LU501430B1 - Enrichment, detection and characterization of circulating tumor cells with susd2 and enpp1 - Google Patents

Abstract

A method for the isolation, or isolation and detection, of circulating tumor cells (CTCs) from blood or lymph, or disseminated tumor cells (DTCs) from bone marrow. SUSD2 or ENPPl is used as a biomarker for the isolation of CTCs or DTCs. Isolation can, for example, be done immunomagnetically using anti-SUSD2 or anti-ENPPl antibodies coupled to magnetic particles.

Description

PAT 1825 LU

ENRICHMENT, DETECTION AND CHARACTERIZATION OF CIRCULATING ~~ LU501430

TUMOR CELLS WITH SUSD2 AND ENPP1

DESCRIPTION BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to an improved method for the isolation, and optionally including detection, of circulating tumor cells (CTCs) from blood or lymph and/or disseminated tumor cells (DTCs) from bone marrow. Isolation of CTCs and/or DTCs allows for a subsequent genetic or other analysis, e.g. for developing therapies.

Description of the Related Art

The metastatic cascade in breast cancer comprises the release of primary breast tumour cells into the blood (circulating tumour cells, CTC) followed by the settlement of such tumour cells at secondary organs (disseminated tumour cells, DTC) and their later metastatic outgrowth.

However, there is still a substantial number of patients who relapse despite negative CTC findings at primary diagnosis in breast cancer.

As used herein, the term CTC relates to a tumor cell in the peripheral blood or lymph and

DTC relates to a tumor cell in bone marrow. CTCs and DTCs are extremely difficult to detect.

They are exceptionally rare and may be difficult to distinguish from healthy cells. Existing approaches for detecting CTCs and DTCs have limitations in sensitivity and/or specificity, leading to many healthy cells being mischaracterized as cancerous, and many cancer cells being missed in the analysis.

The current established and used biomarkers for detection and enrichment of tumor cells from the blood or bone marrow of patients are strongly limited to epithelial properties and downregulated in mesenchymal tumor cells. Detection is done using the “epithelial cell adhesion molecule” (EpCAM) or other epithelial markers like cytokeratin (CK). Among a pool of epithelial CTCs there are tumor cells that have undergone or are undergoing “epithelial-to-mesenchymal transition” (EMT) and lost much of their epithelial phenotype (to allow them to detach from the primary tumor actively). Such CTCs and DTCs are thought to 1

PAT 1825 LU be major drivers in metastasis formation and express epithelial markers only in low levels, LUS01430 making their isolation or detection with these markers difficult. EpCAM is a transmembrane protein with an extracellular domain and is suitable both for the detection and the isolation of

CTCs and DTCs expressing large enough levels of epithelial markers. In contrast, cytokeratins are cytoplasmic proteins and are only suitable for the detection of CTCs and

DTCs, but not for the isolation of the cells.

WO2013036620A1 entitled “Methods and Compositions for Cytometric Detection of Rare

Target Cells in a Sample” discloses cytometric methods for the detection of rare target cells, such as circulating tumor cells (CTCs), in a biological sample such as blood. Aspects of the methods include contacting the sample with first and second binding members that specifically bind to a marker of the rare target cell, and cytometrically assaying the sample for the presence of cells comprising bound first and second binding members to detect the rare target cell in the sample. While this publication describes some general approaches for the detection of rare cells in a sample, it is limited to detection. Samples are analyzed for the presence of the cells. There is no mention of any further processing of the sample, including isolation. In addition, neither Sushi domain-containing protein 2 (SUSD2) or Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) are mentioned here, nor the fact that for isolation of the cells at least one antibody has to bind to the extracellular domain of the target protein. In principle, two antibodies can also bind to a protein that is exclusively present in the cytoplasm. There is no attempt in this patent to differentiate between cytoplasmic (sole detection) and transmembrane (isolation and detection) proteins.

WO 2018185336 A1 entitled “Optofluidic device and method for detecting circulating tumour cells” discloses a microfluidic device for detection and quantification of CTCs. Therefore, the sample is incubated with two labelled probes that target different CTC surface marker.

Signals corresponding to the labelled probes are then detected in the microfluidic device.

SUSD2 is mentioned as a possible mesenchymal lineage marker. However, it is not mentioned that SUSD2 could be used for isolation of CTCs.

It is an object of the invention to provide an improved method for isolation, and preferably isolation and detection, of CTCs and DTCs from blood, lymph or bone marrow. Isolation of

CTCs and DTCs allows for a subsequent genetic or other analysis, e.g., for developing therapies. The biomarker proposed by the invention might function as potential immune target 2

PAT 1825 LU for personalized therapies. For example, highly personalized approaches, like the chimeric LU501430 antigen receptor T-cell (CAR-T) technology, are an option to specifically attack cancer cells while reducing unwanted side effects. CARs usually contain specificity-conferring extracellular antibody single chain variable fragment (scFv), a CD3z-domain and intracellular costimulatory domains. The procedure begins with the isolation of T-cells from the blood of a patient, followed by a genetic modification to recognize the surface protein of interest and re- injection into the donor.

In the histogenesis of breast cancer, different pathways for the development of cancer phenotypes are possible [13]. Stem/progenitor cells (mesenchymal phenotype) differentiate via intermediary cells (epithelial/mesenchymal phenotype) to glandular cells (epithelial phenotype). During these steps, the cells may acquire gene aberrations leading to breast cancer phenotypes varying in their degree of epithelial differentiation and oncoprotein expression. Therefore, an approach is desirable that enables the detection/isolation of representative tumor cells of these phenotypes irrespective of their degree of epithelial differentiation.

It is also an object of the invention to provide a method particularly suited for the isolation and preferably isolation plus detection of cells with epithelial and/or mesenchymal attributes, cells with mesenchymal attributes meaning cells showing only low level of keratin (like cells of the cell line MDA-MB-231) or almost negative for keratin like the DTC cell line from the bone marrow of a breast cancer patient BC-M1 [1]. CTCs or DTCs with mesenchymal or epithelial/mesenchymal phenotype are thought to be major drivers in metastasis formation. As the current established and used biomarkers (EpCAM, cytokeratins) for detection and enrichment of tumor cells from the blood of patients are strongly limited to epithelial properties and downregulated in mesenchymal tumor cells, the present invention aims to close this “detection gap” for tumor cells with mesenchymal attributes by identifying new cell surface biomarker proteins for a broad spectrum of different CTC/DTC subpopulations.

However, the invention is not limited to the enrichment, isolation and/or detection of CTCs or

DTCs with mesenchymal attributes, but is also applicable to the enrichment, isolation and/or detection of CTCs or DTCs with epithelial attributes. 3

PAT 1825 LU

SUMMARY OF THE INVENTION LUS01430

An analysis of a novel CTC-derived breast cancer cell line (CTC-ITB-01, [2]) revealed elevated expression of Sushi-domain-containing protein 2 (SUSD2) and Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPPI) compared with the breast cancer cell line MCF-7 (Fig. 1A). For both proteins a tumor-promoting impact was described in breast cancer [3-5]. Expression of these proteins on different breast cancer cell lines was proven by Western Blot and immunofluorescent analyses. While the expression of SUSD2 was observed by cell lines of different phenotype, ENPPI was also detected in the strongly mesenchymal DTC cell line BC-M1 (Fig. 1B).

To date, SUSD2 and ENPPI were not described in the context of Liquid Biopsy or CTCs.

However, both proteins were detected in solid tumors and their role in different cancer entities was analyzed [3-7]. The invention allows enrichment and isolation, and optionally including detection, of viable SUSD2-positive and ENPP1-positive CTCs or DTCs that express the protein on their cell surface using, for example, a direct magnetic labeling approach of the

MACS® technology. Different indirect magnetic labeling approaches using SUSD2 as target were described in literature to isolate SUSD2-positive mesenchymal stem cells in human palatine tonsil, bone marrow and endometrium [8-10]. Enrichment and isolation of ENPPI- positive cells by means of the MACS® technology were not described.

In order to use transmembrane proteins as specific CTC or DTC biomarkers, they should not be expressed by peripheral blood mononuclear cells (PBMCs). To verify the suitability of the transmembrane proteins SUSD2 and ENPP1 as CTC biomarkers, the expression of these proteins in PBMCs was analyzed. Western Blot analyses showed no signal for SUSD2 and

ENPP1 in PBMCs, confirming their suitability as CTC biomarkers. The expression of ENPP1 and SUSD2 was analyzed on 13 breast cancer cell lines and four prostate cancer cell lines.

While ENPP1 was expressed by breast cancer cell lines with mesenchymal characteristics, the expression of SUSD2 was mainly observed in breast cancer cell lines of different phenotype.

A strikingly high expression of SUSD2 was detected in CTC-ITB-01 and its corresponding mouse xenograft cell line (CTC-ITB-01 MIND), suggesting that this protein could play an important role in the establishment of CTC cell lines. The expression of ENPP1 and SUSD2 was also observed in prostate cancer cell lines. 4

PAT 1825 LU

Experimental results show that peripheral blood mononuclear cells (PBMC) are virtually LUS01430 negative for SUSD2 and ENPPI (Fig. 2), whereas a subpopulation of breast cancer cells is positive for these proteins (Fig. 1). Therefore, these proteins are suitable as novel CTC detection marker proteins, which may expand the number of detectable CTC.

The core of the invention is the enrichment, isolation, detection and/or characterization of CTCs with novel biomarkers. For the first time, expression of

SUSD2 and ENPPI on patient CTCs was proven and thus their suitability as biomarkers in liquid biopsy analyses for the isolation and optionally isolation plus detection of CTCs and DTCs. The invention can, however, also be used in the context of other cancers with a high potential to form metastases, e.g., prostate cancer, pancreatic cancer, head and neck cancer, lung cancer or malignant mesothelioma.

On the one hand, the invention aims to enrich and preferably isolate a specific subpopulation of viable CTCs that can be further analyzed, especially in regards of the metastatic potential of such subpopulations. On the other hand, it can be used to increase the number of enriched CTCs and to isolate CTCs by using, for example, a combination of

MicroBeads targeting commonly used enrichment markers such as EpCAM and new markers like SUSD2 and ENPPI. Furthermore, in combination with commonly used detection markers, SUSD2 and ENPPI could improve the detection rate of CTCs and promote treatment selection by enhancing CTC characterization.

Isolation can, for example, be done via magnetic-activated cell sorting (MACS®), a method for separation of various cell populations depending on their surface antigens (CD molecules), using anti-SUSD2 or anti-ENPPI antibodies. The inventors developed a specific MACS- based system for isolating CTCs or DTCs expressing SUSD2. The present invention also allows for the isolation of CTCs or DTCs expressing ENPP1 by this system. Isolation is, however, not limited to this method, and alternatively isolation can be done, for example, by a specially adapted commercial system called “CELLSEARCH” (see, for example, Coumans F,

Terstappen L., 2015, Detection and Characterization of Circulating Tumor Cells by the

CellSearch Approach, Methods Mol Biol. 1347:263-78, doi: 10.1007/978-1-4939-2990-0 18;

Mesquita B, et al. 2017, Molecular analysis of single circulating tumour cells following long- term storage of clinical samples, Mol Oncol. 11(12):1687-1697. doi: 10.1002/1878- 0261.12113) or by other methods, for example by immune cell isolation methods using

PAT 1825 LU immobilized anti-SUSD2 or anti-ENPPI antibodies (e.g., anti-SUSD2 or anti-ENPPI LU501430 antibody coated matrices) or other binding molecules (e.g., aptamers) specifically binding to

SUSD2 or ENPPI Methods using, for example, antibody-coated microstructures are also encompassed by the invention.

In the case of isolation plus detection the present invention requires the contacting of the cells with at least 2 antibodies, for example: 1. capture with an anti-SUSD2 or anti-ENPPI antibody targeting the extracellular domain of

SUSD2 or ENPPI, followed by detection of the captured cells by a labeled secondary antibody targeting the bound anti-SUSD2 or anti-ENPPI antibody, or 2. capture of the cells with a first anti-SUSD2 or anti-ENPPI antibody targeting the extracellular domain of SUSD2 or ENPPI, followed by the detection of the captured cells by a labeled secondary antibody targeting a second anti-SUSD2 or anti-ENPPI antibody, bound to the intracellular domain of SUSD2 or ENPPI. Alternatively, the second anti-SUSD2 or anti-ENPPI antibody can itself be fluorescently labeled.

An anti-keratin antibody, i.e., an antibody directed against cytokeratin, may additionally be used for detecting cytokeratin-positive cells, thus to distinguish between CTCs and DTCs having a mesenchymal phenotype (mCTC, mDTC), which would not test cytokeratin- positive, and epithelial (eCTCs) and hybrid epithelial/mesenchymal phenotype (emCTCs, emDTCs), which would test cytokeratin-positive.

By way of comparison, U.S. Patent 8,524,493 entitled “Released Cytokeratins as Markers for

Epithelial Cells” teaches a protocol for the EPISPOT assay with the detection of the cells by released keratins. In this case, the cells are cultivated on a synthetic membrane. In contrast,

MACS is performed in liquid solution, which allows subsequent handling of the cells.

Moreover, the present invention 1s particularly suitable for the enrichment, isolation and preferably additionaly detection of cells with different attributes, including cells with mesenchymal attributes, i.e. cells that show only low level of keratin (MDA-MB-231) or are almost negative for keratin (BC-M1). 6

PAT 1825 LU

BRIEF DESCRIPTION OF THE DRAWINGS LU501430

Further details and features of the invention will be described on the basis of the following figures, showing:

Fig. 1A Western blot analysis of CTC biomarker candidates. Investigation of SUSD2 and ENPPlexpression in breast cancer cell lines BC-M1, MDA-MB-468,

MCF-7, CTC-ITB-01, Cama-1, T47D and KPL-1. a-tubulin was used as loading control (n=3),

Fig. 1B Determination of epithelial and mesenchymal characteristics of CTC-ITB-01 and the reference cell line candidates by Western blot analysis. The expression of the epithelial markers E-cadherin, EpCAM, K8, K18 and K19, the mesenchymal markers N-cadherin and vimentin, and the stem-cell marker

CD44 was analyzed in CTC-ITB-01 and the reference cell line candidates. BC-

M1 and MDA-MB-468 were applied as positive controls and a-tubulin was used as loading control. Twenty micrograms (40 ug for EpCAM) of total cell extract of each cell line was applied (n=3). K8, keratin 8; K18, keratin 18;

K19, keratin 19,

Fig. 2 Western blot analysis of SUSD2 and ENPP1 in PBMCs: Expression of SUSD2 and ENPPI in PBMCs was analyzed. Therefore, PBMCs lysates from five different healthy donors were prepared and subjected to SDS PAGE. BC-MI,

MDA-MB-231 B02 and MDA-MB-468 were used as positive controls. Forty micrograms of protein were applied to each lane. a-tubulin was used as loading control,

Fig. 3 Detection of ENPPI on CTCs of patients with breast cancer: Cells were stained with anti-ENPPI antibody on a glass slide. Furthermore, pan-keratin was used as tumor cell marker, CD45 as leukocyte marker and nuclei were visualized using DAPI. A: ENPPI-positive CTC of patient 1. B: ENPPl-negative CTC of patient 1. C: ENPPI - positive CTC of patient 2. D: ENPPI-negative CTC of patient 2. Images were taken at 400 x magnification, 7

PAT 1825 LU

Fig. 4 Detection of SUSD2 expression on CTCs: Representative pictures of CTCs LU501430 enriched from blood of patients with metastatic breast cancer using the

CellSearch ® system. CTCs were identified with the CellSearch® CTC Kit as keratin+ /DAPH /CD45- cells. SUSD2 expression was investigated with the anti-SUSD2 Vio Bright-FITC antibody. À: SUSD2-negative CTC. B and ©: CTCs with low SUSD2 expression. D and E: CTCs with moderate SUSD2 expression. F: CTC with strong SUSD2 expression,

Fig. 5 Domain structure of SUSD2 a) Full-length membrane-bound SUSD2. b)

Predicted final version of membrane-bound SUSD2, cleaved within the extracellular domain and releasing a fragment, which binds via disulfide bonds to the remaining membrane-bound fragment,

Fig. 6 Domain structure of ENPP1 a) Full-length membrane-bound ENPP1. b)

Soluble form of ENPP1, cleaved intracellularly,

Fig. 7 Simplified representation of capture and detection of SUSD2 or ENPP1 positive CTC by an immunomagnetic cell separation method, using two different approaches. SUSD2 is shown here as an example. In both approaches an anti-SUSD2 or ENPP1 antibody (“capturing” antibody), which is directed to the extracellular domain of SUSD2 or ENPP1, was coupled to magnetic nanoparticles. In the first approach (Fig. 7a) a labeled secondary antibody directed against the anti-SUSD2 or ENPP1 antibody or against the complex composed of the anti-SUSD2 or ENPP1 antibody, the magnetic nanoparticle and the components (e.g. biotin and streptavidin) coupling the magnetic nanoparticle and the anti-SUSD2 or ENPP1 antibody, is used to detect

SUSD2" or ENPP1" CTCs, in the second approach (Fig. 7b) a second primary anti-SUSD2 or anti-ENPP1 antibody, directed against the intracellular domain of SUSD2 or ENPP1, and a secondary antibody directed against the second primary anti-SUSD2 or anti-ENPP1 antibody are used to detect SUSD2" or

ENPP1" CTCs,

Fig. 8 Schematic illustration of an example of a cell surface molecule-dependent

MACS isolation procedure. CTCs carrying the cell surface molecule (e.g., a 8

PAT 1825 LU transmembrane protein) are captured by an antibody directed against the cell | LU501430 surface molecule. The antibody is coupled to a magnetic nanoparticle, and the complex of antibody bound to the CTC and the magnetic nanoparticle can be retained in a column by a magnet (Fig. 8a) and subsequently released from the column by removing the magnet (8b),

Fig. 9 Microscopic evaluation by immunofluorescent double staining of the MACS® system using SUSD2 MicroBeads and breast cancer cell line cells spiked into blood of healthy donors as model system. Cytospins were obtained after conducting MACS® with SUSD2 MicroBeads. Slides were stained with anti- mouse lgG to detect the MicroBeads bound tumor cells. Furthermore, pan- keratin was applied as tumor cell marker, CD45 as leukocyte marker and nuclei were visualized with DAPI. A: CTC-ITB-01 cell present in the elution fraction. B: CTC-ITB-01 cell, which was found in the combined flow - through and wash fraction. C: T47D cell in the elution fraction. D: T47D cell in the combined flow-through and wash fraction. White scale bars represent 20 pm.

Images were taken at 400 x magnification,

Fig. 10 Microscopic analysis by immunofluorescent double staining of breast cancer patient CTCs obtained by MACS® using SUSD2 MicroBeads (elution fraction). The cytospin of the elution fraction was stained with anti-mouse IgG to detect CTCs bound by SUSD2 MicroBeads. Furthermore, pan-keratin was applied as tumor cell marker, CD45 as leukocyte marker and nuclei were visualized with DAPI. A: CTC cluster with SUSD2-positive CTCs. B: CTCs with positive and negative mouse-IgG staining. C: Zoom of CTCs with positive mouse IgG staining denoted by arrow C in image B. D: Zoom of

CTCs with negative mouse IgG staining denoted by arrow D in image B.

Images of the CTC cluster were taken at 400 x magnification and images in B at 200 x magnification. White scale bars represent 10 um and blue scale bars correspond to 30 um,

Fig. 11 CLUSTAL multiple sequence alignment of the amino acid sequences from human SUSD2 (SUSD2 HUMAN; UniProtKB Q9UGT4 (sequence version (SV) 1; SEQ ID NO: 01)) with the bioinformatics predicted SUSD2 proteins 9

PAT 1825 LU by sequence homology from canis familiaris (dog; UniProtKB E2RSH4 (SV3); LU501430

SEQ ID NO: 02), cavia porcellus (guinea pig; UniProtKB HOVMCS (SV1);

SEQ ID NO: 03) and equus caballus (horse; UniProtKB F6QE14 (SV2); SEQ

ID NO: 04). The identity of the amino acid sequences compared with human

SUSD2 are 76.59% (pig), 76.77 (dog), 78.56 (horse),

Fig. 12 CLUSTAL multiple sequence alignment of the amino acid sequences from human ENPP1 (ENPP1 HUMAN; UniProtKB P22413 (SV2); SEQ ID NO: 05) with the bioinformatics predicted ENPP1 proteins by sequence homology from canis familiaris (dog, UniProtKB F1PJP0 (SV2); SEQ ID NO: 06), cavia porcellus (guinea pig; UniProtKB HOVSW3 (SV2); SEQ ID NO: 07) and equus caballus (horse; UniProtKB F7ALR7 (SV2); SEQ ID NO: 08). The identity of the amino acid sequences compared with human ENPP1 are 79.22% (pig), 86.57 (dog), 86.49 (horse),

Fig. 13 MS survey scan of SUSD2 peptide CGALDGPCSCHPTCSGLGTCCLDFR (SEQ ID NO: 09) detected in a MCF-7/CTC-ITB-01 SILAC sample. MS survey scans show the peak intensities on the vertical axes and the horizontal axes display the m/z. A: MS survey scan at a retention time of 35.14 minutes.

Some peaks are only annotated for orientation. B: Enlarged section of the MS survey scan in A at a mass range of m/z = 950 — 960, showing the SUSD2 peptide with light masses from CTC-ITB-01. Monoisotopic peak of the light peptide ion 1s shown at m/z = 953.38 [M + 3 H]3+, and

Figure 14: MS survey scan of ENPP1 peptide AEYLHTWGGLLPVISK (SEQ ID NO: 10) detected in a MCF-7/CTC-ITB-01 SILAC sample MS survey scans show the peak intensities on the vertical axes and the horizontal axes display the m/z.

A: MS survey scan at a retention time of 44.87 minutes. Some peaks are only annotated for orientation. B: Enlarged section of the MS survey scan in A at a mass range of m/z = 594 — 600, showing the ENPP1 peptides. MS survey scan contains light masses from CTC-ITB-01 and the 13C6 labeled peptides of

MCF-7 at m/z = 595.33 [M + 3 H]3+ and m/z = 597.33 [M + 3 H]3+, respectively.

PAT 1825 LU

DETAILED DESCRIPTION OF THE INVENTION LUS01430 “Epithelial-to-mesenchymal transition” EMT is a normal process in embryonic development, organogenesis, gastrulation and development of the peripheral nervous system which causes epithelia-mesenchymal plasticity and enables these cells to migrate and form different tissues and organs. The process of EMT plays a relevant role in wound healing and tissue regeneration. Cancer cells from the primary tumor also take advantage of this program to start invasion and migration. As a consequence, metastases occur which are primarily responsible for cancer-related deaths. Motility and invasiveness are essential requirements for metastatic spread of malignant cells.

EMT describes a process by which tumor cells can acquire mesenchymal attributes thereby gain attributes like increased migratory capacity. Alternatively, tumors may originate from cancer stem cells. In breast cancer, cancer stem cells are malignant transformed (adult) breast stem cells. Cancer stem cells retain limitless replication potential and high migratory capacity rendering ideal candidates for the formation of distant metastasis. In addition, cancer stem cells are able to undergo uneven cell division. That means, after cell division one daughter cell is again a cancer stem cell and the other daughter cell can begin cellular differentiation.

During the differentiation, the descendants gradually increase epithelial attributes, with the upregulation of epithelial marker proteins like keratins. Cancer stem cells are tumor cells with strong manifestation of a mesenchymal phenotype and very low expression of epithelial proteins like EpCam or keratins. Interestingly, the protein expression profile of cancer stem cells and EMT passed cells is very similar (see below).

Before detaching from primary tumor, the number of cell-cell contacts of the tumor cells decrease by downregulation of proteins responsible for adherence which in turn results in the loss of the apical-basal polarity. After numerous genetic changes the tumor cells are released from the primary tumor, penetrate the basement membrane and intravasate actively or passively the blood circulation or the lymphatic vessels. The CTCs are exposed to physical stress like shear forces in the blood stream or collision with blood cells. Additionally, they must avoid anoikis, a process which normally leads to apoptosis by the lack of cell. ECM contacts and escape the immune system. After reaching a distant organ, tumor cells extravasate the blood circulation by binding to the endothelium after reaching small capillaries. After extravasation, DTCs die, remain in tumor dormancy or resume proliferation 11

PAT 1825 LU to found the outgrowth of macrometastasis. In breast cancer the outgrowth of metastasis can | LU501430 take several years after removal of the primary tumor. One reason for the late metastatic relapse is the dormancy state of DTCs with low proliferation rates resulting in weak therapy effects in this period followed by resuming signal-triggered proliferation. CTCs and DTCs with mesenchymal attributes or after passing through EMT have advantages in motility but not in proliferation. In general, after complete EMT, the expression of epithelial proteins like

EpCAM or E-Cadherin is downregulated due to the loss of cell-cell connections, cytoskeletal alterations and changes in keratin expression patterns followed by the final upregulation of mesenchymal proteins like Vimentin and N-Cadherin.

ENPPI is a type II transmembrane glycoprotein, hydrolyzing pyrophosphate or phosphodiester bonds and negatively regulating bone mineralization (see, for example, [11],

[12]; UniProtKB P22413-1; SEQ ID NO: 05). In case of single-pass type II transmembrane proteins, the C-terminus is on the extracellular side, whereas for type I transmembrane proteins like SUSD2 the N-terminus is on the extracellular side. Furthermore, ENPP1 inhibits insulin receptor activity and thus is associated with type 2 diabetes. An upregulation of

ENPP1 in breast cancer specimen, and a cancer stem cell-promoting effect, mediated by an induction of the side population fraction, has been observed. Furthermore, ENPP1 facilitates drug resistance by upregulation of the ABC transporter ABCG2 and provides a tumor seeding ability. Also in lung cancer and glioblastoma, ENNP1 was associated with stem cell properties and EMT phenotype.

ENPP1 has an extracellular domain, consisting of 828 amino acids, providing the possibility to isolate or to target CTCs by antibodies against the extracellular domain of ENPP1. No expression of ENPP1 was observed in PBMCs, allowing a specific isolation of CTCs from blood, without unspecifically targeting PBMCs.

Western blot analyses revealed that ENPP1 is predominantly expressed by breast cancer cell lines with mesenchymal characteristics (MDA-MB-231, MDA-MB-231 SA, MDA-MB-231

B02, BC-M1 and BT-549) and only weakly positive in few breast cancer cell lines with epithelial characteristics (MCF-7, KPL-1, T47D). Western blot results for MCF-7, MDA-MB- 231 and MDA-MB-468 are in accordance with previous described data obtained by mRNA and protein analysis. Hence, an isolation of CTCs using an antibody against ENPP1 might 12

PAT 1825 LU enrich CTCs that have undergone EMT, and which can be missed by currently applied CTC ~~ LU501430 enrichment technologies, like the Cell Search® system.

The majority of bone metastases in breast cancer are osteolytic, where the normal bone is destroyed by osteoclasts. ENPP1 hydrolyzes nucleotide triphosphates to nucleoside monophosphates and diphosphates (e.g., pyrophosphate). Pyrophosphate again inhibits bone mineralization by binding to hydroxyapatite, wherefore ENPP1 could play a role in osteolytic metastasis. This assumption is in accordance with the high expression of ENPP1 by breast cancer cell lines with ability to form bone metastasis in mice (MDA-MB-231, MDA-MB- 231-SA and MDA-MB-231-B02) and in BC-M1 which was established from the bone marrow of a breast cancer patient. Since MDA-MB-231 B02 and MDA-MB-231 SA have the predilection for dissemination to bone in mice compared to the parental MDA-MB-231 an upregulation of ENPP1 was expected in both sublines, if ENPP1 was essential for bone metastasis formation.

Molecular analyses of ENPP1-positive CTCs could provide deeper insights in the role of

ENPP1 in tumor cell dissemination. For the isolation or inhibition of CTCs using ENPP1 as target, an antibody against the extracellular domain of ENPP1 is required. Especially, due to the ENPP1 mediated upregulation of ABC transporter, targeting of ENPP1-expressing CTCs could eliminate tumor cells responsible for therapy failure. Furthermore, if the association of

ENPP1 with a stem cell-like population also applies in CTCs, ENPP1 could be used as biomarker to identify the disease driving subpopulation of CTCs.

Currently there are no publications describing the role of ENPP1 in prostate cancer. Western

Blot analyses of 4 prostate cancer cell lines revealed the expression of ENPP1 in LNCap, PC- 3 and PC-E1. PC-3 and PC-E1 are cell lines obtained from bone metastasis and which have a high metastatic potential.

SUSD2 is a type I transmembrane protein, comprising 822 amino acids with an extracellular domain of 758 amino acids (see, for example, [12]; UniprotKB Q9UGT4-1; SEQ ID NO: 01).

The protein consists of a Sushi, von Willebrand factor type D, AMOP and a Somatomedin B domain. Amino acids 1 to 27 represent the signal peptide and the protein is cleaved at the

GDPH sequence. Furthermore, the protein has several predicted N-linked glycosylation sites.

SUSD2 was chosen as a promising target for CTC isolation due to the extracellular domain 13

PAT 1825 LU and its absence in PBMCs, proven by Western blot analysis. SUSD2 was previously LUS01430 described as marker for isolation of mesenchymal stem cells in human palatine tonsil, bone marrow and endometrium. Furthermore, it was shown to play a role in neuronal development.

À tumor related role was first described for the mouse homolog of SUSD2, where a tumor- reversing effect was found. The role of human SUSD2 was investigated in cancer, showing a tumor-promoting impact in breast cancer and tumor inhibiting effects in renal cell carcinoma, lung cancer and high grade serous ovarian cancer. IHC analysis of matched sets of breast cancer and normal breast tissues revealed high SUSD2 expression in all stages of breast cancer, while in benign breast tissue expression was only observed in endothelial cells lining the blood vessels and capillaries. SUSD2 was associated with increased invasion through

Matrigel, suggesting a metastasis-promoting effect. An angiogenesis mediating impact of

SUSD2 was shown to be conveyed by recruitment of tumor associated macrophages via the chemoattractant MCP-1.

The theoretical mass of SUSD2, considering the complete amino acid sequence is 90.2 kDa.

Subtracting the mass of the signal peptide, a mass of 87.5 kDa is predicted for the mature

SUSD2 protein. However, the Western blots showed a dominant band at 55 kDa and another band with lower intensity between 110 — 130 kDa, depending on the cell line. Since at least four N-linked glycosylation sites are described for SUSD2 it was assumed that the band at 110 — 130 kDa is a glycosylated form of the full-length protein. The identity of the 55 kDa band was proven by LC-MS/MS analysis in this work, providing eight unique peptides of

SUSD2. Thus, the 55 kDa signal detected by Western blot analysis is most likely specific for

SUSD2, suggesting posttranslational cleavage or translation at an alternative initiation site.

One of the eight identified SUSD2 peptides found at 55 kDa starts with the amino acid methionine and is located at the N-terminal side of the SUSD2 peptide. As the genetic code for methionine, AUG, also serves as an initiation site for the mRNA translation, a shorter version of SUSD2 might have been generated due to the use of an alternative start codon.

However, if translation started at this methionine (amino acid 85) and ended at the stop codon

ATG, which is located behind the base code for proline 822, a mass of 81.4 kDa is predicted.

Thus, the 55 kDa form of SUSD2 is more likely a cleavage product generated after translation of the complete protein. Nevertheless, translation at an alternative initiation site might still have happened yielding in a signal detected between 110 — 130 kDa after glycosylation. 14

PAT 1825 LU

One of many hurdles for a CTC to survive in the bloodstream is the immune system. Several | LU501430 mechanisms are described for tumor cells to evade the immune system, including abnormal expression of major histocompatibility complex class I proteins, loss and modification of tumor associated antigens or blocking the antitumor function of T-cells. The latter can be mediated by galectin-1, which induces apoptosis in T-cells. An interaction of SUSD2 and galectin-1 was described, where SUSD?2 facilitates the cell surface presentation of galectin-1.

CTC-ITB-01 is strongly SUSD2-positive and might possess immune system evading mechanisms as this cell line originates from CTCs that survived in the bloodstream of a breast cancer patient. However, this attribute was most likely not mediated by galectin-1 since only a very weak galectin-1 expression was observed in CTC-ITB-01.

The cancer stem cell theory includes the hypothesis that cancer stem cells might originate from normal stem cells. The presence of mammary stem cells is supported by the changes occurring in the mammary gland after pregnancy and lactation and in accordance with the cancer stem cell theory, transformation of the mammary stem cells could initiate breast cancer. Since SUSD2 is associated with mesenchymal stem cell-like cells, this protein might further support the stem cell-like properties of CTC-ITB-01 and suggests that the cell line might originated from a mammary stem cell. Prove of this theory requires evidence for

SUSD2 expression on mammary stem cells.

The strikingly high expression of SUSD2 in CTC-ITB-01 and the promising findings in CTC-

ITB-01 MIND, indicated that SUSD2 might be a suitable target for CTC enrichment.

Furthermore, the presence of SUSD2 in cell lines of different phenotypes could enable to target a broad range of different CTC subtypes (epithelial, hybrid epithelial/mesenchymal and mesenchymal). As mentioned above, SUSD2 was utilized as marker for the isolation of mesenchymal stem cell-like cells using FACS or MACS®. To enrich SUSD2-expressing cells by MACS®), these studies applied an indirect system by using PE-conjugated antibodies against SUSD2 and anti-PE MicroBeads.

Referring to SUSD2, the present inventors showed that separation of SUSD2-positive CTCs can be achieved using the MACS® system together with SUSD2 MicroBeads. Customized human SUSD2 MicroBeads were produced by Miltenyi Biotec, where the anti-SUSD2 antibody is directly coupled to the superparamagnetic beads. The system was first tested by spiking experiments using the highly SUSD2-positive CTC-ITB-01 and the SUSD2-negative

PAT 1825 LU

T47D cell lines. Suitability of both cell lines as positive and negative control was proven by | LU501430

FACS analysis, to ensure that the obtained recovery rates are not influenced by a missing or present expression of SUSD2 on the positive or negative control, respectively. FACS analysis showed that 100 % of adherent and 98 % of non-adherent CTC-ITB-01 cells were positive for

SUSD2, while only 0.98 % of T47D cells were SUSD2-positive, proving the suitability of these controls. The MACS® spiking experiments yielded in recovery rates of 70.67 % and 3.33 % for CTC-ITB-01 and T47D, respectively, while 4.67 % of CTC-ITB-01 and 64.00 %

T47D cells were detected in the combined flow-through and wash fraction. These results indicated a specific separation of SUSD2-positive cells with the SUSD2 MicroBeads.

CTC-ITB-01 cells found in the elution fraction showed a strong circular mouse IgG staining in IF analysis, proving the binding of the SUSD2 MicroBeads to the SUSD2-expressing CTC-

ITB-01 cells. Hence, the specific binding of SUSD2 MicroBeads to SUSD2-positive cells was proven. IF analysis of CTC-ITB-01 cells in the combined flow-through and wash fraction showed at most a very weak staining for mouse IgG, suggesting that those cells were not bound by the SUSD2 MicroBeads or in much lower extent compared to those cells detected in the elution fraction. Even though probably all CTC-ITB-01 cells express SUSD2, some cells present lower amount of SUSD2 proteins on their surface, as heterogeneous SUSD2 expression was observed during IF analysis of CTC-ITB-01. The CTC-ITB-01 cells with lower SUSD2 expression provide fewer binding sites for the SUSD2 MicroBeads and were probably therefore not retained in the magnetic field. Furthermore, the accessibility of CTC-

ITB-01 cells to the SUSD2 MicroBeads might have been lowered, if cells were surrounded by many PBMCs.

Subjecting blood from patients with breast cancer to the MACS® system SUSD2-positive

CTCs were enriched in the elution fraction for 3 patients, but also SUSD2-negative CTCs were observed in the same fraction. Furthermore, some SUSD2-positive CTCs were observed in the combined flow-through and wash fraction for one patient with very high CTC count.

This could be due to the fact that the system was optimized for a low number of 50 tumor cells during spiking experiments, as the CTC frequency is usually low (one tumor cell in a background of 10° — 107 blood cells). But this patient had an uncommonly high number of

CTCs, which likely requires a different protocol with higher amount of SUSD2 MicroBeads to label all SUSD2-positive cells with the MicroBeads. This could decrease the appearance of

SUSD2-positive cells in the combined flow-through and wash fraction. Due to the very high 16

PAT 1825 LU number of CTCs, SUSD2-negative cells appeared as background in the elution fraction, LUS01430 which is in accordance with the observation that unlabeled leukocytes are also found in the elution fraction. Due to the high number, leukocytes are not completely removed during the washing step and leave the column during the elution step, appearing together with the labeled cells. Likely, a more intense washing step would have yielded in lower number of SUSD2- negative cells in the elution fraction.

To provide further evidence for SUSD2 expression on CTCs, the CellSearch® system was used. Therefore, the anti-SUSD2-Vio Bright FITC antibody was tested in the Cell Search® system by spiking experiments using CTC-ITB-01 and MDA-MB-468 as positive controls and T47D as negative control. Since results were in accordance with previous obtained data by Western blot and IF analysis, the antibody was proven to be suitable for SUSD2 detection in CellSearch® enriched tumor cells. Of 23 CTC-positive patients, 9 patients harbored at least one SUSD2-positive CTC. These 9 patients showed a heterogenous distribution in the amount of SUSD2-positive CTCs ranging from 0.39 % to 60 %. The majority of the SUSD2-positive

CTCs showed low SUSD2 expression. Two blood samples each were analyzed for four patients. Two of those patients showed SUSD2-positive CTCs only in one blood sample. This might be due to a low specificity of the anti-SUSD2 Vio Bright FITC antibody or the low frequency of SUSD2-positive CTCs in those patients. However, the suitability of the anti-

SUSD2-Vio Bright FITC antibody in the CellSearch® system was proven in spiking experiments and for the other two patients SUSD2-positive CTCs were detected in both blood samples, further indicating a sufficient specificity of the anti-SUSD2 Vio Bright FITC antibody.

Evidence for expression of SUSD2 and ENPPI on patient CTCs was provided by CellSearch analyses and immunofluorescence analyses, respectively (Fig. 3-4, 10). Fig. 3 shows detection of ENPPI on CTCs of patients with breast cancer. Cells were stained with anti-ENPPI antibody on a glass slide. Furthermore, pan-keratin was used as tumor cell marker, CD45 as leukocyte marker and nuclei were visualized using DAPI. A: ENPPI-positive CTC of patient 1. B: ENPPI-negative CTC of patient 1. C: ENPPI - positive CTC of patient 2. D: ENPPI- negative CTC of patient 2. Images were taken at 400 x magnification. Fig. 4 shows detection of SUSD2 expression on CTCs. Representative pictures of CTCs enriched from blood of patients with metastatic breast cancer using the CellSearch ® system are shown. CTCs were identified with the CellSearch® CTC Kit as keratin+ /DAPI+ /CD45- cells. SUSD2 17

PAT 1825 LU expression was investigated with the anti-SUSD2 Vio Bright-FITC antibody. A: SUSD2- LU501430 negative CTC. B and C: CTCs with low SUSD2 expression. D and E: CTCs with moderate

SUSD2 expression. F: CTC with strong SUSD2 expression.

Therefore, these proteins can be used as biomarkers for CTC detection and characterization.

Since SUSD2 and ENPPI are transmembrane proteins, enrichment of CTCs targeting these proteins is possible. Therefore, SUSD2 was targeted for CTC enrichment in this invention.

Since SUSD2 and ENPPI are transmembrane proteins with large extracellular domain, these extracellular regions are directly accessible for anti-SUSD2 and anti-ENPPI antibodies without further manipulation of the cells (e. g. permeabilization). This allows binding of anti-

SUSD2 and anti-ENPPI antibodies to the extracellular domains, in which the antibodies are coupled to magnetic beads (MACS, see below). By this approach, it is possible to isolate viable CTC (or DTC) from the blood of cancer patients. Unlike other approaches, MACS can be up scaled to larger volumes allowing the isolation of CTC from larger blood volumes.

This increases the possibility to isolate tumor cells from the sample so that the number of

CTC positive blood samples of breast cancer patients can be increased.

In addition, the CTC isolation by MACS is a very mild method, which allows the subsequent analysis ofthe isolated intact cells by other methods like DNA mutation analysis. Alternatively, since the cells isolated by MACS are still viable, these cells can be cultured allowing further analyses of their genotypic and phenotypic characteristics.

In Fig. 5 and 6, the structures of the transmembrane proteins SUSD2 or ENPPI (both denoted with reference numeral 1) are schematically depicted, respectively. SUSD2 is anchored in the cell membrane 60 via a transmembrane domain 12 (amino acids 786-806). The N-terminal 15 is located extracellularly, the C-terminal 14 is located at the end of the intracellular (cytoplasmic) domain 13. The extracellular domain 11 of SUSD2 has a GDPH cleavage site 112, such that the extracellular domain 11 can be split into a first truncated membrane-bound extracellular domain fragment 11 and a second fragment 111 which is bound to the first fragment 110 via disulfide bridge (see Fig. 5b). The extracellular domain 11 can, for example, be a target for MACS. Both the extracellular domain 11 and the cytoplasmic domain 13 can be a target for immunofluorescence detection, for example. ENPP1 is also anchored in the cell membrane 60 via a transmembrane domain 12 (amino acids 77-97). In contrast to SUSD2, the 18

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C-terminal 14 of ENPP1 is located extracellularly, whereas the N-terminal 15 is located at the LU501430 end of the intracellular (cytoplasmic) domain 13. The extracellular domain 11 has potentially two cleavage sites 112. ENPP1 is likely cleaved intracellularly such that a soluble fragment 111 can be secreted (see Fig. 6b). The extracellular domain 11 can, for example, be a target for MACS. Both the extracellular domain 11 and the cytoplasmic domain 13 can be a target for immunofluorescence detection, for example. The soluble protein fragment 111 can be a target for an ELISA.

As one example, the method of the invention can be carried out using magnetic-activated cell sorting (MACS®), using magnetic-activated anti-SUSD2 or anti-ENPPI antibodies, i.e., anti-

SUSD2 or anti-ENPPI antibodies coupled to magnetic nanoparticles, as described below in more detail in relation to Figs. 7, 8. However, isolation of CTC and/or DTC can be achieved via an anti-SUSD2 or anti-ENPPI antibody coupled to any suitable substrate or functionalized on any surface.

Fig. 7 shows an example of an embodiment of the method of the invention using magnetic activated cell sorting (MACS®) for the isolation of SUSD2” or ENPPT cells, i.e, SUSD2- positive or ENPPI-positive cells. SUSD2" or ENPPT cells are here represented by SUSD2 or ENPPI| 1 anchored in the cell-membrane 60. In a first step, a CTC or DTC, carrying

SUSD2 or ENPPI on its cell-surface, is captured with a first primary antibody 2 coupled to the magnetic nanoparticle 50 which is directed to the extracellular domain 11 of SUSD2 or

ENPPI 1. The first primary antibody 2 is preferably a polyclonal antibody, particularly preferred a (polyclonal) antibody binding to the part of the extracellular domain 11 of SUSD2 or ENPPI that is not cleaved off, i.e., binding also to the truncated version of SUSD2 or

ENPPI. SUSD2" or ENPPI CTC/DTCs can be isolated via MACS®, as described in relation to Fig. 8. It should, however, be noted, that the isolation can be performed using a modified

CellSearch system (with SUSD2 or ENPPI instead of EpCAM as a marker) or other isolation procedures, e.g., via anti- SUSD2 or anti-ENPPI antibodies bound to a solid phase other than a magnetic nanoparticle 50. For the subsequent detection of CTC/DTCs, two approaches are shown. In a first approach (see Fig. 7a) a fluorescently labeled first secondary antibody 3 directed against the first primary anti-SUSD2 antibody or anti-ENPPI antibody 2, or against the complex composed of the first primary anti-SUSD2 antibody or anti-ENPP1 antibody 2, the magnetic nanoparticle 50 and the components coupling the magnetic nanoparticle 50 to the first primary anti-SUSD2 antibody or anti-ENPP1 body 2, (e.g. biotin and streptavidin), 19

PAT 1825 LU is added in order to detect SUSD2-positive or ENPPI-positive CTC/DTCs. In another LUS01430 approach (see Fig. 7b), a second primary anti-SUSD2 antibody or anti-ENPP1 body 4, directed against a preferably cytoplasmic terminal region of the transmembrane protein 1, i.e. a preferably C-terminal region in case of SUSD2 and a preferably N-terminal region in case of ENPP1, of the intracellular (cytoplasmic) domain 13 of SUSD2 or ENPPI 1, is added and bound to SUSD2 or ENPPI 1, and a fluorescently labeled second secondary antibody 5 directed against the second primary anti-SUSD2 antibody or anti-ENPP1 body 4 is added in order to detect SUSD2-positive or ENPPl-positive CTC/DTCs.

The method of the invention can be adapted to various geometries and topologies. First, the coupling of an anti-SUSD2 antibody or anti-ENPP1 body is not limited to coupling to magnetic nanoparticles, as described above in relation to Fig. 6. Rather, an anti-SUSD2 antibody or anti-ENPP1 body can be coupled by different methods to any suitable material, e.g. any solid phase material used in the prior art for immobilizing antibodies. Such coupling methods may be for example the application of reactive cosslinkers like NHS esters (N- hydroxysuccinimide esters) and imidoesters, which allow the coupling of anti-SUSD2 bodies or anti-ENPP1 bodies to a suitable material or surface. Another option is the covalent conjugation of the anti-SUSD2 antibody or anti-ENPP1 body to fluorescent (non-magnetic or magnetic) nanoparticles. This approach could be used for detection (fluorescent nanoparticles) or isolation/enrichment (magnetic nanoparticles). Coupling of an anti-SUSD2 antibody or anti-ENPP1 body to a fluorescent magnetic nanoparticle is advantageous in that this direct approach would work without a secondary antibody reaction. As already mentioned, different materials/surfaces instead of magnetic nanoparticles to which the anti-SUSD2 antibody or anti-ENPP1 body is coupled can be applied. Such material may be for example Sepharose® (a crossiinked, beaded-form of agarose, or nanoparticles (fluorescence labeled or unlabeled), but other materials or surfaces may also be applied. For example, the cell collector (see US patent US10022109B2) can be adjusted to the capture of SUSD1- or ENPP1-positive CTCs or

DTCs by functionalizing the device with anti-SUSD2 antibody or anti-ENPP1 body. Another example is the detection/isolation by a modified commercial system called “CellSearch”, which is an immunomagnetic technology that uses anti-EpCAM antibodies coupled to ferrofluid nanoparticles for separation of EpCAM-expressing cells. Supplement or substitution of anti-EpCAM antibodies with anti-SUSD2 antibody or anti-ENPP1 body coupled to ferrofluid nanoparticles in the CellSearch approach enriches SUSD2-positive or

ENPPI-positive CTC or improves the number of enriched CTC, respectively.

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Currently, immunomagnetic methods (MACS) are widely-used to isolate tumor cells. For example, the immunomagnetic isolation via the extracellular domain of EpCAM is used to capture EpCAM-positive CTCs and DTCs in metastatic breast cancer. This approach enables the isolation of CTCs or DTCs with an epithelial phenotype. The disadvantage of this approach is the loss of EpCAM-negative CTCs and DTCs. EpCAM-negative tumor cells might have undergone a complete EMT which leads to a mesenchymal phenotype or a partial

EMT which leads to a hybrid epithelial/mesenchymal phenotype. Mesenchymal CTCs are thought to be more invasive than the epithelial counterparts but the hybrid epithelial/mesenchymal phenotype is linked with metastasis formation and metastatic relapse.

These findings indicate that there is need for a cell surface molecule which can be used for isolating CTCs or DTCs, for example a MACS-based method for the isolation of CTCs with mesenchymal or hybrid epithelial/mesenchymal attributes. The inventors have found that the large extracellular domain of SUSD2 and ENPPI is applicable for, for example, MACS- based isolation of CTCs/DTCs with different phenotype (epithelial, mesenchymal or hybrid epithelial/mesenchymal). Due to the fact that different biological variants of SUSD2 and

ENPPI correlate with dissemination and invasion of tumor cells, SUSD2-positive and

ENPPI-positive tumor cells might provide information about metastasis formation-potential and metastasis development.

For the labeling procedure an indirect and a direct variant can be distinguished. The antigen of interest on the cell surface can be hybridized with an antibody or a biotinylated antibody followed by the binding of the super-paramagnetic nanoparticles (MACS-nanobeads, size range: 20-100 nm) which are conjugated to a secondary antibody (recognizing the primary antibody) or to Streptavidin (recognizing the biotinylated antibody). This approach represents the indirect technique. The faster direct approach is represented by the direct binding of

MACS bead-conjugated antibodies to the cell surface antigen of interest. For the isolation procedure a positive and a negative selection can be differentiated. The positive selection enriches the cells harboring the antigen of interest on their cell surface and the cells lacking the cell surface antigen are discarded. For this purpose, the antigen-positive cells are captured with an antibody or other binding agent coupled to a magnetic nanoparticle and retained by a magnetic field. On the other hand, the negative selection isolates all cells lacking the cell surface antigen of interest by capturing them with a binding agent, e.g., antibody, linked to a magnetic nanoparticle, while the cells expressing the cell surface antigen are not captured and 21

PAT 1825 LU released from, for example, a MACS column. After successful labeling with magnetic LU501430 nanoparticles the cells are applied to a ferromagnetic iron-column positioned in a magnetic field. In Fig. 8, the procedure is schematically shown for the positive selection of cells by a cell surface molecule-dependent MACS isolation procedure. Cells 80 are labeled with a biotinylated antibody 2 coupled via streptavidin to a magnetic nanoparticle 50. The antibody 2 is directed against a membrane-bound antigen, e.g. a membrane-bound protein having an extracellular domain, like SUSD2 or ENPPI. Nanoparticle-labeled cells 80 retain in the column 70 by the application of a magnetic field of a magnet 71 and unlabeled or antigen- negative cells 81 pass the column 70 and will be discarded, or collected in a collecting vessel 90. Thereafter (see Fig. 8b), the magnetic column 70 is removed from the magnet 71, the labeled cells 80 are eluted and collected in a collecting vessel 90, and further analysis can be performed. The complex composed of the biotinylated antibody 2 and the Streptavidin- conjugated magnetic nanoparticle 50 recognizes and binds to the target cell 80. The cell- antibody-nanoparticle complex binds to the MACS column 70 placed into a permanent magnet 71. The unlabeled cells 81 or cells negative for the surface protein are discarded after passing the column 70 without binding. After removal of the column 70 from the magnet 71 the labeled cells 80 are collected and represent an enriched population.

In principle, it is possible to carry out the isolation of CTCs or DTCs with only the anti-

SUSD2 antibody or anti-ENPP1 body coupled to the nanoparticles (or similar suitable substrates). The identity of the isolated cells can be confirmed, for example, by immunofluorescence by applying a second anti-SUSD2 antibody or anti-ENPP1 body and a fluorescently labeled secondary antibody directed against the second anti-SUSD2 antibody or anti-ENPP1 body, or a fluorescently labeled secondary antibody directed against the first

SUSD2-antibody or ENPPl-antibody (anti-catching antibody). Additionally, an anti-keratin antibody may be used in combination with the second anti-SUSD2 antibody or anti-ENPP1 body or the anti-catching antibody. Alternatively, the cells can be isolated and transferred to another vessel for subsequent procedures. Such procedures may include a cell lysis step followed by whole genome amplification followed by DNA sequencing of the genome to identify tumor-relevant gene mutations. Such gene mutations might be responsible for therapy resistance if a gene of a therapeutic target is mutated (e. g. EGFR). An alternative evolving approach is the analysis of the isolated cells on proteome level, for example by single cell mass spectrometry. In that case cancer relevant target structures (most therapy targets are proteins) can be directly assessed, which helps the prediction of therapy success of the 22

PAT 1825 LU patients. Depending on the isolation procedure, isolated cells are viable and thus can be used LV501430 for subsequent functional in vitro (e.g., migration and invasion assay) and in vivo (e.g., patient-derived xenograft in zebrafish embryos) assays possibly after expansion in long-term cultures.

As an alternative to magnetic beads (nanoparticles), the anti-SUSD2 antibody or anti-ENPP1 antibody can be coupled to an organic matrix such as Sepharose®, and this can be added to the sample. Sepharose forms a kind of slurry. When you have the complex CTC-anti-SUSD2 antibody-Sepharose or CTC-anti-ENPP1 body-Sepharose in the sample, you can simply let the complex settle down (no centrifugation step necessary) and discard the supernatant. If you want to remove normal cells that got stuck in the slurry, simply dissolve the slurry in a suitable buffer and let it settle down again and discard the supernatant. Accordingly, the invention can be carried out with an organic matrix (e. g. Sepharose) having anti-SUSD2 antibody or anti-ENPPI antibody coupled via reactive functional groups, wherein the separation involves gravitation force, for example by settlement of the CTC-anti-SUSD2 antibody-Sepharose or CTC-anti-ENPP1 body-Sepharose complex or centrifugation.

Further, the distribution of SUSD2 and ENPP1 was investigated in other mammals by database search (Fig. 11, Fig 12). After identification of the corresponding proteins in dog, horse and pig by sequence homology search, we performed a sequence homology analysis.

SUSD2 and ENPP1 are present in these animals with a sequence homology of more than 75%.

The present invention thus uses the Sushi-domain-containing protein 2 (SUSD2) and

Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPPI) as a biomarker for the isolation, preferably the isolation and detection of CTCs or DTCs having an epithelial phenotype (eCTC, eDTC), a mesenchymal phenotype (mCTC, mDTC) or a hybrid epithelial/mesenchymal phenotype (emCTC, emDTC), preferably a mesenchymal or hybrid epithelial/mesenchymal phenotype. It has been found that these biomarkers are, for example, strongly expressed in CTCs from breast cancer. The invention can, however, also be used in the context of other cancers with a high potential to form metastases, e.g., prostate cancer, pancreatic cancer, head and neck cancer, lung cancer or malignant mesothelioma.

The term “isolation” in relation to targeted cells like CTCs or DTCs refers to the separation of these cells from a biological sample containing the cells, for example, from peripheral blood, 23

PAT 1825 LU lymph, bone marrow or the like. The term includes the separation of these cells from other, LUS01430 not targeted cells, e.g., the separation of SUSD2-positive and ENPPI-positive CTCs or DTCs from the biological sample medium and other cells in the same sample, e.g., SUSD2- negative or ENPPl-negative CTCs or DTCs. The term encompasses enrichment of the targeted cells, i.e., increasing the concentration of the targeted cells by removal of fractions not containing targeted cells, or removal of targeted cells to a collection medium, or by accumulating targeted cells in a part or compartment of a container or in an area of an object slide.

The term “detection” in relation to targeted cells like CTCs or DTCs refers to identification of the targeted cells in a biological sample, in a medium enriched with the targeted cells or in a medium at least mainly, preferably exclusively containing the targeted cells. Identification means suitably labeling the cells and qualitatively or quantitatively detecting the presence of the cells.

The term “subject” as used herein refers to a non-human animal, preferably a non-human mammal, for example a cat, dog, horse, cattle, or to a human.

The term “nanoparticle” as used herein relates to particles of a size of 1 to <100 nm. In relation to a plurality of nanoparticles the term means that the particle size of at least half of the particles in the number size distribution of the plurality of nanoparticles is 100 nm or below.

Materials and Methods

To avoid an unwanted isolation of non-tumor cells during CTC isolation by targeting a cell surface protein, an absence of this protein on PBMCs is necessary. Database search declared no expression of SUSD2 or ENPP1 by PBMCs. To approve that the selected proteins are suitable biomarkers for CTC isolation, this information was verified by Western blot analysis using PBMC lysates.

SUSD2: SUSD2 was identified in five replicates by 27 unique peptides, including peptide

CGALDGPCSCHPTCSGLGTCCLDEFR (SEQ ID NO: 09; Fig. 13) in a SILAC-based proteome analysis comparing the CTC-derived cell line CTC-ITB-01 and MCF-7. The 24

PAT 1825 LU monoisotopic peak of the peptide CGALDGPCSCHPTCSGLGTCCLDFR (theoretical LU501430 monoisotopic mass = 2,857.17 Da; SEQ ID NO: 09;) was observed in the MS survey scan at m/z = 953.38 [M + 3 HJ? * (Fig. 13). Due to the large sequence of this peptide, a high number of isotopic peaks is present. Signals present at m/z= 955.38 and 955.71 [M + 3 HP" might result from a corresponding heavy peptide ion, but the signal intensities suggest that these signals are rather isotopic peaks of the light peptide ion. Additionally, data of all analyzed peptides identified for SUSD2 obtained by MaxQuant software indicated no expression of SUSD2 in

MCF-7, further suggesting that signals at m/z = 955.38 and 955.71 [M + 3 H]** are isotopic peaks from the light peptide ion.

ENPP1: ENPP1 was identified in five replicates by 14 unique peptides in a SILAC-based proteome analysis comparing the CTC-derived cell line CTC-ITB-01 and MCF-7 (Table 1).

ENPP1 was expressed by both cell lines, proven by the simultan presence of light and heavy peptide ion signals in the MS survey scans, e.g. observed for the ENPP1 peptide

AEYLHTWGGLLPVISK (SEQ ID NO: 10; Fig. 14). For this peptide, the light peptide ion signal was observed at m/z = 595.33 [M + 3 HJ’, while the heavy peptide ion signal was found at m/z = 597.33 [M + 3 H]** caused by the incorporation of *C¢ lysine. However, the light peptide shows higher signal intensity than the heavy peptide, indicating a higher expression of ENPP1 in CTC-ITB-01 compared to MCF-7.

Table 1: LC-MS/MS results of the CTC biomarker candidates Determination of differential expression of the CTC biomarker candidates SUSD2 and ENNP1, between MCF- 7 and CT CT BOL eos eee : Uniprot Protein Total Number Number | Averageof Standard = p-value® accession short number ofunique of bio- | normalized deviation number name of peptides logical ‘ratio (H/L) : peptides analyzed” repli- : ten AVEO ES rome :Q9UGT4 SUSD2 150 27 5 : onlyinCTC- - ;- : P22413 : ENPP1 | 39 : 14 5 | - 2.93 : 0.052 : 4,697 x 11036 “Total number of all analyzed peptides identified for one protein (unique and not unique).

Same peptides were counted multiple times (once per each fraction and replicate), b Number of peptides exclusively associated with one protein. Number contains only different peptides; © Student's #-test, value of p< 0.05 was considered as significant difference

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CTCs can be enriched in different ways, e.g. by density gradient centrifugation, magnetic cell LU501430 separation or with the CellSearch system. Following enrichment, CTCs can be detected and characterized by immunocytochemistry. Proteins on the cell surface of CTCs can be used for both enrichment and detection. Furthermore, cell surface proteins represent potential targets for therapeutic antibodies. Thus, it was aimed to prove the presence of SUSD2 and ENPP1 on

CTCs, wherefore immunofluorescence (IF) staining protocols were established. Therefore, cytospins were prepared, using healthy donor blood spiked with cancer cell line cells, to mimic a patient sample containing CTCs in the blood.

For the transmembrane proteins ENPP1 and SUSD2 an immunofluorescence staining protocol was established and successfully applied to patient slides. Thus, the IF staining protocol couldbe used in the future for the detection of CTCs in patient samples. Furthermore, a system for magnetic cell separation (MACS®) of SUSD2-positive cells was established, which can potentially be used for the isolation of SUSD2-positive CTCs from the blood of breast cancer patients. For this, evidence for a SUSD2 expression in CTCs of patients was required. Therefore, the CellSearch® system was used to enrich CTCs from the blood of patients with metastatic breast cancer. Subsequently, the enriched CTCs were analyzed for

SUSD2 expression by immunofluorescence staining. SUSD2-expressing CTCs were detected in 9 of 23 CTC-positive patients.

The invention uses a column-based method, which allows magnetic separation of cells based on surface antigens (see Fig. 8). Here, SUSD2 MicroBeads (Miltenyi Biotec, Bergisch

Gladbach, Germany) were used, which are 50-nm superparamagnetic particles that are conjugated to an antibody against SUSD2. This allows the enrichment of SUSD2-positive cells such as CTCs from a biological sample such as blood.

For spiking experiments, PBMCs from healthy donor blood were isolated and 50 CTC-ITB- 01 or 50 T47D (Cell line service, Eppelheim, Germany) cells were added to the PBMCs, as positive or negative control, respectively. The cell pellet was resuspended in 70 ul of

MACS® buffer and 10 ul of FcR Blocking reagent (both Miltenyi Biotec, Bergisch Gladbach,

Germany). The sample was then incubated with 20 pl of SUSD2-Micro Beads for 30 minutes at 4°C in the dark. In the meantime, the LS column, containing ferromagnetic spheres, was placed in the VarioMACS™ Seperator (Miltenyi Biotec, Bergisch Gladbach, Germany), which is a permanent magnet that causes a high-gradient magnetic field within the column. 26

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The column was conditioned with 3 ml of MACS® buffer (Miltenyi Biotec, Bergisch LUS01430

Gladbach, Germany). The sample was diluted with 1 ml of MACS® buffer and then applied onto the column. The following washing step was performed for three times with 3 ml of

MACS® buffer. For elution of labeled cells, the column was removed from the separator and cells were eluted with 3 ml of MACS® buffer and another 2 ml of MACS® buffer using the plunger to flush out all cells. One cytospin was generated from the combined flow-through and first washing fraction and another cytospin was obtained from the elution fraction. Patient samples were subjected to the same steps after PBMCs and tumor cells of patient blood samples were isolated with Ficoll Paque (GE Healthcare, Chalfont St Giles, United

Kingdom). Volumes given above are for up to 107 total cells. When working with more than 107 cells, volumes were adjusted accordingly.

Recovery and detection rates were determined for the elution fraction and the combined flow- through and wash fraction, respectively (Table 2).

Table 2: Recovery and detection rates obtained with MACS® using SUSD2 MicroBeads and

CTC-ITB-01 or T47D cells in spiking experiments (n=3)

Cell line Recovery rate in elution fraction Detection rate in flow-through

Tr em en

Cytospins, which were obtained after conduction of the MACS® spiking experiment using

SUSD2 MicroBeads, were subjected to IF analysis (Fig. 9). As the SUSD2 MicroBeads are detectable with anti-mouse IgG antibody, the cytospins were stained with anti-mouse IgG to detect the SUSD2 MicroBeads bound to the cell surface. For the elution fraction of CTC-ITB- 01 a circular staining was detected using an anti-mouse IgG antibody, indicating proper binding of the anti-SUSD2 MicroBeads to SUSD2 on the tumor cells. It was observed that very few CTC-ITB-01 cells also appear in the flow-through and wash fraction. However, these cells showed almost negative staining for mouse IgG, indicating no or imperfect binding of the MicroBeads to those cells. No signal was detected for T47D cells using anti-mouse IgG antibody, suggesting a high specificity of the SUSD2 MicroBeads. Nevertheless, very few

T47D cells were found to appear in the elution fraction, detected with pan-keratin (Table 2). 27

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Blood samples of advanced stage breast cancer patients were subjected to the invention (SUSD2-MACS technology). Fig. 10 shows IF analysis of breast cancer patient CTCs obtained by MACS® using SUSD2 MicroBeads (elution fraction). Cytospin of the elution fraction was stained with anti-mouse IgG to detect CTCs bound by SUSD2 MicroBeads.

Furthermore, pan-keratin was applied as tumor cell marker, CD45 as leukocyte marker and nuclei were visualized with DAPI. A: CTC cluster with SUSD2-positive CTCs. B: CTCs with positive and negative mouse- IgG staining. C: Zoom of CTCs with positive mouse IgG staining denoted by arrow C in image B. D: Zoom of CTCs with negative mouse IgG staining denoted by arrow D in image B. Images of the CTC cluster were taken at 400 x magnification and images in B at 200 x magnification. White scale bars represent 10 um and blue scale bars correspond to 30 um. In two patient samples enrichment of SUSD2-positive CTCs was achieved. Some CTCs were found to show a positive, circular staining for mouse-IgG, while others were negative (Fig. 10).

Accordingly, the above experimental data show that the inventors have successfully isolated and detected CTCs from the blood of a breast cancer patient with SUSD2 as biomarker using

MACS. The same approach is also applicable to ENPP1. The procedure has been repeated to confirm detection of CTCs by approach 1 which shows that the isolated CTC were coupled to the magnetic nanoparticles confirming that these cells were specifically isolated by the magnetic nanoparticles and not due to unspecific carry-over from the blood sample. With approach 2, the presence of SUSD2 or ENPPI can be confirmed by two different anti-SUSD2 or anti-ENPPI specific antibodies. For CTC catching by MACS a first primary anti-SUSD2 antibody or anti-ENPPI antibody 2 directed against the extracellular domain 11 has to be used and for SUSD2 or ENPPI detection by immunofluorescence a second primary anti-SUSD2 or anti-ENPPI specific antibody (4) directed against the cytoplasmic C-terminal tail of SUSD2 or

ENPPI has to be used.

Detection of keratin positive CTC confirms that the method of the invention is suitable for the specific isolation of CTC by SUSD2 or ENPPI. In addition, the method of the invention is suitable for the isolation of CTCs that are negative for keratin, thus CTC with mesenchymal attributes. It is reasonable that keratin negative cells that were positive for SUSD2 or ENPP1 are CTC with mesenchymal attributes. The invention provides a tool for the detection, isolation and molecular analysis of CTC with different characteristics, including 28

PAT 1825 LU mesenchymal attributes, which are considered as a probable candidate for the actual LU501430 metastasis founding cells in breast cancer.

After confirmation of SUSD2 and ENPPI in CTC of breast cancer patients, the inventors investigated DTC cell lines that were generated from the bone marrow of breast cancer patients. High levels of ENPPI protein was detected in all analyzed DTC cell lines from breast, lung and prostate cancer patients. Notably, these cell lines showed no detectable signals for the epithelial marker proteins of the keratin family and EpCam. EpCam is most commonly used for the isolation of tumor cells from liquid samples by magnetic beads.

Therefore, the analyzed DTC populations would remain undetectable by an anti-EpCam based

MACS approach. Since DTC cell lines were positive for ENPP1, the invention could be used to enrich and isolate ENPPI-positive DTC from bone marrow samples. SUSD2 was detected in cell lines of different phenotype and could therefore be used for enrichment, isolation and detection of usually undetectable CTC/DTC but also of subpopulations that have an epithelial phenotype.

CTC biomarkers candidates as targets for therapeutic antibodies

Monoclonal antibodies are a rapidly growing class of drugs and the most common targets for therapeutic antibodies are cancers and autoimmune diseases. Antibodies can modulate signaling pathways, which promote tumor growth, survival and metastasis. Furthermore, antibodies bound to tumor cells can potentiate the host immune response against tumor cells by mediation of antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity or the induction of T-cell immunity. An ideal target of therapeutic monoclonal antibody in cancer should be a cell surface protein or a secreted protein mediating tumour-promoting effects. The CTC biomarker candidates identified during this work could be used as targets for therapeutic antibodies, since SUSD2 and ENPP1 are cell surface proteins. For cell surface proteins high and stable expression in tumor cells and a low or absent expression in normal tissues is desirable for targets of therapeutic antibodies, as this could decrease unwanted effects on normal cells and increase the concentration of unbound antibodies to affect the tumor cells. Furthermore, the pharmacological response is determined by the concentration of the antibody at the target. Despite an expression of the target by normal cells, a tumor-specific effect might be achieved if the bioavailability of the antibody is higher in the tumor tissue than in normal tissue. Antibodies are large proteins of approximately 150 kDa and might not reach the antigen in normal tissues due to organ-specific barriers but in the tumor tissue 29

PAT 1825 LU through leaky vessels. Therapeutic antibodies are commonly intravenously administered and | LU501430 might be primarily distributed in the blood, wherefore the target should not be expressed by normal blood cells.

SUSD2 is expressed by mesenchymal stem cells of the bone marrow, which might lead to unwanted side effects of therapeutic antibodies targeting SUSD2, as the bone marrow sinusoid capillaries are more permissive due to the fenestrated structure. Furthermore, SUSD2 is expressed in the essential organs like the lung or the kidney. Nevertheless, patients could be screened for SUSD2-positive CTCs with the CellSearch® system prior to application of such an antibody. In patients with high numbers of SUSD2-positive CTCs the antibody would likely bind to SUSD2 on CTCs in the blood stream and is no longer available to effect other cells. Advantageously, ENPP1 shows no or low expression in essential organs like the heart, lung or kidney. High expression of ENPP1 was found in the thyroid and parathyroid glands, which could be removed without life-threatening consequences. Therapeutic antibodies against ENPP1 might target tumor cells involved in therapy resistance (ABC transporter), wherefore the combination of an anti-ENPP1 antibody with existing therapies could improve patient outcome.

References

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Andreas, A.; Muller, V., Wikman, H.; Riethdorf, S., et al. Disseminated Tumor Cells Persist in the Bone Marrow of Breast Cancer Patients through Sustained Activation of the Unfolded

Protein Response. Cancer Res 2015, 75, 5367-5377.

[2] Koch, C., Kuske, A, Joosse, S. A, Yigit, G., et al, Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity. EMBO Molecular Medicine 2020, 12, e11908.

[3] Watson, A. P., Evans, R. L., Egland, K. A, Multiple functions of sushi domain containing 2 (SUSD2) in breast t umorigenesis. Mo/ Cancer Res 2013, 11, 74-85.

Hultgren, E. M, Patrick, M. E., Evans, R. L., Stoos, C. T., Egland, K. A., SUSD2 promotes tumor- associated macrophage recruitment by increasing levels of MCP-1 in breast cancer.

PLoS One 2017, 12, e0177089.

PAT 1825 LU

[4] Takahashi, R. -u., Miyazaki, H., Takeshita, F., Yamamoto, Y., et al., Loss of microRNA- LU501430 27b contributes to breast cancer stem cell generation by activating ENPPI. Nature communications 2015, 6, 1-15.

[5] Lau, W. M., Doucet, M, Stadel, R., Huang, D., et al., Enp pl : a potential facilitator of breast cancer bone metastasis. PLoS One 2013, 8, e66752.

[6] Hu, M., Guo, W., Liao, Y., Xu, D., et al., Dysregulated ENPPI increases the malignancy of human lung cancer by inducing epithelial-mesenchymal transition phenotypes and stem cell features. Am J Cancer Res 2019, 9, 134-144.

[7] Lee, B.-J., Kang, D.-W., Park, H.-Y, Song, J.-S, et al, Isolation and localization of mesenchymal stem cells in human palatine tonsil by W5C5 (SUSD2) . Cellular Physiology and Biochemistry 2016, 38, 83-93.

[9] Sivasubramaniyan, K., Harichandan, A., Schumann, S., Sobiesiak, M, et al., Prospective isolation of mesenchymal stem cells from human bone marrow using novel antibodies directed against Sushi domain containing 2. Stem cells and development 2013, 22, 1944- 1954.

[10] Masuda, H., Anwar, S.S., Buhring, H.J., Rao, J. R., Gargett, C. E., A novel marker of human endometrial mesenchymal stem-like cells. Cell Transplant 2012, 21, 2201-2214.

[11] Kato K., Nishimasu H., Okudaira S., Mihara E., Ishitani R., Takagi J., Aoki J., Nureki

O., 2012, Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling, Proceedings of the National Academy of Sciences 109 (42), 16876-16881, DOI: 10.1073/pnas.1208017109

[12] Onyedibe, K.I.; Wang, M.; Sintim, H.O. ENPP1, an Old Enzyme with New Functions, and Small Molecule Inhibitors—A STING in the Tale of ENPP1. Molecules 2019, 24, 4192, doi/10.3390/molecules24224192

[12] Patrick, MLE; Egland, K.A. SUSD2 Proteolytic Cleavage Requires the GDPH Sequence and Inter-Fragment Disulfide Bonds for Surface Presentation of Galectin-1 on Breast Cancer

Cells. Int. J. Mol. Sci. 2019, 20, 3814. Do0i:/10.3390/1jms20153814

[13] Korsching, E ; Jeffrey, S.S.; Meinerz, W.; Decker, T.; Boecker, W., Buerger H. Basal carcinoma of the breast revisited: an old entity with new interpretations. J Clin Pathol. 2008

May;61(5):553-60. doi: 10.1136/jcp.2008.055475 31

PAT 1825 LU

Reference Numbers LUS01430 1 Transmembrane protein (SUSD2 or ENPPI) 10 signal peptide 11 extracellular domain 110 first (membrane bound) extracellular domain fragment 111 second extracellular domain fragment 112 extracellular domain cleavage site 113 disulfide bond 12 transmembrane domain 13 intracellular (cytoplasmic) domain 14 C-terminal 15 N-terminal 2 1% primary antibody (against SUSD2, ENPPI extracellular domain; biotinylated); capturing antibody 3 1“ secondary antibody (against 1* primary antibody) 4 2" primary antibody (against SUSD2, ENPPI cytoplasmic domain) 2"* secondary antibody (against 2" primary antibody) magnetic nanoparticle (with bound streptavidin) 60 cell membrane (of CTC/DTC) 70 MACS column 71 Magnet 80 CTC/DTC (SUSD2*, ENPP1*) 81 CTC/DTC (SUSD?2”, ENPPI) 90 collecting vessel 32

PAT 1825 LU

SEQUENCE LISTING LU501430 <110> UNIVERSITATSKLINIKUM HAMBURG-EPPENDORF (UKE) <120> ENRICHMENT, DETECTION AND CHARACTERIZATION OF CIRCULATING TUMOR CELLS

WITH SUSD2 AND ENPP1 <130> PAT 1825 LU <160> 10 <170> BiSSAP 1.3.6 <210> 1 <211> 822 <212> PRT <213> Homo sapiens <400> 1

Met Lys Pro Ala Leu Leu Pro Trp Ala Leu Leu Leu Leu Ala Thr Ala 1 5 10 15

Leu Gly Pro Gly Pro Gly Pro Thr Ala Asp Ala Gln Glu Ser Cys Ser

Met Arg Cys Gly Ala Leu Asp Gly Pro Cys Ser Cys His Pro Thr Cys

Ser Gly Leu Gly Thr Cys Cys Leu Asp Phe Arg Asp Phe Cys Leu Glu 60

Ile Leu Pro Tyr Ser Gly Ser Met Met Gly Gly Lys Asp Phe Val Val 65 70 75 80

Arg His Phe Lys Met Ser Ser Pro Thr Asp Ala Ser Val Ile Cys Arg 85 90 95

Phe Lys Asp Ser Ile Gln Thr Leu Gly His Val Asp Ser Ser Gly Gln 100 105 110

Val His Cys Val Ser Pro Leu Leu Tyr Glu Ser Gly Arg Ile Pro Phe 115 120 125

Thr Val Ser Leu Asp Asn Gly His Ser Phe Pro Arg Ala Gly Thr Trp 130 135 140

Leu Ala Val His Pro Asn Lys Val Ser Met Met Glu Lys Ser Glu Leu 145 150 155 160

Val Asn Glu Thr Arg Trp Gln Tyr Tyr Gly Thr Ala Asn Thr Ser Gly 33

PAT 1825 LU 165 170 175 LU501430

Asn Leu Ser Leu Thr Trp His Val Lys Ser Leu Pro Thr Gln Thr Ile 180 185 190

Thr Ile Glu Leu Trp Gly Tyr Glu Glu Thr Gly Met Pro Tyr Ser Gln 195 200 205

Glu Trp Thr Ala Lys Trp Ser Tyr Leu Tyr Pro Leu Ala Thr His Ile 210 215 220

Pro Asn Ser Gly Ser Phe Thr Phe Thr Pro Lys Pro Ala Pro Pro Ser 225 230 235 240

Tyr Gln Arg Trp Arg Val Gly Ala Leu Arg Ile Ile Asp Ser Lys Asn 245 250 255

Tyr Ala Gly Gln Lys Asp Val Gln Ala Leu Trp Thr Asn Asp His Ala 260 265 270

Leu Ala Trp His Leu Ser Asp Asp Phe Arg Glu Asp Pro Val Ala Trp 275 280 285

Ala Arg Thr Gln Cys Gln Ala Trp Glu Glu Leu Glu Asp Gln Leu Pro 290 295 300

Asn Phe Leu Glu Glu Leu Pro Asp Cys Pro Cys Thr Leu Thr Gln Ala 305 310 315 320

Arg Ala Asp Ser Gly Arg Phe Phe Thr Asp Tyr Gly Cys Asp Met Glu 325 330 335

Gln Gly Ser Val Cys Thr Tyr His Pro Gly Ala Val His Cys Val Arg 340 345 350

Ser Val Gln Ala Ser Leu Arg Tyr Gly Ser Gly Gln Gln Cys Cys Tyr 355 360 365

Thr Ala Asp Gly Thr Gln Leu Leu Thr Ala Asp Ser Ser Gly Gly Ser 370 375 380

Thr Pro Asp Arg Gly His Asp Trp Gly Ala Pro Pro Phe Arg Thr Pro 385 390 395 400

Pro Arg Val Pro Ser Met Ser His Trp Leu Tyr Asp Val Leu Ser Phe 405 410 415

Tyr Tyr Cys Cys Leu Trp Ala Pro Asp Cys Pro Arg Tyr Met Gln Arg 420 425 430

Arg Pro Ser Asn Asp Cys Arg Asn Tyr Arg Pro Pro Arg Leu Ala Ser 435 440 445

Ala Phe Gly Asp Pro His Phe Val Thr Phe Asp Gly Thr Asn Phe Thr 450 455 460

Phe Asn Gly Arg Gly Glu Tyr Val Leu Leu Glu Ala Ala Leu Thr Asp 465 470 475 480

Leu Arg Val Gln Ala Arg Ala Gln Pro Gly Thr Met Ser Asn Gly Thr 485 490 495 34

PAT 1825 LU

Glu Thr Arg Gly Thr Gly Leu Thr Ala Val Ala Val Gln Glu Gly Asn LU501430 500 505 510

Ser Asp Val Val Glu Val Arg Leu Ala Asn Arg Thr Gly Gly Leu Glu 515 520 525

Val Leu Leu Asn Gln Glu Val Leu Ser Phe Thr Glu Gln Ser Trp Met 530 535 540

Asp Leu Lys Gly Met Phe Leu Ser Val Ala Ala Gly Asp Arg Val Ser 545 550 555 560

Ile Met Leu Ala Ser Gly Ala Gly Leu Glu Val Ser Val Gln Gly Pro 565 570 575

Phe Leu Ser Val Ser Val Leu Leu Pro Glu Lys Phe Leu Thr His Thr 580 585 590

His Gly Leu Leu Gly Thr Leu Asn Asn Asp Pro Thr Asp Asp Phe Thr 595 600 605

Leu His Ser Gly Arg Val Leu Pro Pro Gly Thr Ser Pro Gln Glu Leu 610 615 620

Phe Leu Phe Gly Ala Asn Trp Thr Val His Asn Ala Ser Ser Leu Leu 625 630 635 640

Thr Tyr Asp Ser Trp Phe Leu Val His Asn Phe Leu Tyr Gln Pro Lys 645 650 655

His Asp Pro Thr Phe Glu Pro Leu Phe Pro Ser Glu Thr Thr Leu Asn 660 665 670

Pro Ser Leu Ala Gln Glu Ala Ala Lys Leu Cys Gly Asp Asp His Phe 675 680 685

Cys Asn Phe Asp Val Ala Ala Thr Gly Ser Leu Ser Thr Gly Thr Ala 690 695 700

Thr Arg Val Ala His Gln Leu His Gln Arg Arg Met Gln Ser Leu Gln 705 710 715 720

Pro Val Val Ser Cys Gly Trp Leu Ala Pro Pro Pro Asn Gly Gln Lys 725 730 735

Glu Gly Asn Arg Tyr Leu Ala Gly Ser Thr Ile Tyr Phe His Cys Asp 740 745 750

Asn Gly Tyr Ser Leu Ala Gly Ala Glu Thr Ser Thr Cys Gln Ala Asp 755 760 765

Gly Thr Trp Ser Ser Pro Thr Pro Lys Cys Gln Pro Gly Arg Ser Tyr 770 775 780

Ala Val Leu Leu Gly Ile Ile Phe Gly Gly Leu Ala Val Val Ala Ala 785 790 795 800

Val Ala Leu Val Tyr Val Leu Leu Arg Arg Arg Lys Gly Asn Thr His 805 810 815

Val Trp Gly Ala Gln Pro

PAT 1825 LU 820 LU501430 <210> 2 <211> 795 <212> PRT <213> Canis lupus familiaris <400> 2

Met Gly Ser Gly Gln Ile Ser Ser Ala Ala Pro Pro Pro Gly Ala Gln 1 5 10 15

Gly Ser Cys Ser His Arg Cys Gly Asp Arg Asp Gly Ser Cys Ser Cys

His Pro Thr Cys Ser Gly Leu Gly Thr Cys Cys Ser Asp Phe Arg Asp

Phe Cys Leu Glu Val Ser Pro Tyr Ser Gly Ser Met Met Gly Gly Lys 60

Asp Phe Gln Val Gln His Leu Thr Trp Phe Ile Pro Thr Asp Gly Val 65 70 75 80

Ile Cys Arg Phe Lys Glu Ser Ile Gln Thr Leu Gly Tyr Val Asp Ser 85 90 95

Ser Gly His Val His Cys Val Ser Pro Leu Leu Tyr Glu Thr Gly His 100 105 110

Ile Pro Phe Thr Leu Ser Met Asp Asn Gly Arg Ser Phe Pro Arg Ser 115 120 125

Gly Thr Trp Leu Ala Val His Pro Ser Lys Val Ser Glu Thr Glu Lys 130 135 140

Ser Gln Leu Glu Asn Glu Thr Leu Trp Gln Tyr Tyr Gly Thr Ala Gly 145 150 155 160

Thr Thr Asp Asn Leu Thr Val Thr Trp Glu Pro Ser Ala Leu Pro Thr 165 170 175

Arg Ser Val Thr Ile Glu Leu Trp Gly Tyr Glu Glu Thr Gly Lys Pro 180 185 190

Tyr Ser Gly Glu Trp Thr Ala Lys Trp Ser Tyr Leu Tyr Ser Leu Ala 195 200 205

Thr Ser Ile Pro Asn Ser Gly Phe Phe Thr Phe Thr Pro Lys Pro Ala 210 215 220

Pro Pro Asn Phe Gln Arg Trp Glu Val Gly Ala Leu Arg Ile Ile Ser 225 230 235 240

Ser Arg Tyr Tyr Ala Gly Glu Lys Asp Val Gln Ala Leu Trp Ser Asn 245 250 255 36

PAT 1825 LU

Glu His Ala Leu Ala Trp His Leu Gly Glu Asp Phe Arg Ala Asp Pro LU501430 260 265 270

Lys Ala Trp Ala Arg Ala Gln Cys Leu Ala Trp Glu Gln Leu Glu Asp 275 280 285

Gln Leu Pro Asn Phe Leu Glu Glu Leu Pro Asp Cys Pro Cys Thr Leu 290 295 300

Ala Gln Ala Arg Ala Asp Ser Gly Arg Phe His Val Ser Pro Pro Pro 305 310 315 320

Pro Pro Pro Ala Pro Glu Asn Gln Glu Gly Lys Gly Gly Glu Gln Cys 325 330 335

Cys Tyr Thr Glu Ala Gly Ser Leu Leu Leu Thr Ala Asp Ser Thr Gly 340 345 350

Gly Ser Thr Pro Asp Arg Gly His Asp Trp Gly Ala Pro Pro Phe Arg 355 360 365

Thr Pro Pro Arg Val Pro Gly Leu Ser His Trp Leu Tyr Asp Val Val 370 375 380

Ser Phe Tyr His Cys Cys Leu Trp Ala Pro Glu Cys Ser Arg Tyr Met 385 390 395 400

Arg Arg Arg Pro Ser Ser Asp Cys Arg Ser Tyr Arg Pro Ser Arg Leu 405 410 415

Ala Ser Ala Phe Gly Asp Pro His Phe Ile Thr Phe Asp Gly Ala Ser 420 425 430

Phe Ser Phe Asn Gly Arg Gly Glu Tyr Val Leu Leu Glu Ala Glu Leu 435 440 445

Thr Asn Leu Arg Val Gln Gly Arg Ala Gln Pro Arg Thr Thr Pro Glu 450 455 460

Gly Pro Gln Asp Arg Gly Thr Gly Leu Thr Ala Val Ala Val Gln Glu 465 470 475 480

Gly Asn Ser Asp Val Val Glu Val Arg Leu Ala Gly Gly Ala Gly Val 485 490 495

Leu Gln Val Leu Leu Asn Gln Glu Val Leu Ser Phe Thr Glu Gln Ser 500 505 510

Trp Met Asp Leu Lys Gly Met Phe Leu Ser Val Ala Ala Gly Asp Ser 515 520 525

Val Ser Ile Met Leu Ser Ser Gly Ala Gly Leu Glu Val Ser Val Gln 530 535 540

Gly Pro Phe Leu Ser Val Thr Val Leu Leu Pro Glu Lys Phe Leu Thr 545 550 555 560

His Thr Gln Gly Leu Leu Gly Thr Leu Asn Asp Asp Pro Thr Asp Asp 565 570 575

Phe Val Leu Arg Asp Gly Asn Val Leu Pro Pro Lys Ala Ser Ser Arg 37

PAT 1825 LU 580 585 590 LU501430

Glu Leu Phe Arg Phe Gly Gly Leu Trp Thr Leu Gly Ala Val Arg Asn 595 600 605

Thr Ser Ser Leu Leu Thr Tyr Asp Ser Trp Phe Leu Val Asn Asn Phe 610 615 620

Leu Tyr Gln Pro Lys His Asp Tyr Thr Phe Gln Pro Leu Phe Pro Glu 625 630 635 640

Glu Thr Thr Pro Asn Pro Ser Gln Ala Ala Glu Ala Ala Lys Leu Cys 645 650 655

Gly Asp Asn Tyr Phe Cys Ile Phe Asp Val Met Ala Thr Gly Ser Leu 660 665 670

Ser Val Gly Asn Ala Thr Arg Met Ala His Gln Trp His Gln His Arg 675 680 685

Ala Gln Ser Leu Gln Pro Val Thr Ser Cys Gly Trp Leu Ala Pro Pro 690 695 700

Pro Asn Gly Arg Lys Glu Gly Thr Arg Tyr Leu Ser Gly Ser Thr Val 705 710 715 720

Tyr Phe His Cys Asp Ser Gly Tyr Ser Leu Val Gly Ala Glu Val Ser 725 730 735

Thr Cys Gln Ala Asp Gly Ile Trp Ser Arg Pro Thr Pro Met Cys Gln 740 745 750

Pro Ala Arg Ser Tyr Thr Val Leu Leu Ser Ile Ile Phe Gly Gly Leu 755 760 765

Ala Val Val Ala Val Val Ala Leu Val Tyr Val Leu Leu Arg His Arg 770 775 780

Lys Gly Asn Met Ala Ile Trp Gly Ser Gln Pro 785 790 795 <210> 3 <211> 823 <212> PRT <213> Cavia porcellus <400> 3

Met Lys Leu Ala Leu Leu Pro Trp Val Leu Leu Leu Leu Ala Thr Val 1 5 10 15

Pro Gly Pro Gly Pro Arg Pro Thr Ala Asp Ala Gln Asp Ser Cys Ser

Leu Arg Cys Gly Asn Gln Asn Gly Thr Cys Ser Cys His Pro Thr Cys 38

PAT 1825 LU

Ser Gly Leu Gly Thr Cys Cys Pro Asp Phe Arg Asp Phe Cys Leu Glu LU501430 60

Ile Ser Pro Phe Ser Gly Ser Met Met Gly Gly Lys Asp Phe Val Val 65 70 75 80

Arg His Phe Lys Trp Ser Thr His Thr Glu Gly Val Ile Cys Arg Phe 85 90 95

Lys Glu Ser Ile Gln Ile Leu Gly His Val Asp Ser Leu Gly Gln Val 100 105 110

His Cys Val Ser Pro Leu Leu Tyr Glu Thr Gly Arg Ile Pro Phe Thr 115 120 125

Ile Ser Met Asp Asn Gly Arg Ser Phe Leu Arg Ala Gly Thr Trp Leu 130 135 140

Ala Val His Pro Asn Lys Val Ser Gln Ser Glu Lys Gly Gln Leu Val 145 150 155 160

Asn Glu Thr Arg Trp Gln Tyr Tyr Gly Thr Pro Gly Thr Thr Thr Gly 165 170 175

Asn Leu Ser Val Thr Trp Asp Thr Ser Val Leu Ser Thr Gln Thr Val 180 185 190

Asn Ile Glu Leu Trp Gly Tyr Glu Glu Thr Gly Thr Pro Tyr Ser Gly 195 200 205

Asn Trp Ile Ala Lys Trp Ser Tyr Leu Tyr Ser Leu Ala Thr Asn Ala 210 215 220

Pro Asn Ser Gly Phe Phe Thr Phe Ile Pro Glu Pro Ala Leu Ala Ser 225 230 235 240

Tyr Gln Lys Trp Arg Val Gly Ala Leu Arg Ile Thr Ala Ser Lys His 245 250 255

Tyr Gln Gly Glu Lys Asp Val Leu Ala Ile Trp Thr Asn Glu His Ala 260 265 270

Leu Ala Trp His Leu Gly Asp Asp Phe Arg Lys Asp Ser Val Ser Trp 275 280 285

Ala Arg Ala Gln Cys Leu Asp Trp Glu Glu Leu Glu Asp Asn Leu Pro 290 295 300

Asp Phe Leu Lys Glu Leu Pro Asp Cys Pro Cys Asn Leu Val Gln Ala 305 310 315 320

Arg Ala Asp Thr Gly Arg Phe Phe Thr Asp Tyr Gly Cys Asp Ile Glu 325 330 335

Gln Asn Ser Thr Cys Thr Tyr His Pro Gly Ala Val His Cys Val Arg 340 345 350

Ser Val Gln Ala Ser Pro Arg Phe Ala Ser Gly Gln Gln Cys Cys Tyr 355 360 365

Arg Lys Asp Gly Ser Gln Leu Leu Thr Ala Asp Ser Ile Gly Gly Ser 39

PAT 1825 LU 370 375 380 LU501430

Thr Pro Asp Arg Gly His Asp Trp Gly Ala Pro Pro Tyr Arg Thr Pro 385 390 395 400

Pro Arg Val Pro Thr Leu Ser His Trp Leu Tyr Asp Val Met Ser Phe 405 410 415

Tyr Tyr Cys Cys Leu Trp Ala Pro Glu Cys Ser Arg Tyr Met Gln Arg 420 425 430

Arg Pro Ser Ser Asp Cys Arg Thr Tyr Arg Pro Pro Arg Leu Ala Ser 435 440 445

Val Phe Gly Asp Pro His Phe Val Thr Phe Asp Gly Thr Lys Phe Thr 450 455 460

Phe Asn Gly Arg Gly Glu Tyr Val Leu Leu Glu Gly Glu Ala Asp Gly 465 470 475 480

Lys Asp Leu Arg Val Gln Ala Arg Ala Glu Pro Thr Thr Leu Thr Ser 485 490 495

Gly Thr Gln Ala Arg Gly Thr Gly Leu Thr Ala Val Val Val Gln Glu 500 505 510

Asp Ser Ser Asp Val Val Glu Ala Arg Leu Ala His Arg Pro Gly Val 515 520 525

Leu Glu Val Leu Leu Asn Ser Glu Val Leu Ser Phe Ala Glu Gln Ser 530 535 540

Trp Met Asp Leu Asn Gly Leu Phe Leu Ser Val Ala Gly Glu Asp Lys 545 550 555 560

Val Ser Ile Met Leu Ser Ser Gly Ala Gly Leu Glu Val Ser Ile Gln 565 570 575

Gly Pro Phe Leu Ser Val Ala Val Leu Leu Pro Glu Lys Phe Gln Asn 580 585 590

His Thr Arg Gly Leu Leu Gly Thr Leu Asn Asn Asn Pro Ala Asp Asp 595 600 605

Phe Thr Leu Arg Ser Gly Met Val Leu Pro Ser Asn Ala Ser Ala Gln 610 615 620

Glu Leu Phe Gln Phe Gly Ala Glu Trp Ala Val His Asn Ser Ser Ser 625 630 635 640

Leu Phe Thr Tyr Asp Ser Trp Ser Leu Val Tyr Asn Phe Leu Tyr Arg 645 650 655

Pro Lys His Asp Ser Thr Phe Lys Pro Leu Phe Ser Asp Glu Ile Val 660 665 670

Leu Ser Pro Asp Gln Ala Glu Glu Val Ala Lys Leu Cys Gly Asp Asp 675 680 685

His Phe Cys Lys Phe Asp Val Ala Val Thr Gly Asn Leu Ile Val Gly 690 695 700

PAT 1825 LU

Asn Val Thr Arg Ala Ala His Gln Met His Gln Glu Arg Leu Gln Ser LU501430 705 710 715 720

Leu Gln Pro Val Val Ser Cys Gly Trp Leu Ala Ala Pro Ser Asn Gly 725 730 735

His Lys Glu Asp Phe Lys Tyr Leu Val Gly Ser Thr Val Arg Phe His 740 745 750

Cys Glu Ser Gly Tyr Ser Leu Ala Gly Ala Asp Thr Ser Thr Cys Gln 755 760 765

Ala Asp Gly Thr Trp Ser Ala Pro Thr Pro Glu Cys Gln Leu Gly Arg 770 775 780

Ser Tyr Thr Val Leu Leu Ser Ile Ile Phe Gly Gly Leu Ala Val Val 785 790 795 800

Ala Leu Val Ala Ile Ile Tyr Leu Leu Leu Arg Arg Arg Lys Asn Met 805 810 815

Asn Leu Trp Arg Ser His Pro 820 <210> 4 <211> 821 <212> PRT <213> Equus caballus <400> 4

Met Lys Leu Leu Leu Leu Pro Trp Ala Leu Leu Leu Leu Val Thr Ala 1 5 10 15

Pro Ser Pro Gly Arg Trp Pro Ala Ala Asp Ala Gln Glu Ser Cys Ser

His Arg Cys Gly Glu Trp Asn Glu Leu Cys Ser Cys His Pro Thr Cys

Phe Gly Leu Asp Ser Cys Cys Lys Asp Phe Arg Asp Phe Cys Leu Glu 60

Ile Leu Pro Tyr Ser Gly Ser Met Met Gly Gly Lys Asp Phe Val Val 65 70 75 80

Gln His Leu Asn Trp Phe Ser Pro Thr Asp Gly Val Ile Cys Arg Phe 85 90 95

Asn Glu Ser Val Gln Thr Arg Gly His Val Asp Ser Leu Gly His Val 100 105 110

His Cys Val Ser Pro Leu Leu Tyr Glu Thr Gly Arg Ile Pro Phe Thr 115 120 125

Leu Ser Met Asp Asn Gly Ser Ser Phe Pro Arg Ser Gly Thr Trp Leu 41

PAT 1825 LU 130 135 140 LU501430

Ala Val His Pro Ser Lys Val Ser Gln Thr Glu Lys Leu Gln Leu Glu 145 150 155 160

Asn Glu Thr Arg Trp Gln Tyr Tyr Gly Thr Ala Gly Thr Lys Gly Asn 165 170 175

Leu Thr Leu Thr Trp Asn Thr Ala Ala Leu Pro Thr Gln Ser Val Thr 180 185 190

Ile Glu Leu Trp Gly Tyr Glu Glu Thr Gly Lys Pro Tyr Ser Glu Asp 195 200 205

Trp Thr Ala Lys Trp Ser Tyr Leu Tyr Ala Leu Ala Thr Asn Val Ser 210 215 220

Asn Ser Gly Phe Phe Thr Phe Thr Pro Glu Pro Ala Pro Gln Asn Tyr 225 230 235 240

Gln Arg Trp Glu Val Gly Ala Leu Arg Ile Ile Asn Ser Glu Tyr His 245 250 255

Ser Gly Glu Lys Asp Val His Ala Leu Trp Ser Asn Glu His Ala Leu 260 265 270

Ala Trp His Leu Gly Asp Asp Phe Arg Ala Asp Pro Val Ala Trp Ala 275 280 285

Arg Thr Gln Cys Leu Ala Trp Glu Glu Leu Glu Asp Gln Leu Pro Asn 290 295 300

Phe Leu Glu Glu Leu Pro Asp Cys Pro Cys Thr Leu Ala Gln Ala Arg 305 310 315 320

Ser Asp Ser Gly Arg Phe His Thr Asp Tyr Gly Cys Asp Ile Glu His 325 330 335

Gly Ser Glu Cys Thr Tyr His Pro Gly Ala Val His Cys Val Arg Ser 340 345 350

Val Gln Ala Ser Pro Gln Tyr Ala Ser Gly Gln Gln Cys Cys Tyr Thr 355 360 365

Ala Ala Gly Thr Gln Leu Leu Thr Ala Asp Ser Met Gly Gly Ser Thr 370 375 380

Pro Asp Arg Gly His Asp Trp Gly Ala Pro Pro Phe Arg Ala Pro Pro 385 390 395 400

Arg Val Pro Gly Leu Ser His Trp Leu Tyr Asp Val Ile Ser Phe Tyr 405 410 415

His Cys Cys Leu Trp Ala Pro Glu Cys Ser Arg Tyr Met Arg Arg Arg 420 425 430

Pro Ser Ser Asp Cys Arg Ser Tyr Arg Pro Pro Arg Leu Ala Ser Ala 435 440 445

Phe Gly Asp Pro His Phe Val Thr Phe Asp Gly Ala Asn Phe Thr Phe 450 455 460 42

PAT 1825 LU

Asn Gly His Gly Glu Tyr Val Leu Leu Glu Ala Ser Leu Thr Asn Leu LU501430 465 470 475 480

Thr Val Gln Ala Arg Ala Gln Pro Asp Thr Thr Pro Glu Gly Thr Gln 485 490 495

Ala Arg Gly Thr Gly Leu Thr Ala Val Ala Val Gln Glu Gly Asp Ser 500 505 510

Asp Val Val Glu Val Arg Leu Ala Gly Gly Ala Gly Val Leu Gln Val 515 520 525

Leu Leu Asn Gln Glu Val Leu Ser Phe Ala Glu Gln Ser Trp Met Asp 530 535 540

Leu Lys Gly Met Phe Leu Ser Val Ala Ala Glu Asp Arg Val Ser Ile 545 550 555 560

Met Leu Ser Ser Gly Ala Gly Leu Glu Val Ser Ile Gln Gly Pro Phe 565 570 575

Leu Ser Val Thr Val Leu Leu Pro Glu Lys Phe Leu Thr Tyr Thr Gln 580 585 590

Gly Leu Leu Gly Thr Leu Asn Asp Asn Phe Thr Asp Asp Phe Thr Leu 595 600 605

Arg Ser Gly Gln Val Leu Pro Pro Asp Ala Ser Ser Gln Glu Leu Phe 610 615 620

Gln Phe Gly Ala Asp Trp Ala Val Glu Asn Ala Ser Ser Leu Leu Thr 625 630 635 640

Tyr Asp Ser Gln Leu Leu Val Asn Asn Phe Leu Tyr Gly Pro Lys His 645 650 655

Asp Pro Thr Phe Arg Pro Leu Phe Pro Asp Glu Ile Thr Pro Asn Ser 660 665 670

Ser Gln Thr Thr Glu Val Ala Lys Leu Cys Gly Asp Asn His Phe Cys 675 680 685

Ser Phe Asp Val Ala Ala Thr Gly Ser Leu Ser Val Gly Asn Ala Thr 690 695 700

Arg Met Ala Tyr Gln Leu His Gln Arg Arg Val Gln Ser Leu Gln Ser 705 710 715 720

Val Val Ser Cys Gly Trp Leu Ala Pro Pro Leu Asn Gly His Lys Asp 725 730 735

Gly Met Arg Tyr Leu Val Gly Ser Thr Ile His Phe His Cys Asp Thr 740 745 750

Gly Tyr Ser Leu Ala Gly Ala Glu Ala Ser Thr Cys Gln Ala Asn Gly 755 760 765

Thr Trp Ser Thr Pro Thr Pro Thr Cys Gln Pro Arg Arg Ser His Thr 770 775 780

Val Leu Leu Ser Ile Ile Phe Gly Gly Leu Ala Val Val Ala Leu Met 43

PAT 1825 LU 785 790 795 800 LU501430

Ala Val Val Phe Val Val Leu Arg Arg Arg Lys Gly Asn Met Ala Ile 805 810 815

Trp Gly Ser Gln Pro 820 <210> 5 <211> 925 <212> PRT <213> Homo sapiens <400> 5

Met Glu Arg Asp Gly Cys Ala Gly Gly Gly Ser Arg Gly Gly Glu Gly 1 5 10 15

Gly Arg Ala Pro Arg Glu Gly Pro Ala Gly Asn Gly Arg Asp Arg Gly

Arg Ser His Ala Ala Glu Ala Pro Gly Asp Pro Gln Ala Ala Ala Ser

Leu Leu Ala Pro Met Asp Val Gly Glu Glu Pro Leu Glu Lys Ala Ala 60

Arg Ala Arg Thr Ala Lys Asp Pro Asn Thr Tyr Lys Val Leu Ser Leu 65 70 75 80

Val Leu Ser Val Cys Val Leu Thr Thr Ile Leu Gly Cys Ile Phe Gly 85 90 95

Leu Lys Pro Ser Cys Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys 100 105 110

Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val Glu 115 120 125

Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu Pro Glu 130 135 140

His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr 145 150 155 160

Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp Cys 165 170 175

Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu Lys Ser Trp Val Glu 180 185 190

Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala Gly Phe Glu 195 200 205

Thr Pro Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe Arg Ala Glu Tyr 210 215 220 44

PAT 1825 LU

Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys Leu Lys Lys LU501430 225 230 235 240

Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro Val Tyr Pro Thr Lys Thr 245 250 255

Phe Pro Asn His Tyr Ser Ile Val Thr Gly Leu Tyr Pro Glu Ser His 260 265 270

Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro Lys Met Asn Ala Ser Phe 275 280 285

Ser Leu Lys Ser Lys Glu Lys Phe Asn Pro Glu Trp Tyr Lys Gly Glu 290 295 300

Pro Ile Trp Val Thr Ala Lys Tyr Gln Gly Leu Lys Ser Gly Thr Phe 305 310 315 320

Phe Trp Pro Gly Ser Asp Val Glu Ile Asn Gly Ile Phe Pro Asp Ile 325 330 335

Tyr Lys Met Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Leu Ala 340 345 350

Val Leu Gln Trp Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe Tyr 355 360 365

Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr Gly Pro 370 375 380

Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp Gly Met Val 385 390 395 400

Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu His Arg Cys Leu 405 410 415

Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu Gln Gly Ser Cys Lys 420 425 430

Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys Asn Ile Lys 435 440 445

Val Ile Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser Asp Val Pro Asp 450 455 460

Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala Arg Asn Leu Ser Cys 465 470 475 480

Arg Glu Pro Asn Gln His Phe Lys Pro Tyr Leu Lys His Phe Leu Pro 485 490 495

Lys Arg Leu His Phe Ala Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe 500 505 510

Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg Lys 515 520 525

Tyr Cys Gly Ser Gly Phe His Gly Ser Asp Asn Val Phe Ser Asn Met 530 535 540

Gln Ala Leu Phe Val Gly Tyr Gly Pro Gly Phe Lys His Gly Ile Glu

PAT 1825 LU 545 550 555 560 LU501430

Ala Asp Thr Phe Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu 565 570 575

Leu Asn Leu Thr Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn 580 585 590

His Leu Leu Lys Asn Pro Val Tyr Thr Pro Lys His Pro Lys Glu Val 595 600 605

His Pro Leu Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp Asn Leu 610 615 620

Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr 625 630 635 640

Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile Lys His Glu Thr 645 650 655

Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys Glu Asn Thr Ile Cys 660 665 670

Leu Leu Ser Gln His Gln Phe Met Ser Gly Tyr Ser Gln Asp Ile Leu 675 680 685

Met Pro Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn Asp Ser Phe Ser 690 695 700

Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp Phe Arg Ile Pro Leu 705 710 715 720

Ser Pro Val His Lys Cys Ser Phe Tyr Lys Asn Asn Thr Lys Val Ser 725 730 735

Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile 740 745 750

Tyr Ser Glu Ala Leu Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser 755 760 765

Phe Gln Val Ile Trp Arg Tyr Phe His Asp Thr Leu Leu Arg Lys Tyr 770 775 780

Ala Glu Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Val Phe Asp 785 790 795 800

Phe Asp Tyr Asp Gly Arg Cys Asp Ser Leu Glu Asn Leu Arg Gln Lys 805 810 815

Arg Arg Val Ile Arg Asn Gln Glu Ile Leu Ile Pro Thr His Phe Phe 820 825 830

Ile Val Leu Thr Ser Cys Lys Asp Thr Ser Gln Thr Pro Leu His Cys 835 840 845

Glu Asn Leu Asp Thr Leu Ala Phe Ile Leu Pro His Arg Thr Asp Asn 850 855 860

Ser Glu Ser Cys Val His Gly Lys His Asp Ser Ser Trp Val Glu Glu 865 870 875 880 46

PAT 1825 LU

Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val Glu His Ile Thr LU501430 885 890 895

Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp Ile Leu 900 905 910

Lys Leu Lys Thr His Leu Pro Thr Phe Ser Gln Glu Asp 915 920 925 <210> 6 <211> 916 <212> PRT <213> Canis lupus familiaris <400> 6

Met Glu Arg Gly Gly Gln Ala Gly Leu Gly Ser Arg Glu Gly Pro Ala 1 5 10 15

Gly Asn Gly Pro Asp Pro Gly Cys Ala Arg Ala Ala Ala Ala Pro Gly

Asp Gly Gln Ala Ala Ala Ala Leu Leu Ala Pro Met Asp Leu Gly Glu

Glu Pro Leu Glu Lys Ala Ala Arg Ala Arg Pro Ala Lys Asp Pro Asn 60

Thr Tyr Lys Val Leu Ser Leu Val Leu Ser Val Cys Val Leu Thr Thr 65 70 75 80

Ile Leu Gly Cys Ile Phe Gly Leu Lys Pro Ser Cys Ala Lys Glu Val 85 90 95

Lys Ser Cys Lys Gly Arg Cys Phe Glu Arg Thr Phe Gly Asn Cys Arg 100 105 110

Cys Asp Val Ala Cys Val Asp Leu Gly Asn Cys Cys Leu Asp Tyr Gln 115 120 125

Glu Thr Cys Ile Glu Pro Glu Arg Ile Trp Thr Cys Ser Lys Phe Arg 130 135 140

Cys Gly Glu Lys Arg Leu Ser Arg Ser Leu Cys Ser Cys Ser Asp Asp 145 150 155 160

Cys Arg Asp Lys Gly Asp Cys Cys Val Asn Tyr Ser Ser Val Cys Leu 165 170 175

Gly Glu Lys Ser Trp Val Glu Glu Thr Cys Glu Ser Ile Asp Glu Pro 180 185 190

Gln Cys Pro Ala Gly Phe Glu Met Pro Pro Thr Leu Leu Phe Ser Leu 195 200 205

Asp Gly Phe Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro 47

PAT 1825 LU 210 215 220 LU501430

Val Ile Ser Lys Leu Lys Asn Cys Gly Thr Tyr Thr Lys Asn Met Arg 225 230 235 240

Pro Val Tyr Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr 245 250 255

Gly Leu Tyr Pro Glu Ser His Gly Ile Ile Asp Asn Lys Ile Tyr Asp 260 265 270

Pro Lys Met Asn Ala Phe Phe Ala Leu Lys Ser Lys Glu Lys Phe Asn 275 280 285

Pro Glu Trp Tyr Lys Gly Glu Pro Ile Trp Leu Thr Thr Lys Tyr Gln 290 295 300

Gly Leu Lys Ser Gly Thr Phe Phe Trp Pro Gly Ser Asp Val Glu Ile 305 310 315 320

Lys Gly Ile Leu Pro Asp Ile Tyr Lys Met Tyr Asn Gly Ser Ile Pro 325 330 335

Phe Glu Glu Arg Ile Leu Ala Val Leu Lys Trp Leu Gln Leu Pro Lys 340 345 350

Asp Glu Arg Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser 355 360 365

Ser Gly His Ser Tyr Gly Pro Val Ser Ser Glu Val Ile Arg Ala Leu 370 375 380

Gln Arg Val Asp Asn Met Val Gly Met Leu Met Asp Gly Leu Lys Gly 385 390 395 400

Leu Asn Leu His Arg Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly 405 410 415

Met Glu Gln Gly Ser Cys Lys Lys Tyr Val Tyr Leu Asn Lys Tyr Leu 420 425 430

Gly Asp Val Lys Asp Ile Lys Ile Val Tyr Gly Pro Ala Ala Arg Leu 435 440 445

Arg Pro Ser Asp Val Pro Asn Lys Tyr Phe Ser Phe Asn Tyr Glu Asp 450 455 460

Leu Ala Lys Asn Leu Ser Cys Arg Glu Pro Asn Gln His Phe Lys Pro 465 470 475 480

Tyr Leu Lys His Phe Leu Pro Lys Arg Met His Phe Ala Lys Asn Asp 485 490 495

Arg Ile Glu Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala 500 505 510

Leu Asn Pro Ser Glu Arg Lys His Cys Gly Ser Gly Phe His Gly Ser 515 520 525

Asp Asn Leu Phe Ser Asn Met Gln Ala Leu Phe Ile Gly Tyr Gly Pro 530 535 540 48

PAT 1825 LU

Gly Phe Lys His Asn Ile Glu Val Asp Ser Phe Glu Asn Ile Glu Val LU501430 545 550 555 560

Tyr Asn Leu Met Cys Asp Leu Leu Asn Leu Thr Pro Ala Pro Asn Asn 565 570 575

Gly Thr His Gly Ser Leu Asn His Leu Leu Lys Asn Pro Val Tyr Thr 580 585 590

Pro Lys Asn Pro Lys Glu Val His Pro Met Val Gln Cys Pro Phe Thr 595 600 605

Arg Ala Pro Arg His Asn Leu Asp Cys Ser Cys Asp Pro Ser Val Leu 610 615 620

Pro Val Thr Asp Phe Glu Thr Gln Leu Asn Leu Thr Val Ala Glu Glu 625 630 635 640

Lys Val Ile Lys Arg Gly Thr Leu Pro Tyr Gly Arg Pro Arg Val Ile 645 650 655

Gln Lys Asn Thr Thr Val Cys Leu Leu Tyr Gln His Gln Phe Val Ser 660 665 670

Gly Tyr Ser His Asp Ile Leu Met Pro Leu Trp Thr Ser Tyr Thr Val 675 680 685

Asp Arg Asn Asp Ser Phe Ser Thr Glu Asp Phe Ser Asn Cys Leu Tyr 690 695 700

Gln Asp Leu Arg Ile Pro Leu Ser Pro Ile His Lys Cys Ser Phe Tyr 705 710 715 720

Lys Asn Asn Ala Glu Leu Ser Tyr Gly Phe Leu Tyr Pro Pro Gln Leu 725 730 735

Asn Lys Gly Ser Asn Gln Leu Tyr Ser Glu Ala Leu Leu Ser Thr Asn 740 745 750

Ile Val Pro Met Tyr Gln Ser Phe Gln Val Ile Trp His Tyr Phe His 755 760 765

Gly Thr Leu Leu Gln Arg Tyr Val Glu Glu Arg Asn Gly Val Asn Val 770 775 780

Val Ser Gly Pro Val Phe Asp Ser Asp Tyr Asp Gly Arg Tyr Asp Ser 785 790 795 800

Ser Glu Thr Leu Arg Gln Asn Ser Arg Leu Ile His Asn Gln Glu Ile 805 810 815

Leu Ile Pro Thr His Phe Phe Ile Val Leu Thr Asn Cys Lys Asp Thr 820 825 830

Phe Gln Thr Pro Ser Gln Cys Asp Asp Leu Asp Ser Leu Ala Phe Ile 835 840 845

Leu Pro His Arg Ala Asp Asn Ser Glu Ser Cys Val His Gly Lys His 850 855 860

Glu Ser Leu Trp Val Glu Glu Leu Leu Arg Leu His Arg Ala Arg Ile 49

PAT 1825 LU 865 870 875 880 LU501430

Thr Asp Val Glu His Leu Thr Gly Leu Ser Phe Tyr Gln Glu Arg Arg 885 890 895

Glu Ser Ile Ser Asp Ile Leu Lys Leu Lys Thr Gln Leu Pro Ala Phe 900 905 910

Asp Gln Glu Asp 915 <210> 7 <211> 900 <212> PRT <213> Cavia porcellus <400> 7

Met Glu Gln Arg Asp Gly Gly Asp Ala Gly Arg Ala Gly Pro Ala Gly 1 5 10 15

Asp Pro Gln Ala Ala Ala Ala Leu Leu Thr Pro Met Glu Leu Gly Glu

Glu Pro Leu Glu Lys Ala Ala Arg Ala Arg Thr Ala Lys Asp Pro Asn

Thr Tyr Lys Val Leu Ser Leu Val Leu Ser Val Cys Val Leu Thr Thr 60

Ile Leu Gly Cys Ile Phe Gly Leu Lys Pro Ser Cys Ser Arg Glu Val 65 70 75 80

Lys Ser Cys Lys Gly Arg Cys Phe Glu Arg Thr Phe Gly Asn Cys Arg 85 90 95

Cys Asp Ser Ala Cys Val Asp Phe Gly Asn Cys Cys Leu Asp Tyr Gln 100 105 110

Glu Thr Cys Leu Glu Pro Ala His Ile Trp Thr Cys Asn Lys Phe Arg 115 120 125

Cys Gly Glu Lys Arg Val Pro His Asn Leu Cys Ser Cys Ser Asp Asp 130 135 140

Cys Lys Asp His Asp Asp Cys Cys Val Asn Tyr Ser Ser Met Cys Gln 145 150 155 160

Asp Glu Lys Ser Trp Leu Glu Ala Pro Cys Glu Arg Ile Asn Thr Pro 165 170 175

Gln Cys Pro Ala Gly Phe Asp Ala Pro Pro Thr Leu Leu Phe Ser Leu 180 185 190

Asp Gly Phe Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro 195 200 205 50

PAT 1825 LU

Val Ile Ser Lys Leu Lys Asn Cys Gly Thr Tyr Thr Lys Asn Met Arg LU501430 210 215 220

Pro Val Tyr Pro Thr Lys Thr Phe Pro Asn His Tyr Thr Ile Val Thr 225 230 235 240

Gly Leu Tyr Pro Glu Ser His Gly Ile Ile Asp Asn Arg Met Phe Asp 245 250 255

Pro Gln Met Asn Ala Ser Phe Ser Phe Lys Thr Lys Gln Lys Phe Asn 260 265 270

Pro Gln Trp Tyr Gln Gly Glu Pro Ile Trp Leu Thr Ala Lys Tyr Gln 275 280 285

His Leu Ile Ser Gly Thr Phe Phe Trp Pro Gly Ser Asp Val Glu Ile 290 295 300

Asn Gly Asn Phe Pro Asp Phe Tyr Glu Val Tyr Asn Ser Ser Ile Pro 305 310 315 320

Phe Glu Lys Arg Ile Leu Gly Val Leu Asp Trp Leu Gln Leu Pro Gln 325 330 335

Gly Glu Arg Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser 340 345 350

Ala Gly His Phe Tyr Gly Pro Val Ser Gly Glu Val Ile Lys Ala Leu 355 360 365

Gln Arg Val Asp Ser Met Val Gly Met Leu Met Glu Gly Leu Arg Glu 370 375 380

Leu Lys Leu Asp Lys Cys Leu Asn Leu Leu Leu Val Ser Asp His Gly 385 390 395 400

Met Glu Gln Asp Ser Cys Lys Lys Tyr Val Tyr Leu Asn Lys Tyr Leu 405 410 415

Gly Asp Val Thr Asn Ile Lys Val Thr Tyr Gly Pro Ala Ala Arg Leu 420 425 430

Arg Leu Ser Asp Val Leu Asn Glu Tyr Ser Thr Phe Asp Tyr Glu Gly 435 440 445

Leu Ala Arg Asn Leu Ser Cys Arg Glu Pro Lys Gln His Phe Lys Pro 450 455 460

Tyr Leu Lys Gln Phe Leu Pro Lys Arg Phe His Phe Ala Asn Asn Asp 465 470 475 480

Arg Ile Glu Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala 485 490 495

Leu Asp Pro Thr Glu Arg Lys His Cys Gly Gly Gly Phe His Gly Ser 500 505 510

Asp Asn Ile Phe Ser Asn Met Gln Ala Leu Phe Ile Gly Tyr Gly Pro 515 520 525

Gly Phe Lys His Gly Val Glu Val Asp Thr Phe Glu Asn Ile Glu Leu 51

PAT 1825 LU 530 535 540 LU501430

Tyr Asn Leu Met Cys Asp Leu Leu Lys Leu Thr Pro Ala Pro Asn Asn 545 550 555 560

Gly Thr His Gly Ser Leu Asn His Leu Leu Lys His Pro Val Tyr Thr 565 570 575

Pro Lys His Pro Lys Glu Ala Gln Ser Leu Gly Lys Cys Pro Ile Thr 580 585 590

Gly Thr Pro Arg Asp Gly Leu Gly Cys Ser Cys Asp Pro Ala Ile Ser 595 600 605

Pro Val Glu Asp Phe Gln Ile Gln Phe Asn Leu Thr Arg Thr Glu Glu 610 615 620

Lys Asn Ile Asp Arg Gly Ala Leu Pro Tyr Gly Arg Pro Arg Leu Val 625 630 635 640

Gln Arg Lys Gly Ser Val Cys Leu Leu Tyr His His Gln Phe Val Ser 645 650 655

Gly Tyr Ser Leu Asp Ile Leu Met Pro Leu Trp Ala Ser Tyr Thr Val 660 665 670

Asp Lys Asn Asp Arg Phe Ser Val Gly Asp Phe Ser Asn Cys Val Phe 675 680 685

Gln Asp Leu Arg Ile Pro Leu Ser Pro Leu His Lys Cys Ser Phe Tyr 690 695 700

Lys Asn Asn Ala Lys Leu Ser Tyr Gly Phe Leu Ala Pro Pro Gln Leu 705 710 715 720

Ser Lys Tyr Ala Ser Gln Ile Tyr Ser Glu Ala Leu Leu Thr Thr Asn 725 730 735

Ile Val Pro Met Tyr Pro Ser Phe Gln Val Ile Trp Arg Tyr Leu His 740 745 750

Asp Thr Leu Leu Pro Arg Leu Ala Lys Glu Lys Asn Gly Ile Asn Val 755 760 765

Val Ser Gly Pro Val Phe Asp Ser Asp Phe Asp Gly His Ser Asp Thr 770 775 780

Ala Glu Thr Leu Lys Gln Asn Arg Arg Phe Ile Arg Asn Gln Glu Val 785 790 795 800

Leu Ile Pro Thr His Phe Phe Met Val Leu Thr Ser Cys Lys Asn Thr 805 810 815

Leu Gly Thr Pro Leu Tyr Cys Thr Glu Leu Asp Thr Met Ala Phe Ile 820 825 830

Leu Pro His Arg Pro Asp Asn Ser Glu Ser Cys Thr His Glu Lys Ser 835 840 845

Glu Ser Ser Trp Ile Glu Glu Leu Leu Arg Met His Arg Ala Arg Val 850 855 860 52

PAT 1825 LU

Ile Asp Val Glu Ala Ile Thr Gly Leu Ser Phe Tyr Gln Glu Arg Lys LU501430 865 870 875 880

Gln Pro Val Ser Asp Ile Leu Lys Leu Lys Thr His Leu Pro Ile Phe 885 890 895

Ser Glu Glu Asp 900 <210> 8 <211> 1043 <212> PRT <213> Equus caballus <400> 8

Met Ala Cys Ala Asp His Thr Leu Arg Phe Gln Ser Arg Pro Arg Arg 1 5 10 15

Gln Ala Thr Gln Gly Asp Ser Cys Lys Cys Ala Asp Gly Ala Gln Gly

Ala Ser Arg Gly Gly Trp Gly Glu Gly Gln Asp Gly Pro Thr Gln Arg

Thr Arg Asn Asn Arg His Ala Gly Ser Pro Lys Ser Asp Arg Lys Pro 60

Asn Asn Pro Gly Arg Arg Val Ala Phe Pro Ser Leu Gly Gly Arg Gly 65 70 75 80

Ala Gly Ala Gly Pro Gly Val Arg Arg Ala Gly Pro Gly Arg Ala Gly 85 90 95

Arg Gly Gly Arg Leu Gly Leu Leu Lys Ala Arg Arg Gly Gly Gly Pro 100 105 110

Ser Arg Val Ser Met Glu Arg Asp Gly Cys Ala Gly Gly Gly Ser His 115 120 125

Gly Gly Gly Cys Glu Gly Gly Arg Gly Ser Arg Glu Asp Pro Ala Gly 130 135 140

Asn Gly Arg Asp Pro Ser Arg Gly His Ala Ser Pro Ala Pro Gly Asp 145 150 155 160

Pro Gln Ala Ala Ala Ser Leu Leu Ala Pro Met Asp Val Gly Glu Glu 165 170 175

Pro Leu Glu Lys Ala Ala Arg Ala Gly Ala Ala Lys Asp Pro Asn Thr 180 185 190

Tyr Lys Val Leu Ser Leu Val Leu Ser Val Cys Val Leu Thr Val Ile 195 200 205

Leu Gly Cys Ile Phe Gly Leu Lys Pro Ser Cys Ala Lys Glu Val Lys 53

PAT 1825 LU 210 215 220 LU501430

Ser Cys Lys Gly Arg Cys Phe Glu Arg Thr Phe Gly Asn Cys Arg Cys 225 230 235 240

Asp Ser Ala Cys Ala Asp Leu Gly Asn Cys Cys Leu Asp Tyr Gln Glu 245 250 255

Thr Cys Ile Glu Pro Glu Arg Ile Trp Thr Cys Ser Lys Phe Arg Cys 260 265 270

Gly Glu Lys Arg Leu Ser Arg Ser Leu Cys Ser Cys Ser Asp Asp Cys 275 280 285

Arg Asp Lys Asp Asp Cys Cys Ile Asn Tyr Ser Ser Ala Cys Leu Gly 290 295 300

Glu Lys Ser Trp Val Gln Glu Thr Cys Glu Asn Ile Asn Glu Pro Gln 305 310 315 320

Cys Pro Ala Gly Phe Glu Met Pro Pro Thr Leu Leu Phe Ser Leu Asp 325 330 335

Gly Phe Arg Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro Val 340 345 350

Ile Asn Lys Leu Lys Thr Cys Gly Thr Tyr Ala Lys Asn Met Arg Pro 355 360 365

Val Tyr Pro Thr Lys Thr Phe Pro Asn His Tyr Ser Ile Val Thr Gly 370 375 380

Leu Tyr Pro Glu Ser His Gly Ile Ile Asp Asn Lys Met Tyr Asp Pro 385 390 395 400

Lys Met Asn Ala Ser Phe Ala Leu Lys Ser Lys Glu Lys Phe Asn Pro 405 410 415

Lys Trp Tyr Lys Gly Glu Pro Ile Trp Leu Thr Ala Lys Tyr Gln Gly 420 425 430

Leu Lys Ala Gly Thr Phe Phe Trp Pro Gly Ser Asp Val Lys Ile Asn 435 440 445

Gly Leu Phe Pro Asp Ile Tyr Lys Ile Tyr Asn Gly Ser Val Pro Phe 450 455 460

Glu Glu Arg Ile Leu Ala Val Leu Lys Trp Leu Gln Leu Pro Lys Asp 465 470 475 480

Glu Arg Pro His Phe Tyr Thr Leu Tyr Leu Glu Glu Pro Asp Ser Ser 485 490 495

Gly His Ser His Gly Pro Val Ser Ser Glu Val Ile Arg Ala Leu Gln 500 505 510

Arg Val Asp Asn Met Val Gly Met Leu Met Asp Gly Leu Lys Gly Leu 515 520 525

Asn Leu His Arg Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly Met 530 535 540 54

PAT 1825 LU

Glu Gln Ala Ser Cys Lys Lys Tyr Ala His Leu Asp Lys Tyr Leu Gly LU501430 545 550 555 560

Asp Val Lys Asp Ile Lys Leu Val Tyr Gly Pro Ala Ala Arg Leu Arg 565 570 575

Pro Ser Asp Val Pro Asp Lys Tyr Tyr Ser Phe Asn Tyr Glu Gly Leu 580 585 590

Ala Arg Asn Leu Ser Cys Arg Glu Leu Asn Gln His Phe Lys Pro Tyr 595 600 605

Leu Lys His Phe Leu Pro Lys Arg Leu His Phe Ala Lys Asn Asp Arg 610 615 620

Ile Glu Pro Leu Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu Ala Leu 625 630 635 640

Asn Pro Pro Ala Glu Lys His Cys Gln Gly Gly Phe His Gly Ser Asp 645 650 655

Asn Val Phe Ser Asn Met Gln Ala Leu Phe Ile Gly Tyr Gly Pro Gly 660 665 670

Phe Gln His Gly Ala Glu Val Asp Ser Phe Glu Asn Ile Glu Val Tyr 675 680 685

Asn Leu Met Cys Asp Leu Leu Asn Leu Ile Pro Ala Ser Asn Asn Gly 690 695 700

Thr His Gly Ser Leu Asn His Leu Leu Lys Asn Pro Ile Tyr Thr Pro 705 710 715 720

Thr His Pro Lys Glu Val Ser Ser Leu Val Gln Cys Pro Phe Thr Arg 725 730 735

Thr Pro Arg Asn Asn Leu Gly Cys Ser Cys Asn Pro Ser Ile Leu Pro 740 745 750

Ile Val Asp Leu Gln Thr Gln Leu Asn Leu Thr Met Ala Glu Glu Asn 755 760 765

Ile Ile Lys Arg Gly Thr Leu Pro Tyr Gly Arg Pro Arg Val Ile Gln 770 775 780

Lys Asn Ser Thr Val Cys Leu Leu Tyr Gln His Gln Phe Val Ser Gly 785 790 795 800

Tyr Ser His Asp Ile Leu Met Pro Leu Trp Thr Ser Tyr Thr Val Asp 805 810 815

Arg Asn Asp Ile Phe Ser Thr Glu Asp Phe Ser Asn Cys Leu Tyr Gln 820 825 830

Asp Leu Arg Val Ser Leu Ser Pro Val His Glu Cys Ser Phe Tyr Lys 835 840 845

Asn Asn Ala Lys Leu Ser Tyr Gly Phe Leu Ser Pro Pro Gln Leu Asn 850 855 860

Lys Gly Ser Ser Lys Ile Asn Ser Glu Ala Leu Leu Thr Thr Asn Ile

PAT 1825 LU 865 870 875 880 LU501430

Val Pro Met Tyr Gln Ser Phe Gln Val Ile Trp His Tyr Phe His Gly 885 890 895

Thr Leu Leu Gln Arg Tyr Ala Gln Glu Arg Asn Gly Val Asn Val Val 900 905 910

Ser Gly Pro Val Phe Asp Ser Asp Phe Asp Gly Arg Tyr Asp Ser Ser 915 920 925

Glu Thr Leu Lys Gln Asn Ser Arg Arg Val Arg Asn Gln Glu Val Leu 930 935 940

Ile Pro Thr His Phe Phe Ile Val Leu Thr Ser Cys Lys Asp Pro Asp 945 950 955 960

Gln Ala Pro Ser Gln Cys Glu Asn Leu Asp Ala Ser Ala Phe Ile Leu 965 970 975

Pro His Arg Thr Asp Asn Ser Glu Ser Cys Val His Gly Lys Gln Glu 980 985 990

Ser Ser Trp Val Glu Glu Leu Leu Arg Leu His Arg Ala Arg Ile Thr 995 1000 1005

Asp Val Glu Gln Ile Thr Gly Leu Ser Phe Tyr Gln Glu Arg Lys Glu 1010 1015 1020

Pro Val Ser Asp Ile Leu Lys Leu Lys Thr His Leu Pro Ile Phe Asn 1025 1030 1035 1040

Gln Glu Asp <210> 9 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> SUSD2 peptide <400> 9

Cys Gly Ala Leu Asp Gly Pro Cys Ser Cys His Pro Thr Cys Ser Gly 1 5 10 15

Leu Gly Thr Cys Cys Leu Asp Phe Arg <210> 10 <211> 16 56

PAT 1825 LU <212> PRT LU501430 <213> Artificial Sequence <220> <223> ENPP1 peptide <400> 10

Ala Glu Tyr Leu His Thr Trp Gly Gly Leu Leu Pro Val Ile Ser Lys 1 5 10 15 57

Claims (19)

PAT 1825 LU Claims LU501430

1. A method for isolation of circulating tumor cells (CTCs) and/or disseminated tumor cells (DTCs) from a blood, lymph or bone marrow sample of a subject, comprising: a) obtaining from the subject a sample containing cells to be isolated, b) exposing the cells to anti-SUSD2 or anti-ENPPI antibodies conjugated to particles or a matrix having a separation functionality for a time sufficient for CTCs and/or DTCs to attach to the anti-SUSD2 or anti-ENPPI antibody conjugated particles or matrix, c) separating the particles or matrix from the sample using the separation functionality of the particles or matrix, thereby isolating CTCs and/or DTCs.

2. The method of claim 1, wherein the particles are magnetic nanoparticles, and wherein separating involves a magnetic field.

3. The method of claim 2, wherein the anti-SUSD2 or anti-ENPPI antibodies conjugated to the magnetic nanoparticles are labelled with or contain at least one fluorophor.

4. The method of claim 1, wherein anti-SUSD2 or anti-ENPPI antibodies are against full length SUSD2 or ENPPI.

5. The method of claim 1, wherein anti-SUSD2 or anti-ENPPI antibodies are against truncated length SUSD2 or ENPPI.

6. The method of claim 1, wherein the matrix is an organic matrix having anti-SUSD2 or anti-ENPPI antibodies coupled via reactive functional groups, and wherein separation involves sedimentation by gravity or centrifuge.

7. The method of claim 6, wherein the organic matrix is agarose.

8. The method as in claim 1, the CTCs or DTCs having a mesenchymal phenotype, wherein the CTCs or DTCs having a mesenchymal phenotype (MCTC, mDTC) have a predominant or exclusively mesenchymal phenotype, or a hybrid epithelial/mesenchymal phenotype (emCTC, emDTC). 58

PAT 1825 LU

9. The method of claim 1, wherein the anti-SUSD2 or anti-ENPPI antibody conjugated ~~ LU501430 particles are anti-SUSD2 antibody conjugated particles.

10. The method of claim 1, wherein the anti-SUSD2 or anti-ENPPI antibody conjugated particles are anti-ENPPI antibody conjugated particles.

11. A method for the isolation and detection of circulating tumor cells (CTCs) and/or disseminated tumor cells (DTCs) from a blood, lymph or bone marrow sample of a subject, comprising: a) obtaining a blood, lymph or bone marrow sample from the subject, b) exposing the cells to a first anti-SUSD2 or anti-ENPPI antibody, c) isolating any bound SUSD2 or ENPPI positive cells from the sample, d) exposing bound SUSD2 or ENPPI positive cells to a second antibody for detection of bound SUSD2 or ENPPI positive cells.

12. The method according to claim 11, wherein the second antibody 1s a labeled secondary antibody directed against the first anti-SUSD2 or anti-ENPPI antibody, a second anti-SUSD2 or anti-ENPPI antibody, or a labeled second anti-SUSD2 or anti-ENPPI antibody, or a labeled secondary antibody directed against a second bound anti-SUSD2 or anti-ENPPI antibody.

13. The method according to claim 11, further comprising isolation of T-cells from the blood of the subject, genetic modification of the T-cells to recognize SUSD2 or ENPPI surface protein and re-injection of the genetic modified T-cells into the subject.

14. The method according to claim 11, wherein isolation is done immunomagnetically using anti-SUSD2 or anti-ENPP1 antibodies coupled directly or indirectly to magnetic nanoparticles.

15. The method according to claim 14, wherein cells labeled with anti-SUSD2 or anti- ENPPI antibodies coupled to magnetic nanoparticles are applied to a ferromagnetic iron- column positioned in a magnetic field, wherein labeled cells are retained in the column and unlabeled or antigen-negative cells pass the column and are discarded, and wherein the ferromagnetic iron-column is removed from the magnetic field and the labeled cells are eluted and available for further analysis. 59

PAT 1825 LU LU501430

16. The method according to claim 11, wherein the circulating tumor cells (CTCs) and/or disseminated tumor cells (DTCs) have a mesenchymal phenotype, or a hybrid epithelial/mesenchymal phenotype.

17. The method of claim 11, wherein the anti-SUSD2 or anti-ENPPI antibody is anti- SUSD2 antibody.

18. The method of claim 11, wherein the anti-SUSD2 or anti-ENPPI antibody is anti- ENPP1 antibody.

19. A method for the isolation and detection of circulating tumor cells (CTCs) and/or disseminated tumor cells (DTCs) from a blood, lymph or bone marrow sample of a subject, comprising: a) obtaining a sample containing cells to be tested, b) exposing the cells to a first anti-SUSD2 or anti-ENPPI antibody for capturing of the cells, c) isolating any bound SUSD2 or ENPPI positive cells from the sample, d) exposing bound SUSD2 or ENPPI positive cells to a second anti-SUSD2 or anti- ENPPI antibody and/or an anti-keratin antibody for the immunofluorescent detection of bound SUSD2 or ENPPI positive cells, wherein the anti-SUSD2 or anti-ENPPI antibody is labelled with a different fluorophor from the anti-keratin antibody, e) classifying SUSD2 or ENPPl/keratin double positive isolated cells as eCTC or emCTC and SUSD2 or ENPPI positive/keratin negative cells as MCTC. 60