ARTC1 LIGANDS FOR CANCER TREATMENT

Abstract

The present invention relates to a non-agonist ligand of ARTC1, which inhibits the ADP-ribosyltransferase activity of ARTC1, or an inhibitor nucleic acid sequence capable of downregulating or inhibiting expression of a target nucleic acid sequence encoding ARTC1, for use in prevention or treatment of cancer. The invention also relates to a method for diagnosis of cancer.

Claims

1. A method for treating a cancer, comprising: administering to a subject in need thereof a non-agonist ligand of ARTC1, thereby treating the cancer.

2. The method of claim 1, wherein said ligand inhibits the ADP-ribosyltransferase activity of ARTC1.

3. The method of claim 1, wherein said ligand is selected from an antibody, an antibody-like molecule, an aptamer, an antibody fragment, particularly wherein said non-agonist ligand is selected from an antibody and an antibody-like molecule.

4. The method of claim 1, wherein said 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 wherein a. LCDR1 is a CDR1 comprised in a sequence selected from SEQ ID NO 006, SEQ ID NO 009, SEQ ID NO 012, SEQ ID NO 015 and SEQ ID NO 018; b. LCDR2 is a CDR2 comprised in a sequence selected from SEQ ID NO 006, SEQ ID NO 009, SEQ ID NO 012, SEQ ID NO 015 and SEQ ID NO 018; c. LCDR3 is a CDR3 comprised in a sequence selected from SEQ ID NO 006, SEQ ID NO 009, SEQ ID NO 012, SEQ ID NO 015 and SEQ ID NO 018; d. HCDR1 is a CDR1 comprised in a sequence selected from SEQ ID NO 005, SEQ ID NO 008, SEQ ID NO 011, SEQ ID NO 014 and SEQ ID NO 017; e. HCDR2 is a CDR2 comprised in a sequence selected from SEQ ID NO 005, SEQ ID NO 008, SEQ ID NO 011, SEQ ID NO 014 and SEQ ID NO 017; and f. HCDR3 is a CDR3 comprised in a sequence selected from SEQ ID NO 005, SEQ ID NO 008, SEQ ID NO 011, SEQ ID NO 014 and SEQ ID NO 017.

5. The method of claim 4, wherein said 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 wherein a. said LCDR1 is comprised in, particularly is identical to an LCDR1 reference sequence selected from SEQ ID NO 020, SEQ ID NO 021, SEQ ID NO 022, SEQ ID NO 023 and SEQ ID NO 024 or wherein said LCDR1 is derived from any one of said LCDR1 reference sequences by the substitution rules given below; b. said LCDR2 is comprised in, particularly is identical to an LCDR2 reference sequence selected from SEQ ID NO 025, SEQ ID NO 026, SEQ ID NO 027, SEQ ID NO 028 and SEQ ID NO 029 or wherein said LCDR2 is derived from any one of said LCDR2 reference sequences by the substitution rules given below, particularly said LCDR2 is comprised in, particularly is identical to an LCDR2 reference sequence selected from SEQ ID NO 050, SEQ ID NO 051, SEQ ID NO 052, SEQ ID NO 053 and SEQ ID NO 054 or wherein said LCDR2 is derived from any one of said LCDR2 reference sequences by the substitution rules given below; c. said LCDR3 is comprised in, particularly is identical to an LCDR3 reference sequence selected from SEQ ID NO 030, SEQ ID NO 031, SEQ ID NO 032, SEQ ID NO 033 and SEQ ID NO 034 or wherein said LCDR3 is derived from any one of said LCDR3 reference sequences by the substitution rules given below; d. said HCDR1 is comprised in, particularly is identical to a HCDR1 reference sequence selected from SEQ ID NO 035, SEQ ID NO 036, SEQ ID NO 037, SEQ ID NO 038 and SEQ ID NO 039 or wherein said HCDR1 is derived from any one of said LCDR1 reference sequences by the substitution rules given below; e. said HCDR2 is comprised in, particularly is identical to a HCDR2 reference sequence selected from SEQ ID NO 040, SEQ ID NO 041, SEQ ID NO 042, SEQ ID NO 043 and SEQ ID NO 044 or wherein said HCDR2 is derived from any one of said HCDR2 reference sequences by the substitution rules given below, particularly said HCDR2 is comprised in, particularly is identical to a HCDR2 reference sequence selected from SEQ ID NO 055, SEQ ID NO 056, SEQ ID NO 057, SEQ ID NO 058 and SEQ ID NO 059 or wherein said HCDR2 is derived from any one of said HCDR2 reference sequences by the substitution rules given below; and f. said HCDR3 is comprised in, particularly is identical to a HCDR3 reference sequence selected from SEQ ID NO 045, SEQ ID NO 046, SEQ ID NO 047, SEQ ID NO 048 and SEQ ID NO 049 or wherein said HCDR3 is derived from any one of said HCDR3 reference sequences by the substitution rules given below; and wherein the substitution rules for deriving said LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 sequences from their respective reference sequence are: a. glycine (G) and alanine (A) are interchangeable; valine (V), leucine (L), and isoleucine (I) are interchangeable, A and V are interchangeable; b. tryptophan (W) and phenylalanine (F) are interchangeable, tyrosine (Y) and F are interchangeable; c. serine (S) and threonine (T) are interchangeable; d. aspartic acid (D) and glutamic acid (E) are interchangeable e. asparagine (N) and glutamine (Q) are interchangeable; N and S are interchangeable; N and D are interchangeable; E and Q are interchangeable; f. methionine (M) and Q are interchangeable; g. cysteine (C), A and S are interchangeable; h. proline (P), G and A are interchangeable; i. arginine (R) and lysine (K) are interchangeable; particularly wherein at most two amino acids are exchanged, more particularly wherein at most one amino acid is exchanged by the substitution rules given above.

6. The method of claim 4, wherein a. said LCDR1 is selected from SEQ ID NO 020, SEQ ID NO 021, SEQ ID NO 022, SEQ ID NO 023 and SEQ ID NO 024; b. said LCDR2 is selected from SEQ ID NO 025, SEQ ID NO 026, SEQ ID NO 027, SEQ ID NO 028 and SEQ ID NO 029, particularly said LCDR2 is selected from SEQ ID NO 050, SEQ ID NO 051, SEQ ID NO 052, SEQ ID NO 053 and SEQ ID NO 054; c. said LCDR3 is selected from SEQ ID NO 030, SEQ ID NO 031, SEQ ID NO 032, SEQ ID NO 033 and SEQ ID NO 034; d. said HCDR1 is selected from SEQ ID NO 035, SEQ ID NO 036, SEQ ID NO 037, SEQ ID NO 038 and SEQ ID NO 039; e. said HCDR2 is selected from SEQ ID NO 040, SEQ ID NO 041, SEQ ID NO 042, SEQ ID NO 043 and SEQ ID NO 044, particularly said HCDR2 is selected from SEQ ID NO 055, SEQ ID NO 056, SEQ ID NO 057, SEQ ID NO 058 and SEQ ID NO 059; and f. said HCDR3 is selected from SEQ ID NO 045, SEQ ID NO 046, SEQ ID NO 047, SEQ ID NO 048 and SEQ ID NO 049.

7. The method of claim 4, comprising a. said LCDR1 is of sequence SEQ ID NO 020, said LCDR2 is of sequence SEQ ID NO 025 or SEQ ID NO 050, said LCDR3 is of sequence SEQ ID NO 030, said HCDR1 is of sequence SEQ ID NO 035, said HCDR2 is of sequence SEQ ID NO 040 or SEQ ID NO 055, and said HCDR3 is of sequence SEQ ID NO 045, b. said LCDR1 is of sequence SEQ ID NO 021, said LCDR2 is of sequence SEQ ID NO 026 or SEQ ID NO 051, said LCDR3 is of sequence SEQ ID NO 031, said HCDR1 is of sequence SEQ ID NO 036, said HCDR2 is of sequence SEQ ID NO 041 or SEQ ID NO 056, and said HCDR3 is of sequence SEQ ID NO 046, c. said LCDR1 is of sequence SEQ ID NO 022, said LCDR2 is of sequence SEQ ID NO 027 or SEQ ID NO 052, said LCDR3 is of sequence SEQ ID NO 032, said HCDR1 is of sequence SEQ ID NO 037, said HCDR2 is of sequence SEQ ID NO 042 or SEQ ID NO 057, and said HCDR3 is of sequence SEQ ID NO 047, d. said LCDR1 is of sequence SEQ ID NO 023, said LCDR2 is of sequence SEQ ID NO 028 or SEQ ID NO 053, said LCDR3 is of sequence SEQ ID NO 033, said HCDR1 is of sequence SEQ ID NO 038, said HCDR2 is of sequence SEQ ID NO 043 or SEQ ID NO 058, and said HCDR3 is of sequence SEQ ID NO 048, or e. said LCDR1 is of sequence SEQ ID NO 024, said LCDR2 is of sequence SEQ ID NO 029 or SEQ ID NO 054, said LCDR3 is of sequence SEQ ID NO 034, said HCDR1 is of sequence SEQ ID NO 039, said HCDR2 is of sequence SEQ ID NO 044 or SEQ ID NO 059, and said HCDR3 is of sequence SEQ ID NO 049.

8. The method of claim 4, comprising a. a first sequence at least 90% identical, particularly ≥94%, ≥96% or even ≥98% identical to one of SEQ ID NO 006, SEQ ID NO 009, SEQ ID NO 012, SEQ ID NO 015 and SEQ ID NO 018; and b. a second sequence at least 90% identical, particularly ≥94%, ≥96% or even ≥98% identical to one of SEQ ID NO 005, SEQ ID NO 008, SEQ ID NO 011, SEQ ID NO 014 and SEQ ID NO 017.

9. The method of claim 4, comprising 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.

10. The method of claim 4, characterized in being able to prevent ADP-ribosylation of RR, RG, GR, RXR and GXXXXR motifs.

11. The method of claim 4, characterized in that it is specifically reactive against a polypeptide encoded by any one of SEQ 001, SEQ 002, SEQ 003 or SEQ 004.

12. A method for treating a cancer, comprising administering to a subject in need thereof an inhibitor nucleic acid sequence capable of downregulating or inhibiting expression of a target nucleic acid sequence encoding ARTC1, thereby treating the cancer.

13. The method of claim 12, wherein said inhibitor nucleic acid sequence is an antisense oligonucleotide, an siRNA, an shRNA, an sgRNA or an miRNA.

14. The method of claim 1, wherein said 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, particularly wherein said cancer is selected from breast cancer, colon cancer, lung cancer, liver cancer, glioma, kidney cancer, testis cancer, pancreas cancer, sarcoma, melanoma, and prostate cancer, more particularly wherein said cancer is selected from breast cancer, lung cancer, kidney cancer and glioma, even more particularly wherein said cancer is breast cancer, more particularly wherein said cancer is triple-negative breast cancer.

15. 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 providing an isolated sample of said patient; determining the expression level of ARTC1 in said isolated sample; and assigning a likelihood of having or developing cancer to said patient, or assigning a likelihood of prognosis to said patient; assigning the patient to an outcome group or assigning the patient to treatment with an anticancer treatment, particularly an anticancer treatment comprising administration of a ligand or nucleic acid as specified in claim 1.

16. The method according to claim 15, wherein a high likelihood of having or developing cancer is assigned to said patient, or a more severe prognosis is assigned to said patient; or treatment with an anticancer treatment is assigned to said patient; 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.

17. The method of claim 1, wherein a high likelihood of having or developing cancer has been assigned to said subject.

18. The method of claim 12, wherein said 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, particularly wherein said cancer is selected from breast cancer, colon cancer, lung cancer, liver cancer, glioma, kidney cancer, testis cancer, pancreas cancer, sarcoma, melanoma, and prostate cancer, more particularly wherein said cancer is selected from breast cancer, lung cancer, kidney cancer and glioma, even more particularly wherein said cancer is breast cancer, more particularly wherein said cancer is triple-negative breast cancer.

19. The method of claim 12, wherein a high likelihood of having or developing cancer is assigned to said subject.

Description

DESCRIPTION OF THE FIGURES

[0141] FIG. 1 shows that ARTC1-expression correlates with reduced survival of cancer patients. Organ-centric tumor TMAs were stained for human ARTC1. The staining was evaluated according to a 3 stage score (negative, weak and strong staining) and correlated with anonymous post-mortem patient data (weak and strong staining were combined as positive). Cases with bad tissue quality or missing patient data were omitted form the analysis. (A) Survival of breast cancer patients (all entities). (B) Survival of kidney cancer patients (all entities). (C) Survival of patients with lung adenocarcinoma. (D) Examples of stainings of individual tissue punches.

[0142] FIG. 2 shows that ARTC1-expression correlates with more severe subtypes of cancer. Tumor TMAs were stained and evaluated as explained in FIG. 1. (A) Correlation of survival and ARTC1-expression in patients with invasive ductal BrCa. (B) Correlation of survival and ARTC1-expression in patients with triple-negative BrCa, the most severe form of BrCa. (C) Correlation of ARTC1-expression and the tumor grade in patients with brain cancer.

[0143] FIG. 3 shows treatment with an antibody blocking the enzymatic function of ARTC1. Human cancer cell lines were transduced with human ARTC1, with an empty vector (as control) or with an shRNA construct to knock down endogenous ARTC1 expression. (A) 0.5 Mio MDA-MB-231 cells 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. The control group received vehicle (PBS) only. Tumor size was measured with a caliper. (B) 1 Mio A549 cells 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 or a scFv version of rat A3 fused to a rabbit IgG-Fc in a molar amount equivalent to 6.75 mg/kg of a full IgG antibody. (C) MDA-MB-231 (left) and A549 (right) transduced with either empty vector or with full-length human ARTC1 were grown in CB17-Scid mice.

[0144] FIG. 4 shows sections from a collection of rare tumor types were stained for human ARTC1 (Abcam ab185293). All pictures were taken with a 10× objective.

[0145] FIG. 5 shows (A) A549 (0.5 Mio) cells transduced to express human ARTC1 were inoculated subcutaneously into CB17-Scid animals and treated in the same scheme as in FIG. 3A. (B) SW620 (0.5 Mio) cells transduced to express human ARTC1 were inoculated subcutaneously into CB17-Scid animals and treated in the same scheme as in FIG. 3A. (C) The antibody clone A197 was recombinantly produced in the form of rat-mouse chimeric antibody and was named MA197ximo2a. 4T1 (10E4 cells) transduced to express mouse ARTC1 (mARTC1) were inoculated subcutaneously into BALB/c animals and treated in the same scheme as in FIG. 3A but with antibody MA197ximo2a. (D) MC38 murine colon carcinoma cells were either control transduced (EV) or transduced with mARTC1. For the experimental metastasis model, 0.3 Mio cells were injected intravenously and the animal's lung weights were determined after 3 weeks.

[0146] FIG. 6 shows (A) MDA-MB-231 cells were transduced with enzymatically inactive mutant human ARTC1 (hARTC1-mut) and were inoculated orthotopically into CB17-Scid animals. Treatment was as in FIG. 3A. (B) Human ARTC1-expressing MDA-MB-231 cells were inoculated into CB17-Scid or NOD-Scid-yc (NSG) animals and the animals were treated twice weekly with the indicated doses. (C) MDA-MB-231 cells which were either control transduced (EV), knockdown for ARTC1 (KD) or overexpress ARTC1 (OE) were grown and treated as in FIG. 3A. At the end of the experiment, tumors were prepared for mass spectrometric analysis of ADP-ribosyl modifications. The graph shows the number of unique arginine ADP-ribosylation sites. (D) Tumors from the same experiment as in 6C were prepared for histological analysis. Sections were stained with the Af1521-Fc reagent. (E) Human ARTC1-expressing MDA-MB-231 cells were inoculated into CB17-Scid and the animals were treated as in FIG. 3A (PBS or HA003ximo2a). At the end of the experiment, tumors were prepared for histology and section were stained for the indicated markers. (F) Section of the same samples as in 6D were stained for CD31 and the area of DAB staining was measured with Fiji software.

[0147] FIG. 7 shows that antibody-dependent cellular cytotoxicity (ADCC) assays were performed with PBMCs from three different donors either at a fixed effector:target ratio of 50:1 or at titrating ratios. A549 that were either control transduced (EV) or transduced with human ARTC1 served as targets. A human IgG1 Fc chimeric form of HA003 or an isotype-matched control (IMC) was used at 10 ug/ml.

[0148] FIG. 8 shows that wildtype C57BL/6 (WT-B6) or ARTC1-deficient (ARTC1-KO) animals were injected with a single dose of MA197ximo2a at day 0 i.p. and were bled on the indicated days. Blood plasma was analysed for creatine kinase (CK) and alanine aminotransferase (ALT).

EXAMPLES

Example 1: ARTC1-Expression in Tumor Tissue Microarrays (TMA)

[0149] 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).

[0150] Cancers with positive ARTC1 staining are: breast, colon, lung, liver, glioma, kidney, testis, pancreas, sarcoma, melanoma, and prostate cancer.

[0151] 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.

[0152] 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 (FIG. 4).

Example 2: Correlation of Patient Data with ARTC1-Positivness

[0153] 2.1 ARTC1-Expression Correlates with Reduced Survival.

[0154] 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 (FIG. 1).

[0155] 2.2 ARTC1-Expression Correlates with More Severe Tumor Subtypes.

[0156] 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) (FIGS. 2A and B). The proportion of ARTC1-positive cases increases from ‘all BrCa’ over ‘invasive ductal BrCa’ until the most severe and currently least treatable form, the TNBC (48% vs 55% vs 74%, respectively). For TNBC, the difference in the 100-month survival expectancy is most pronounced among BrCa (88% for ARTC1-negative and 47% for ARTC1-positive cases). An analogous correlation between ARTC1-expression and tumor severity grade was observed for brain tumors (FIG. 2C). While low grade tumors (pilostystic astrocytoma (grade 1) and low-grade astrocytoma (grade 2) are nearly negative for ARTC1, anaplastic astrocytoma (grade 3) shows 60% mainly weak staining. In contrast, glioblastoma (grade 4) are 100% positive with 80% strong staining.

Example 3: Absence of ARTC1-Expression in Normal Tissue

[0157] 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.

Example 4: Treatment Data

[0158] 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 (FIG. 3A). This data shows that patients can be treated by a therapy targeted to ARTC1 either by an antibody, RNA interference or a small molecule inhibitor.

[0159] 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).

[0160] 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 (FIG. 3B). A second group received once weekly a scFv version of rat A3 fused to a rabbit IgG-Fc in a molar amount equivalent to 6.75 mg/kg of a full IgG antibody. The data shows that a treatment with 3 mg/kg antibody once weekly had no effect, while an equivalence dose of 6.75 mg/kg already slightly delayed tumor growth.

[0161] FIG. 3C shows that ectopic expression of human ARTC1 in human cancer cell lines leads to an enhanced tumor growth in CB17-Scid mice (FIG. 3C, left MDA-MB-231, right A549). Thus, FIG. 3A (knockdown) and FIG. 3C prove that ARTC1 is a drive of tumor growth.

[0162] 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 (FIGS. 5A and 5B).

[0163] 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 (FIG. 5C). The results show that the ARTC1-targeted treatment also works in a fully syngeneic system and in the presence of a full immune system.

[0164] 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 (FIG. 5D). Lungs were harvested on day 21 and treated with Bouin's solution. Metastases nodules (not shown) and lung weights were determined. These experiments revealed that ARTC1 contributes to disease severity by enhancing metastatic seeding. Antibody treatment led to a reduction of metastatic burden.

Example 5: MOA

[0165] The mechanism of action is twofold: 1. by inhibition of ADP-ribosylation (ADPR), and 2. via antibody-dependent cellular cytotoxicity (ADCC).

[0166] 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).

[0167] Furthermore, upon antibody treatment or shRNA knockdown, these proteins are not ADP-ribosylated anymore.

[0168] 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 (FIG. 6A, hARTC1-mut). The experiment shows that a very efficient treatment effect can be achieved, without the need to block the enzymatic function. The ADCC mediated by the mouse IgG2a Fc part of the HA003ximo2a antibody is sufficient for a treatment effect in this experimental setting. The results show that the tumor growth can be reduced also if the inhibitory function of the antibody is irrelevant, indicating that NK cell and macrophage-mediated cellular are also engaged by the antibody.

[0169] 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 (FIG. 6B). The results indicate that the antibody treatment has an effect even in the complete absence of immune cells, while the ARTC1-inhibitory capacity is retained in this system.

[0170] 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 (FIG. 6C). The reduced extent of ADP-ribosylation was furthermore confirmed by staining histological section of the respective tumors with the Af1521-Fc reagent which binds ADP-ribose and can be visualized by anti-Fc staining. The histological data confirm the mass spectrometry data as a strongly reduced ADP-ribosylation staining was obtained in anti-ARTC1 treated tumors (FIG. 6D).

[0171] Histological analysis of treated and untreated MDA-MB-231 tumors from treatment experiments in CB17-Scid mice revealed an influence on immune cell infiltration (FIG. 6E). CD11 b is present on all myeloid cells including the myeloid derived suppressor cells (M DSC) which are frequently populating immunologically cold tumors. CD11b+ cells could be detected throughout the ARTC1-expressing control tumors while CD11b+ cells were confined to the tumor borders in antibody-treated tumors. F4/80 is expressed by macrophages and could be detected at a moderate frequency within control tumors. In contrast, strong vessel-associated F4/80 staining was detected in antibody-treated tumors. The natural killer cell receptor NCR1/NKp46 characterizes NK cells. While hardly any NK cells could be detected in control tumors, a strongly enhanced NK cell infiltration was detectable in antibody-treated tumors although with a low signal intensity. The inventors had identified ARTC1-dependent ADP-ribosylation marks also on molecules of the vascularization pathway (data not shown). To determine whether tumor vascularization was affected by anti-ARTC1 antibody treatment, histological section of treated and untreated MDA-MB-231 tumors from treatment experiments in CB17-Scid mice were stained for PECAM-1/CD31 which is expressed on early and mature endothelial cells. Human ARTC1-expressing tumor contained the highest amount of CD31 staining suggesting that ARTC1 activity promotes neovascularization within tumors. Antibody treatment or knockdown of ARTC1 was associated with reduced CD31 staining, suggesting that reduced tumor growth is in part due to reduced neovascularization owing to reduced or absent ARTC1 activity (FIG. 6F).

[0172] 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 (FIG. 7, left and middle, two donors). Furthermore, the data shows that the ADCC is also effector cell dependent (FIG. 7 right).

Example 6: Safety Data

[0173] 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) (FIG. 8).

[0174] Amino Acid Sequence of Human ARTC1

[0175] The amino acid sequence of human ARTC1 (hARTC1) is provided in the Uniprot database (www.uniprot.org) under identifier P52961.

TABLE-US-00001 hARTC1 (SEQ ID NO 001): MQMPAMMSLLLVSVGLMEALQAQSHPITRRDLFS QEIQLDMALASFDDQYAGCAAAMTAALPDLNHTE FQANQVYADSWTLASSQWQERQARWPEWSLSPTR PSPPPLGFRDEHGVALLAYTANSPLHKEFNAAVR EAGRSRAHYLHHFSFKTLHFLLTEALQLLGSGQR PPRCHQVFRGVHGLRFRPAGPRATVRLGGFASAS LKHVAAQQFGEDTFFGIWTCLGAPIKGYSFFPGE EEVLIPPFETFQVINASRLAQGPARIYLRALGKH STYNCEYIKDKKCKSGPCHLDNSAMGQSPLSAVW SLLLLLWFLVVRAFPDGPGLL

[0176] 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.

TABLE-US-00002 hARTC1 [160-200] SEQ ID NO 002: EALQLLGSGQRPPRCHQVFRGVHGLRFRPAGPRATVRLGGF hARTCI [180-220] SEQ ID NO 003: GVHGLRFRPAGPRATVRLGGFASASLKHVAAQQFGEDTFFG hARTCI [220-260] SEQ ID NO 004: GIWTCLGAPIKGYSFFPGEEEVLIPPFETFQVINASRLAQG

TABLE-US-00003 TABLE 1 Rat antibodies used in this study Antibody SEQ ID NO type RG4-A111 005 Heavy chain 006 Light chain 007 recombinant scFV R19-A3 008 Heavy chain 009 Light chain 010 recombinant scFV R17254- 011 Heavy chain A271 012 Light chain 013 recombinant scFV R17254- 014 Heavy chain A327 015 Light chain 016 recombinant scFV R17254- 017 Heavy chain A197 018 Light chain 019 recombinant scFV

TABLE-US-00004 TABLE 2 CDR sequences KABAT LCDR1 LCDR2 LCDR3 (SEQ ID (SEQ ID (SEQ ID NO) NO) NO) RG4- RASSSVSYMY ETSKLAS QQWNYPSCT A111 (020) (025) (030) R19- RASSGVSYMA GTSKLAS QQCCSTPLT A3 (021) (026) (031) A271 KSSQSLLSSG YASTRQS LQHYISPFT NQKNYLA( 022) (027) (032) A327 KTSQNVDYYG EGSNLPS QQSKDYPWT ITYMH (023) (028) (033) A197 RASSSVSYMY DTSKLAS QQWSSSPSMT (024) (029) (034) KABAT LCDR2 long(SEQ ID NO) RG4- ETSKLASGVPD (050) A111 R19- GTSKLASGVPN (051) A3 A271 YASTRQSGVPD (052) A327 EGSNLPSGIPA (053) A197 DTSKLASGVPN (054) HCDR1 HCDR2 HCDR3 KABAT (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) RG4- NFAMA SISYDGVN RGAAPFDF A111 (035) TYYRD (040) (045) R19- DYAMV TITYDGSR QGGYTTDY A3 (036) IYYRD (041) YYVMDA (046) A271 DFPMA TISTGGGTT PLYYSNYV (037) YYRG (042) GNVMDA (047) A327 INYWD HINYSGGT EGAGGLDY (038) NYNP (043) (048) A197 NFPMA WSISGGAT GDGSSRGYYFDY (039) YYRD (044) (049) KABAT HCDR2 long (SEQ ID NO) RG4- SISYDGVNTYYRDSVRG (055) A111 R19- TITYDGSRIYYRDSVRG (056) A3 A271 TISTGGGTTYYRGSVRG (057) A327 HINYSGGTNYNPSLRS (058) A197 WSISGGATYYRDSVRG (059)