Patent Application
20230151118
This application claims the benefit of European Patent Applications EP20168333.1, filed 6 Apr. 2020, and EP20177397.5, filed 29 May 2020, all of which are incorporated herein by reference.
The present invention relates to a non-agonist ligand of ARTC1, particularly a ligand that inhibits the ADP-ribosyltransferase activity of ARTC1 for use in prevention or treatment of cancer. Alternatively, an inhibitor nucleic acid sequence capable of downregulating or inhibiting expression of a target nucleic acid sequence encoding ARTC1 is provided for use in prevention or treatment of cancer.
ADP-ribosylation is a post-translational protein modification that has been shown to regulate protein function and gene expression under both physiological conditions and in diseases such as cancer and in inflammation. Protein ADP-ribosylation exists in various forms (monomeric, polymeric) and is added to the peptide/protein in the context of different sequence motifs.
ADP-ribosylation is catalyzed within cells by ARTD family members (structurally related to Diphtheria toxin). The modification at the plasma membrane or in the extracellular space is regulated by ARTC family members (structurally related to Clostridium toxin). Four human (ARTC1, 3, 4, 5, formerly also hART1-5) and six mouse ARTC genes (ARTC1, 2.1, 2.2, 3, 4, 5, formerly also mART1-5) have been identified. For both species, ARTC1 is expressed mainly in skeletal and heart muscles, non-lactated mammary gland, brown adipocytes, epithelial cells or activated granulocytes. Although ARTC1 is highly active in vitro, identification of ARTC1 targets in vivo and subsequent characterization of ARTC1-regulated cellular processes on the proteome level has been challenging and only a few ADP-ribosylated targets are known thus far. Therefore, ARTC1-target sites in proteins known to be involved in the regulation of diseases are highly relevant targets for therapeutic intervention of various diseases.
The objective of the present invention is to provide means and methods to treat or prevent cancer. This objective is attained by the subject-matter of the independent claims of the present specification.
The inventors found the extra-cellular ADP-ribosyl transferase (ART) ARTC1 to be overexpressed in a number of human cancer types (including breast, lung, colon and brain tumors, and many more). Furthermore, they found that ARTC1 modifies proteins on the surface of the tumor and in its microenvironment by transferring ADP-ribose to specific target sites on those proteins. The target proteins include proteins that are involved in tumor growth (growth factor and receptors), vascularization, metastasis and immune regulation (cytokines and their receptors). Thus, the inventors hypothesize that ARTC1 modifies these proteins and thereby their function and thereby generates a microenvironment that favors tumor growth and metastasis. Inhibition of the enzymatic function of ARTC1 or its expression in tumors and at the surface of tumor cells would lead to reduced tumor growth.
A first aspect of the invention relates to a non-agonist ligand of ARTC1 for use in prevention or treatment of cancer.
A second aspect of the invention relates to a nucleic acid molecule for use in prevention or treatment of cancer, comprising, or consisting of, an inhibitor nucleic acid sequence capable of downregulating or inhibiting expression of a target nucleic acid sequence encoding ARTC1.
A third aspect of the invention relates to a method for diagnosis of cancer in a patient, or a method of determining the prognosis of a cancer patient, or a method of assigning a patient to an outcome group, or a method of assigning a patient to a treatment regimen, said method comprising the steps of
A fourth aspect of the invention relates to the non-agonist ligand according to the first aspect of the invention, or the nucleic acid molecule of the second aspect of the invention, for use in prevention or treatment of cancer, wherein a high likelihood of having or developing cancer is assigned to said patient according to the method of the third aspect of the invention.
In another embodiment, the present invention relates a pharmaceutical composition comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.
For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.
The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”
As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.
The term adenosine-diphosphate-ribosyl modification (also: ADP-ribosyl modification, ADP-ribosylated peptide, ADP-ribosylated protein) in the context of the present specification relates to a post translational modification (PTM), wherein an ADP-ribose (ADPr) moiety is covalently coupled to an amino acid. This type of post-translational modification is involved in the regulation of various cellular processes including cell signalling, gene regulation, DNA repair and apoptosis.
The term triple-negative breast cancer in the context of the present specification relates to a tumor type not having detectable expression of either one of estrogen receptors, progesterone receptors, or HER2. These tumors are especially difficult to treat, because none of the treatment strategies targeting the estrogen receptor or the progesterone receptor, or approaches making use of HER2-antagonists, can be employed for treatment.
The term mono-ADP-ribosyl modification specifically relates to modifications consisting in a single ADP-ribosyl unit per modification site.
The term specifically reactive in the context of the present invention refers to a property of ligands that bind to their target with a certain affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A ligand specifically reactive to a protein has a dissociation constant KD of ≤10−7 mol/L from its target, but a KD at least four orders of magnitude higher (i.e. having lower affinity) to a target of similar global chemical characteristics, but a significantly different protein sequence.
The term aptamer relates an oligonucleotide or peptide molecule that binds to a specific target molecule. Aptamers can be created by selecting them from a large random sequence pool. Nucleic acid aptamers can be generated through repeated rounds of in-vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to molecular targets such as small molecules, proteins or nucleic acids through non-covalent interactions. Aptamers offer molecular recognition properties that rival that of antibodies.
The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The term “polypeptides” and “protein” are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.
The term peptide in the context of the present specification relates to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more particularly 8 to 15amino acids, that form a linear chain wherein the amino acids are connected by peptide bonds.
Amino acid residue sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rd ed. p. 21). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, Y), Tyrosine (Tyr, Y), and Valine (Val, V).
The term gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.
The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes—and products thereof—of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.
The term Nucleotides in the context of the present specification relates to nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with RNA or DNA oligomers on the basis of base pairing. The term nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymine), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogues of nucleic acids such as phosphotioates, 2′O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2′O, 4′C methylene bridged RNA building blocks). Wherever reference is made herein to a hybridizing sequence, such hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.
The term siRNA (small/short interfering RNA) in the context of the present specification relates to an RNA molecule capable of interfering with the expression (in other words: inhibiting or preventing the expression) of a gene comprising a nucleic acid sequence complementary or hybridizing to the sequence of the siRNA in a process termed RNA interference. The term siRNA is meant to encompass both single stranded siRNA and double stranded siRNA. siRNA is usually characterized by a length of 17-24 nucleotides. Double stranded siRNA can be derived from longer double stranded RNA molecules (dsRNA). According to prevailing theory, the longer dsRNA is cleaved by an endo-ribonuclease (called Dicer) to form double stranded siRNA. In a nucleoprotein complex (called RISC), the double stranded siRNA is unwound to form single stranded siRNA. RNA interference often works via binding of an siRNA molecule to the mRNA molecule having a complementary sequence, resulting in degradation of the mRNA. RNA interference is also possible by binding of an siRNA molecule to an intronic sequence of a pre-mRNA (an immature, non-spliced mRNA) within the nucleus of a cell, resulting in degradation of the pre-mRNA.
The term shRNA (small hairpin RNA) in the context of the present specification relates to an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi).
The term sgRNA (single guide RNA) in the context of the present specification relates to an RNA molecule capable of sequence-specific repression of gene expression via the CRISPR (clustered regularly interspaced short palindromic repeats) mechanism.
The term miRNA (microRNA) in the context of the present specification relates to a small non-coding RNA molecule (containing about 22 nucleotides) that functions in RNA silencing and post-transcriptional regulation of gene expression.
The term antisense oligonucleotide in the context of the present specification relates to an oligonucleotide having a sequence substantially complimentary to, and capable of hybridizing to, an RNA. Antisense action on such RNA will lead to modulation, particular inhibition or suppression of the RNA's biological effect. If the RNA is an mRNA, expression of the resulting gene product is inhibited or suppressed. Antisense oligonucleotides can consist of DNA, RNA, nucleotide analogues and/or mixtures thereof. The skilled person is aware of a variety of commercial and non-commercial sources for computation of a theoretically optimal antisense sequence to a given target. Optimization can be performed both in terms of nucleobase sequence and in terms of backbone (ribo, deoxyribo, analogue) composition. Many sources exist for delivery of the actual physical oligonucleotide, which generally is synthesized by solid state synthesis.
Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).
One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.
Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).
In the context of the present specification, the term hybridizing sequence encompasses a polynucleotide sequence comprising or essentially consisting of RNA (ribonucleotides), DNA (deoxyribonucleotides), phosphothioate deoxyribonucleotides, 2′-O-methyl-modified phosphothioate ribonucleotides, LNA and/or PNA nucleotide analogues.
In the context of the present specification, the term antibody refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen binding fragment or single chains thereof and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region of IgG is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Similarly, the term encompasses a so-called nanobody or single domain antibody, an antibody fragment consisting of a single monomeric variable antibody domain.
In the context of the present specification, the term humanized antibody refers to an antibody originally produced by immune cells of a non-human species, the protein sequences of which have been modified to increase their similarity to antibody variants produced naturally in humans. The term humanized antibody as used herein includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences as well as within the CDR sequences derived from the germline of another mammalian species.
The term antibody-like molecule in the context of the present specification refers to a molecule capable of specific binding to another molecule or target with high affinity/a Kd≤10E-8 mol/l. An antibody-like molecule binds to its target similarly to the specific binding of an antibody. The term antibody-like molecule encompasses a repeat protein, such as a designed ankyrin repeat protein (Molecular Partners, Zurich), an engineered antibody mimetic protein exhibiting highly specific and high-affinity target protein binding (see US2012142611, US2016250341, US2016075767 and US2015368302, all of which are incorporated herein by reference). The term antibody-like molecule further encompasses, but is not limited to, a polypeptide derived from armadillo repeat proteins, a polypeptide derived from leucine-rich repeat proteins and a polypeptide derived from tetratricopeptide repeat proteins.
The term antibody-like molecule further encompasses a specifically binding polypeptide derived from a protein A domain, a fibronectin domain FN3, a consensus fibronectin domain, a lipocalin (see Skerra, Biochim. Biophys. Acta 2000, 1482(1-2):337-50), a polypeptide derived from a Zinc finger protein (see Kwan et al. Structure 2003, 11(7):803-813), a Src homology domain 2 (SH2) or Src homology domain 3 (SH3), a PDZ domain, a gamma-crystallin, ubiquitin, a cysteine knot polypeptide or a knottin, cystatin, Sac7d, a triple helix coiled coil (also known as alphabodies), a Kunitz domain or a Kunitz-type protease inhibitor and a carbohydrate binding module 32-2.
The term specific binding in the context of the present invention refers to a property of ligands that bind to their target with a certain affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reactive ligand has a dissociation constant of ≤10−7 mol/L when binding to its target, but a dissociation constant at least three orders of magnitude higher in its interaction with a molecule having a globally similar chemical composition as the target, but a different three-dimensional structure.
A polymer of a given group of monomers is a homopolymer (made up of a multiple of the same monomer); a copolymer of a given selection of monomers is a heteropolymer constituted by monomers of at least two of the group.
As used herein, the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.
As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).
As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow.
A first aspect of the invention relates to a non-agonist ligand of ARTC1 for use in prevention or treatment of cancer.
In certain embodiments, the ligand inhibits the ADP-ribosyltransferase activity of ARTC1.
In certain embodiments, the ligand is selected from an antibody, an antibody-like molecule, an aptamer, and an antibody fragment.
In certain embodiments, the non-agonist ligand is selected from an antibody and an antibody-like molecule.
In certain embodiments, the ligand is an antibody or antibody-like molecule and comprises a light chain variable region comprising LCDR1, LCDR2 and LCDR3 and a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and
In certain embodiments, the antibody or said antibody-like molecule comprises a light chain variable region comprising LCDR1, LCDR2 and LCDR3 and a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and
Substitution Rules
The substitution rules for deriving said LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 sequences from their respective reference sequence are:
In particular embodiments, at most two amino acids are exchanged. In more particular embodiments, at most one amino acid is exchanged by the substitution rules given above.
For example, if the LCDR1 of RG4-A111 (RASSSVSYMY) is modified by the substitution rules given above, the resulting modified LCDR1 of RG4-A111 may be RGSSSVSYMY (A to G) or RASTSVSYMY (S to T) or any other sequence resulting from those substitution rules.
In certain embodiments,
In certain embodiments, the non-agonist ligand comprises LCDR and HCDR sequences defined as follows:
In certain embodiments, the non-agonist ligand comprises
In certain embodiments, the non-agonist ligand comprises a sequence at least 90% identical, particularly ≥94%, ≥96% or even ≥98% identical to one of SEQ ID NO 007, SEQ ID NO 010, SEQ ID NO 013, SEQ ID NO 016 and SEQ ID NO 019.
In certain embodiments, the non-agonist ligand is characterized in being able to prevent ADP-ribosylation of RR, RG, GR, RXR and GXXXXR motifs.
In certain embodiments, the non-agonist ligand is characterized in that it is specifically reactive against a polypeptide encoded by any one of SEQ 001, SEQ 002, SEQ 003 or SEQ 004.
A second aspect of the invention relates to a nucleic acid molecule for use in prevention or treatment of cancer. The nucleic acid molecule according to this aspect of the invention comprises, or consists of, an inhibitor nucleic acid sequence capable of downregulating or inhibiting expression of a target nucleic acid sequence encoding ARTC1.
In certain embodiments, the inhibitor nucleic acid sequence is an antisense oligonucleotide, an siRNA, an shRNA, an sgRNA or an miRNA.
In certain embodiments of the first or second aspect, the ligand (particularly the antibody or antibody-like molecule) or nucleic acid is administered in treatment of a cancer is selected from breast cancer, colon cancer, lung cancer, liver cancer, glioma, kidney cancer, testis cancer, pancreas cancer, sarcoma, melanoma, prostate cancer, stomach cancer, ovary cancer, bladder cancer, uterus cancer, endometrioid adenocarcinoma, thyroid papillary carcinoma, cervix squamous carcinoma, esophageal cancer, Ewing sarcoma, thyroid anaplastic carcinoma, chordoma, chondrosarcoma, ocular melanoma, pseudomyxoma peritonei, and urachal carcinoma.
In certain particular embodiments, the treatment is for cancer selected from breast cancer, colon cancer, lung cancer, liver cancer, glioma, kidney cancer, testis cancer, pancreas cancer, sarcoma, melanoma, and prostate cancer.
In certain particular embodiments, the treatment is for cancer selected from breast cancer, lung cancer, kidney cancer and glioma. In certain more particular embodiments, the cancer is breast cancer. In certain even more particular embodiments, the cancer is triple-negative breast cancer.
A third aspect of the invention relates to a method for diagnosis of cancer in a patient, or a method of determining the prognosis of a cancer patient, or a method of assigning a patient to an outcome group, or a method of assigning a patient to a treatment regimen. The method according to this aspect of the invention comprises the steps of
In certain embodiments,
if more than 0.5%, particularly more than 1%, more particularly more than 2%, most particularly more than 5% of the cells in said isolated sample of said patient are stained positive for ARTC1.
The term more severe in the context of the present specification relates to a prognosis of a higher likelihood of having or developing a more aggressive form of cancer.
The term stained positive for ARTC1 in the context of the present specification relates to expression of an antigen assayed by an antibody, which can be detected. This may be done by, but is not limited to, staining of a histological tissue slice with an ARTC1 antibody and a labelled secondary antibody.
A fourth aspect of the invention relates to the non-agonist ligand of the first aspect or the nucleic acid molecule of the second aspect for use in prevention or treatment of cancer, wherein a high likelihood of having or developing cancer is assigned to said patient according to the method of the third aspect.
Medical Treatment, Dosage Forms and Salts
Similarly, within the scope of the present invention is a method of treating cancer in a patient in need thereof, comprising administering to the patient a ligand (particularly an antibody or antibody-like molecule) or a nucleic acid molecule according to the above description.
In certain embodiments, the non-agonist ARTC1 polypeptide ligand is an antibody, antibody fragment, an antibody-like molecule or a protein A domains derived polypeptide.
In some embodiments, the non-agonist ARTC1 polypeptide ligand is an immunoglobulin consisting of two heavy chains and two light chains. In some embodiments, the non-agonist anti-ARTC1 polypeptide ligand is a single domain antibody, consisting of an isolated variable domain from a heavy or light chain. In some embodiments, the non-agonist anti-ARTC1 polypeptide ligand is a heavy-chain antibody consisting of only heavy chains such as antibodies found in camelids.
In certain embodiments, the non-agonist ARTC1 polypeptide ligand is an antibody fragment. In certain embodiments, the non-agonist ARTC1 polypeptide ligand is a Fab fragment, i.e. the antigen-binding fragment of an antibody, or a single-chain variable fragment, i.e. a fusion protein of the variable region of heavy and the light chain of an antibody connected by a peptide linker.
Similarly, a dosage form for the prevention or treatment of cancer is provided, comprising a non-agonist ligand or antisense molecule according to any of the above aspects or embodiments of the invention.
Dosage forms may be for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Enteral administration forms are encompassed where appropriate, such as nasal, buccal, rectal, transdermal or oral administration forms, or as an inhalation form or suppository. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.
Method of Manufacture and Method of Treatment According to the Invention
The invention further encompasses, as an additional aspect, the use of a non-agonist ARTC1 ligand or a nucleic acid molecule as identified herein, or its pharmaceutically acceptable salt, as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of cancer.
Similarly, the invention encompasses methods of treatment of a patient having been diagnosed with cancer. This method entails administering to the patient an effective amount of a non-agonist ARTC1 ligand or a nucleic acid molecule as identified herein, or its pharmaceutically acceptable salt, as specified in detail herein.
The invention further encompasses the use of determining an expression level of ARTC1 identified herein for use in the manufacture of a kit for the detection of cancer. This aspect of the invention relates to a system for performing the method for diagnosis of cancer in a patient, or the method of determining the prognosis of a cancer patient, or the method of assigning a patient to an outcome group, or the method of assigning a patient to a treatment regimen according to the third aspect.
Similarly, the invention encompasses a method for treating a tissue sample, using a ARTC1 polypeptide ligand (particularly an antibody) as specified herein. Such method may allow subsequent analysis of the sample with regard to its likelihood of being associated with a medical condition or a favourable outcome of a treatment as described herein, without making the actual step of medical analysis.
Wherever alternatives for single separable features such as, for example, a ligand type or medical indication are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a ligand type may be combined with any of the alternative embodiments of a medical indication mentioned herein.
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
Tumor tissue overview array were obtained from the Pathology Department, University Hospital Zurich, and were stained for human ARTC1 by using a commercially available antibody that works on FFPE tissue (Abcam ab185293).
Cancers with positive ARTC1 staining are: breast, colon, lung, liver, glioma, kidney, testis, pancreas, sarcoma, melanoma, and prostate cancer.
A commercial multi cancer TMA (MC5003d from US Biomax) was stained for human ARTC1 by using a commercially available antibody that works on FFPE tissue (Abcam ab185293). Additional cancers with positive ARTC1 staining are: carcinomas of stomach, ovary, bladder, and uterus; melanoma, endometrioid adenocarcinoma, thyroid papillary carcinoma, cervix squamous carcinoma, Esophagus adenocarcinoma.
Sections from rare tumor types (provided by a CRO and selected by an oncologist) were stained for human ARTC1 by using a commercially available antibody that works on FFPE tissue (Abcam ab185293). The following cancer types stained positive for ARTC1: Ewing sarcoma, thyroid anaplastic carcinoma, chordoma, chondrosarcoma, ocular melanoma, pseudomyxoma peritonei, and urachal carcinoma (
2.1 ARTC1-Expression Correlates with Reduced Survival.
Organ-centric tumor TMAs were obtained from the Pathology Department, University Hospital Zurich, and were stained for human ARTC1 by using a commercially available antibody that works on FFPE tissue (Abcam ab185293). The ARTC1 staining was evaluated and correlated with anonymous post-mortem patient data. The analysis revealed that for all tumor types with sufficient patient data there is a negative correlation between ARTC1-expression and survival (
2.2 ARTC1-Expression Correlates with More Severe Tumor Subtypes.
The data from ARTC1-stained organ-centric TMAs were analysed for a correlation of ARTC1-expression with disease severity. Among the breast cancer cases, the negative correlation between ARTC1-expression and survival was clearly more pronounced among the more severe subtypes of breast cancer (BrCa): invasive ductal and triple-negative breast cancer (TNBC) (
Staining of normal control tissue TMAs (commercial and from Department of Pathology, University Hospital Zurich) reveals absence of ARTC1 expression in most normal tissues with the exception of very weak expression in skeletal and heart muscle (data not shown). These protein expression data confirm previous reports that were only based on RNA expression.
To show that ARTC1 is a therapeutic target in human cancers, the inventors established in vivo cancer models of human cancer cell lines grown in CB17-Scid mice. As for the moment, the inventors have not yet identified established cancer cell lines that express detectable ARTC1 at the cell surface in vitro, thus, they transduced cell lines with full length human ARTC1 or as control with an shRNA to knockdown endogenous ARTC1. The human TNBC cell line MDA-MB-231 (0.5 Mio) were injected orthotopically into the mammary fat pads. Starting from day 7 after tumor inoculation, the treatment group received twice weekly by intraperitoneal injection 15 mg/kg of the enzyme-neutralizing anti-human ARTC1 antibody HA003ximo2a (VH and VL domains of the original rat A3 clone were fused to murine IgG2a/K constant regions). The data shows that with an antibody treatment targeted to extracellular ARTC1, the tumor growth can be reduced as efficiently as with ARTC1 knock-down (
In another experimental setting, the inventors treated with twice weekly 5 mg/kg anti-human ARTC1 antibody HA003ximo2a and obtained a nearly as pronounced treatment effect as with 15 mg/kg (data not shown).
In another treatment experiment, human lung adenocarcinoma A549 cells (1 Mio) were injected s.c. into the flanks of CB17-Scid mice. Starting from day 7 after tumor inoculation, the treatment group received once weekly by intraperitoneal injection 3 mg/kg of the enzyme-neutralizing anti-human ARTC1 antibody HA003ximo2a (
In another treatment experiment, human A549 lung or SW620 colon carcinoma cells, respectively, were injected subcutaneously in CB17-Scid mice and animals were treated from day 7 on with 15 mg/kg HA003ximo2a i.p. twice weekly (
In another treatment experiment, murine syngeneic 4T1 breast cancer cells were injected orthotopically into BALB/c mice and animals were treated from day 7 on with 15 mg/kg MA197ximo2a i.p. twice weekly (
In another treatment experiment, murine syngeneic MC38 colon carcinoma cells were injected i.v. in wildtype C57BL/6 animals and animals were treated from day 0 on with 15 mg/kg MA197ximo2a i.p. twice weekly (
The mechanism of action is twofold: 1. by inhibition of ADP-ribosylation (ADPR), and 2. via antibody-dependent cellular cytotoxicity (ADCC).
Evidence for a role of inhibition of ADPR via blockade of ARTC1 by the therapeutic antibody comes from mass spectrometry analyses of ADP-ribosylation in human tumor cells and samples of solid tumors derived from the above described experimental treatment models (MDA-MB-231 (TNBC) and A549 (LuCa)). The inventors identified ADP-ribosylated proteins that are involved in tumor growth (growth factor and receptors), vascularization, metastasis and immune regulation (cytokines and their receptors).
Furthermore, upon antibody treatment or shRNA knockdown, these proteins are not ADP-ribosylated anymore.
Evidence for a role of ADCC in the MOA of the antibody therapy comes from the experiment in which MDA-MB-231 cells expressing an enzymatically inactive form of human ARTC1 (
In another treatment experiment, the inventors compared the efficacy of treating ARTC1-expressing MDA-MB-231 cells inoculated either in CB17-Scid or NOD-Scid-γc (NSG) mice and treated the animals with different doses of HA003ximo2a twice weekly starting from day 7 (
To determine the extent of enzyme inhibition in vivo upon antibody treatment, the inventors isolated treated and untreated MDA-MB-231 tumors from treatment experiments in CB17-Scid mice and analyzed the ADP-ribosylome using published mass spectrometry methods (Nowak et al., Nat Commun 11(1):5199). The analysis shows that while in parental (WT) or ARTC1-knockdown (KD) MDA-MB-231 tumors, only very few Arginine-specific ADP-ribosylation (R-ADPr) sites could be detected, a large number of peptides modified at R-ADPr sites were detected in ARTC1 overexpressing (OE) MDA-MB-231 cells and that the number of detected sites is strongly reduced upon antibody treatment (
Histological analysis of treated and untreated MDA-MB-231 tumors from treatment experiments in CB17-Scid mice revealed an influence on immune cell infiltration (
To confirm that indeed anti-ARTC1 antibodies can mediate antibody-dependent cellular cytotoxicity (ADCC), the authors performed in vitro ADCC assays with human PBMC as effector cells. The data shows that the presence of ARTC1 on MDA-MB-231 cells allows the antibody HA003ximo2a (10 μg/mL) to induce strong ADCC at an effector:target ratio of 50:1 (
A preliminary safety evaluation was performed by injection of different doses of anti-mouse ARTC1 antibody MA197ximo2a into naïve wildtype C57BL/6 animals or ARTC1-deficient mice. Blood plasma was sampled at days 3, 7, 10 and 14 and the levels of creatine kinase (CK) and alanine aminotransferase (ALT) were determined on a Beckman-Coulter SYNCHRON DxC800 system as measures of muscle and liver toxicity, respectively. No antibody-dependent toxicity was observed in neither of the doses. The blood CK and ALT levels in treated C57BL/6 animals were not elevated and were comparable both to treated ARTC1-deficient animals (in which no target engagement can occur) and normal C57BI/6 values from the literature (Mamm Genome 15:768) (
Amino Acid Sequence of Human ARTC1
The amino acid sequence of human ARTC1 (hARTC1) is provided in the Uniprot database (www.uniprot.org) under identifier P52961.
The human ARTC1 comprises an NAD+-binding catalytic site that is characterized by the catalytic triad RSE built by the three amino acids Arg179, Ser202 and Glu238 of SEQ ID NO 001.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20168333.1 | Apr 2020 | WO | international |
| 20177397.5 | May 2020 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/058907 | 4/6/2021 | WO |
