scope of protection as defined in the appended claims.

Figure legend

Figure 1. Differential splicing events. Relative frequency of splicing aberrations in A549 and SUM159 cells treated with indisulam (IN) versus DMSO (lanes IN vs DMSO). The relative frequency of the possible splicing alterations are represented.

Figure 2. Indisulam increases tumor infiltration of CD4 and Granzyme B positive cells. A) Treatment schedule, B) Tumor growth, C) plasma concentration, each line represents one mouse and D) immune histochemical staining of CD4 and Granzyme B.

Examples

Example 1

The effects of indisulam on mRNA splicing have been studied by performing transcriptome analysis (RNAseq) on A549 lung cancer cells and SUM159 breast cancer cells following treatment with indisulam (3 pM for 16 hours).

The results are shown in Figure 1. These data suggest that treatment of cancer cells with indisulam will result in a significant fraction of cells expressing Frame Shift Proteins (FSPs) due to errors in mRNA splicing. As such, indisulam and other small molecules that interfere with mRNA splicing, will enhance the expression of neo-antigens on the tumor cells in a transient fashion: as long as the drug is given, the neo-antigens will be expressed. Discontinuation of the drug treatment will end expression of the neo-antigens.

Example 2

To address whether indisulam attracts inflammatory cells to tumors in vivo, human MDA-MB- 231 breast cancer cells were injected into the mammary gland of nude mice. After tumors had reached a size of 200 mm3, animals were treated with 20 mg/kg of indisulam daily for 14 days. After this, animals were sacrificed and stained for CD4 (T cell marker) and granzyme B (T cell marker).

Figure 2 shows that indisulam treatment resulted in a significant increase in CD4+ and granzyme B positive cells.

Claims

Claims

1. A compound that induces aberrant mRNA splicing in a tumor cell for use in the treatment of a cancer, wherein in the treatment, the compound is combined with an immune checkpoint therapy.

2. A compound for a use according to claim 1 , wherein the aberrant mRNA splicing in the tumor cell alters the transcriptome profile of the tumor cell by increasing the level of aberrant mRNAs by at least one of: i) alternative 3’splice site usage; ii) alternative 5’splice site usage; iii) intron retention; iv) exon skipping; v) exon retention; and vi) presence of alternative exons, wherein preferably the transcriptome profile can be determined in an RNAseq experiment, preferably the RNAseq experiment of example 1 , and wherein the increase in the level of aberrant mRNAs is preferably at least a 2-fold increase as compared to the level of the same aberrant mRNAs in a control tumor cell not treated with the compound.

3. A compound for a use according to claim 1 or 2, wherein the compound is a small molecule.

4. A compound for a use according to any one of the preceding claims, wherein the compound is an aryl-sulfonamide, preferably selected from the group consisting of indisulam, NSC 719239 (E7820), NSC 339004 (CQS) and tasisulam.

5. A compound for a use according to claim 3 or 4, wherein the compound is indisulam or a functional equivalent thereof.

6. A compound for a use according to any one of the preceding claims, wherein the immune checkpoint therapy comprises the use of an immune checkpoint blocking agent that blocks at least one of PD-1 , PD-L1 and CTLA-4, wherein preferably the immune checkpoint blocking agent is at least one of ipilimumab, nivolumab, pembrolizumab, antibody BGB-A31 and atezolizumab.

7. A compound for a use according to any one of the preceding claims, wherein the compound is administered as a pretreatment of the immune checkpoint therapy, and wherein preferably treatment with the compound is continued during the immune checkpoint therapy.

8. A compound for a use according to any one of the preceding claims, wherein the compound is administered simultaneously, separately or sequentially with the immune checkpoint therapy.

9. A compound for a use according to any one of the preceding claims, wherein the compound is for a use in the prevention of resistance to immune checkpoint therapy or for a use in the treatment of a cancer comprising tumor cells that are resistant to immune checkpoint therapy.

10. A pharmaceutical composition comprising a compound as defined in any one of claims 1 - 5 and an immune checkpoint blocking agent as defined in claim 6, or a kit of parts comprising a pharmaceutical composition comprising a compound as defined in any one of claims 1 - 5 and a pharmaceutical composition comprising an immune checkpoint blocking agent as defined in claim 6, wherein optionally the kit of parts comprises a leaflet with instructions for use.

11. A pharmaceutical composition or a kit of parts according to claim 10 for use in the treatment of a cancer, wherein preferably the composition or the kit is used to prevent resistance to immune checkpoint therapy or to treat a cancer comprising tumor cells that are resistant to immune checkpoint therapy.

12. An agent for identifying a subject that benefits from immune checkpoint therapy, wherein the agent binds to a mutated splicing factor, or binds to a sequence encoding a mutated splicing factor, wherein preferably the agent is a nucleic acid molecule, an antibody or an antigenbinding fragment thereof.

13. A method for identifying a subject that benefits from immune checkpoint therapy, comprising a step of detecting the presence of a mutated splicing factor in a tumor sample, wherein preferably the method is an ex vivo method, wherein preferably the mutated splicing factor is at least one of RBM39, SF3B1 , SRSF2 and U2AF1 .

14. A method for treating a cancer in a subject comprising administering to the subject an effective amount of a compound that induces aberrant mRNA splicing as defined in any one of claims 1 - 5, wherein in the treatment, the compound is combined with an immune checkpoint therapy, wherein preferably the immune checkpoint therapy is a therapy as defined in claim 6.

15. The method of claim 14, wherein the method reduces the risk of developing resistance to the immune checkpoint therapy or wherein the cancer comprises tumor cells that are resistant to immune checkpoint therapy.