Phospholipid ether analogs for identification and isolation of circulating tumor cells.

Abstract

Phospholipid ether analogs for identification and isolation of circulating tumor cells: The present invention is directed to a method for identifying, isolating, and enabling downstream analysis of circulating tumor cells comprising placing a blood or blood serum sample from an individual in contact with a composition comprising a phospholipid ether analog linked to a luminescent molecule or a magnetic sphere and subjecting the individual's blood or blood serum sample to fluorescence microscopy, flow cytometry, or magnetic isolation.

Description

1 / 31 “Phospholipid ether analogs for identification and isolation of circulating tumor cells” CROSS-REFERENCE TO RELATED REQUEST(S)

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 349,713, filed June 14, 2016, the entire contents of which are incorporated herein by reference. BACKGROUND

[0002] Circulating tumor cells (CTCs) are blood-based markers that hypothetically possess predictive and prognostic value in cancer detection and progression. Specifically, CTCs are theorized to be a minimally invasive source of tumor cells from both the primary tumor and metastatic sites. (Alix Panabiens C., et al., Challenges in circulating tumor cell research, Nat Rev Cancer, September 2014, 14(9), 623-31 and Yap T., et al., Circulating tumor cells: a multifunctional biomarker, Clin Cancer Res, May 15, 2014, 20(10), 255-368). Many cancer types are known or predicted to give rise to CTCs, including multiple myeloma. Paiva B., et al., Detailed characterization of multiple myeloma circulating tumor cells shows unique phenotypic, cytogenetic, functional, and circadian distribution profile, Blood, 21 November 2013, 122(22), 3591-3598.Furthermore, tumor stem cells (TSCs), another possible type of tumor cell predicted to have prognostic value, are theorized to be a subpopulation of CTCs. Scatena R. et al., Circulating tumor cells and cancer stem cells: a role for proteomics in defining the interrelationships. Petition 870190036514, dated 04 / 16 / 2019, page 11 / 81 2 / 31 between function, phenotype and differentiation with potential clinical applications, Biochim Biophys Acta, April 2013; 1835(2), 129-143; Faltas B., Cornering metastases: therapeutic targeting of circulating tumor cells and stem cells, Front Oncol, July 3, 2012, (2)68. By definition, a CTC is a nucleated cell, positive for CD45, positive for epithelial cell adhesion molecule (EpCAM) and positive for pancytokeratin. However, the identification and isolation of CTCs from whole blood is technically challenging, since CTCs are extremely rare and can be found in very low concentrations, on the order of 1 cell in 7.5 milliliters (mL) of blood (i.e., 1 in several billion cells).

[0003] Currently, the only accepted and approved indication for CTCs is enumeration as a prognostic marker of cancer progression. The CellSearch® system (CellSearch is a registered trademark of Johnson & Johnson Corp.) is currently the only assay approved by the Food and Drug Administration (FDA) to enumerate CTCs. (Ignatiadas M., et al., Circulating tumor cells and circulating tumor DNA for precision medicine: dream or reality?, Ann Oncol, December 2014, 25(12), 2304-13 and Toss A., et al., CTC enumeration and characterization: moving toward personalized medicine, Ann Transl Med, November 2014, 2(11):108, 116). Unfortunately, CellSearch® is only approved for metastatic breast, colorectal, and prostate cancer. (Hayes DF, et al., Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival, Clin Cancer Petition 870190036514, dated 04 / 16 / 2019, page 12 / 81 3 / 31 Res, 15 de Julho de 2006, 12(1), 4218-24; Cristofanilli M, et al. , Circulating tumor cells, disease progression, and survival in metastatic breast cancer, N Engl J Med, 19 de Agosto de 2004, 351(8), 781-91; De Bono J. S., et al. , Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer, Clin Cancer Res, 01 de Outubro de 2008, 14(19), 63029 e Cohen S. J., et al., Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer, J Clin Oncol, 1 de Julho de 2008, 26(19), 3213-21).

[0004] There are still challenges in identifying CTCs in accordance with the current definition. Firstly, there is potential for false-positive results due to circulating epithelial cells being positive for EpCAM. Secondly, false-negative results may occur due to tumor cells undergoing epithelial-mesenchymal transition, resulting in reduced expression of epithelial markers (Yu M., et al., Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition, Science, February 1, 2013, 339(6119), 580-4). Thirdly, there is growing evidence in the literature that not all CTCs express EpCAM and that certain types of cancer, such as renal cancer, have low or heterogeneous expression of EpCAM (Eichelberg C, et al., Epithelial cell adhesion molecule is an independent prognostic marker in clear cell renal carcinoma, Int J Cancer, July 15, 2013, 132(12), 2948-55 and Spizzo G., et al., EpCAM expression in primary tumor tissues and. Petition 870190036514, dated 04 / 16 / 2019, page 13 / 81 4 / 31 metastases: an immunohistochemical analysis, J Clin Pathol, May 2011, 64(5):415-20. Thus, there is a clinical need for an economical and robust assay using a broad tumor marker that can potentially identify all CTCs without bias. Cancer-targeted alkylphosphocholine (APC) analogs offer a novel process for identifying and isolating CTCs from a wide range of different cancer types. SUMMARY OF THE INVENTION

[0005] The present invention is directed to a process for identifying one or more circulating tumor cells, a process comprising: (i) placing a blood or blood serum sample from an individual in contact with a composition comprising a phospholipid ether (PLE) analogue linked to an article selected from the group consisting of a luminescent molecule and a magnetic sphere; and (ii) subjecting the blood or blood serum sample from an individual to fluorescence microscopy or flow cytometry, wherein, if the article is a magnetic sphere, the article is linked to the PLE by means of a ligand.

[0006] The present invention is further directed to a process for isolating one or more circulating tumor cells, a process comprising the steps of: (i) administer a composition that includes a PLE analogue linked to a selected article from the group Petition 870190036514, dated 04 / 16 / 2019, p. 14 / 81 5 / 31 consisting of a luminescent molecule and a magnetic sphere, to an individual; and (ii) subjecting an individual's blood or blood serum sample to flow cytometry or a magnetic field, wherein, if the article is a magnetic sphere, the article is linked to the PLE via a ligand, and an individual's blood or blood serum sample is subjected to a magnetic field, and wherein, if the article is a luminescent molecule, then an individual's blood or blood serum sample is subjected to flow cytometry.

[0007] In one embodiment, one or more circulating tumor cells comprise tumor stem cells. For the preferred age, the analogue of the present invention is a compound comprising: , formula (I); 2CH2N(CH3)3, formula (II);

[0008] In a PLE linked to an article selected from X-(CH2)n -opoch2ch2% X II (CH2)n-OPOCH II X-(CH2)nO-CH2CHCH2O-P-OCH2CH2N(CH3)3 and 4 O , formula (III); and X ο θ (CH2)n -O -GH2C HGH2O -p - OCH2CHM CH3)3I IY% formula (IV), where: n is an integer from 16 to 30, preferably 18; Petition 870190036514, dated 04 / 16 / 2019, p. 15 / 81 6 / 31 Y is selected from the group comprising -H, OH, -OR1, -C(O)OH, and -OC(O)R1, where R1 is an alkyl group; and X is either a luminescent molecule or a magnetic sphere.

[0009] In a preferred embodiment, the luminescent molecule is a fluorophore, more preferably the fluorophore is selected from the group comprising: independently selected from H, CH3, C2H5 and C3H7, most preferably:

[0010] In another preferred embodiment, the magnetic sphere is selected from the group comprising nanomagnetic spheres, micromagnetic spheres, paramagnetic spheres and superparamagnetic spheres, wherein the magnetic sphere is linked to the PLE analog via a ligand selected from the group comprising a biotin-streptavidin ligand, an azetidinone ligand Petition 870190036514, dated 04 / 16 / 2019, page 16 / 81 7 / 31 and a ligand of amine, azide, alkyne, carboxyl and hydroxyl groups and combinations thereof.

[0011] In another preferred embodiment, one or more circulating tumor cells of the present invention are selected from the group comprising breast cancer, lung cancer, thyroid cancer, cervical cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer and a colorectal cancer cell, a multiple myeloma cell, a tumor stem cell, preferably breast cancer, lung cancer, thyroid cancer, cervical cancer, squamous cell carcinoma, prostate cancer, a pancreatic cancer cell and a colorectal cancer cell and more preferably a prostate cancer cell or a pancreatic cancer cell.In another preferred embodiment, the processes of the present invention can be used in additional downstream data acquisition technologies including, but not limited to, protein isolation, RNA isolation, DNA isolation, gene translocation and / or amplification analysis, and fluorescence in situ hybridization. BRIEF DESCRIPTION OF THE FIGURE

[0012] Figure 1 - Plot of the analysis of CD45-, CD34- cells isolated from patient 108 (colorectal cancer). Panel A shows cells stained with all markers except CD14. Panel B shows cells stained with all markers. The upper left quadrant indicates CD14- / CLR1501+ cells, the upper right quadrant indicates CD14+ / CLR1501+ cells, the lower right quadrant indicates Petition 870190036514, dated 04 / 16 / 2019, page 17 / 81 8 / 31 cells CD14+ / CLR1501- and the lower left quadrant indicates cells CD14- / CLR1501. DETAILED DESCRIPTION OF THE INVENTION

[0013] PLE analogs have the ability to identify, isolate, and enable downstream analysis of CTCs of all types. Tumor cells have five to ten times more lipid rafts compared to healthy cells. Lipid rafts are specialized regions of the phospholipid bilayer membrane that contain high concentrations of cholesterol and sphingolipids, and serve to organize the cell surface and intracellular signaling molecules (e.g., growth factors and cytokine receptors, the phosphatidylinositol 3-kinase (PI3K) / Akt survival pathway). Data suggest that lipid rafts serve as entry portals for PLEs. The marked selectivity of these compounds for tumor cells versus non-tumor cells is attributed to the high affinity of PLEs for cholesterol and the abundance of cholesterol-rich lipid rafts in tumor cells.The fundamental role played by lipid rafts is highlighted by the fact that disruption of the lipid raft architecture suppresses the uptake of lipid emulsions (LEAs) in tumor cells. It has been shown that LEA uptake is reduced by 60% when lipid rafts are prevented from forming.

[0014] Preliminary results obtained in more than 55 xenograft and spontaneous tumor models universally showed that CLR1404 undergoes selective uptake and prolonged retention in tumors. Weichert, JP et al., Alkylphosphocholine analogs for broad-spectrum cancer Petition 870190036514, dated 04 / 16 / 2019, page 18 / 81 9 / 31 Imaging and Therapy, Sci Transl Med, June 11, 2014, 6(240ra75). What was not known previously was whether PLE analogs were capable of being absorbed by CTCs to the extent that CTCs could be identified and isolated.

[0015] The present invention is directed to a process for identifying one or more circulating tumor cells, a process comprising: (i) placing a blood or blood serum sample from an individual in contact with a composition comprising a phospholipid ether (PLE) analogue linked to an article selected from the group consisting of a luminescent molecule and a magnetic sphere; and (ii) subjecting the blood or blood serum sample from an individual to fluorescence microscopy or flow cytometry, wherein, if the article is a magnetic sphere, the article is linked to the PLE via a ligand.

[0016] The present invention is further directed to a process for isolating one or more circulating tumor cells, a process comprising the steps of: (i) placing a blood or blood serum sample from an individual in contact with a composition that includes a PLE analogue linked to an article selected from the group consisting of a luminescent molecule and a magnetic sphere; and (ii) subjecting a blood or blood serum sample from an individual to flow cytometry, preferably Petition 870190036514, dated 04 / 16 / 2019, page 19 / 81 10 / 31 screening of cells activated by fluorescence or a magnetic field, wherein, if the item is a magnetic sphere, the item is linked to the PLE through a ligand and the blood or blood serum sample from an individual is subjected to a magnetic field and wherein, if the PLE is linked to a luminescent molecule, then the blood or blood serum sample from an individual is subjected to flow cytometry. For the preferred age, the analogue of the present invention is a compound comprising: , formula (I); ©2CH2N(CH3)3, formula (II);

[0017] In a PLE linked to an article selected from X-(CH2)nθ ® -opoch2ch2nάθ X II (CH2)n-OPOCH °© II ·1? X-(CH3Jn-O-CH2CHCH2O-P-OCH£CH2N(CH3}3 1, formula (III); and OΟΘ -P-OCH2CH^N(CH3)3 Q ® , formula (IV) , where: n is an integer from 16 to 30, preferably 18; Y is selected from the group comprising -H, OH, -OR1, -0(0) OH, and -00(0)Hχ, where R1 is an alkyl group; and X is either a luminescent molecule or a magnetic sphere. Petition 870190036514, dated 04 / 16 / 2019, p. 20 / 81 11 / 31

[0018] In a preferred embodiment, the most preferably luminescent molecule is a fluorophore. where each R is independently selected from H, CH3, C2H5 and C3H7, most preferably

[0019] In another preferred embodiment, the magnetic sphere is selected from the group comprising nanomagnetic spheres, micromagnetic spheres, paramagnetic spheres and superparamagnetic spheres, wherein the magnetic sphere is linked to the PLE analog by means of a ligand selected from the group comprising a biotin-streptavidin ligand, an azetidinone ligand and an amine, azide, alkyne, carboxyl and hydroxyl group ligand and combinations thereof. Petition 870190036514, dated 04 / 16 / 2019, page 21 / 81 12 / 31

[0020] In another preferred embodiment, one or more circulating tumor cells of the present invention are selected from the group comprising breast cancer, lung cancer, thyroid cancer, cervical cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer and a colorectal cancer cell, and a tumor stem cell and a malignant plasma cell, preferably a prostate tumor cell or a pancreatic tumor cell.

[0021] In another preferred embodiment, the processes of the present invention can be used in additional downstream data acquisition technologies including, but not limited to, protein isolation, RNA isolation, DNA isolation, gene translocation and / or amplification analysis and fluorescence in situ hybridization. Definitions

[0022] In general, reference to “a circulating tumor cell” is intended to refer to a single cell, while reference to “circulating tumor cells” or “cluster of circulating tumor cells” is intended to refer to more than one tumor cell. However, a person skilled in the art would understand that reference to “circulating tumor cells” is intended to include a population of circulating tumor cells, including one or more circulating tumor cells, while reference to “a circulating tumor cell” may include more than one circulating tumor cell. Petition 870190036514, dated 04 / 16 / 2019, page 22 / 81 13 / 31

[0023] The term “circulating tumor cell” or “circulating tumor cells,” as used herein, refers to any tumor cell or cluster of tumor cells found in the blood or blood serum sample of an individual. CTCs may also contain or comprise a tumor stem cell or cluster of tumor stem cells found in an individual’s blood or blood serum sample.

[0024] As used herein, the term “tumor stem cell” refers to a tumor cell capable of self-renewal and differentiation into different types of tumor cells found in a malignant tumor.

[0025] The term “cancer,” as used herein, refers to, but is not limited to, a variety of cancer types, including breast cancer, including male breast cancer, digestive / gastrointestinal cancers, including anal cancer, appendix cancer, extrahepatic bile duct cancer, gastrointestinal carcinoid tumor, colon cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumors (GISTs), islet cell tumors, primary liver cancer in adults, liver cancer in infants, pancreatic cancer, rectal cancer, small bowel cancer, and stomach (gastric) cancer; Endocrine and neuroendocrine cancers, including pancreatic adenocarcinoma, adrenocortical carcinoma, pancreatic neuroendocrine tumors, Merkel cell carcinoma, non-small cell lung neuroendocrine tumor, small cell lung neuroendocrine tumor, parathyroid gland cancer, Petition 870190036514, dated 04 / 16 / 2019, p. 23 / 81 14 / 31 pheochromocytoma, pituitary tumor and thyroid cancer; eye cancers, including intraocular melanoma and retinoblastoma; genitourinary cancer, including bladder cancer, kidney cancer (renal cells), penile cancer, prostate cancer, transitional cell renal pelvis cancer and ureter cancer, testicular cancer, urethral cancer and Wilms' tumor; germ cell cancers, including central nervous system cancer in infants, extracranial germ cell tumor in infants, extragonadal germ cell tumor, ovarian germ cell tumor and testicular cancer; gynecological cancers, including cervical cancer, endometrial cancer, gestational trophoblastic tumor, epithelial ovarian cancer, ovarian germ cell tumor, uterine sarcoma, vaginal cancer and vulvar cancer;Head and neck cancers, including hypopharyngeal cancer, laryngeal cancer, lip cancer and oral cavity cancer, metastatic squamous cell carcinoma with primary occult cancer, oral cancer, nasopharyngeal cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, pharyngeal cancer, salivary gland cancer and throat cancer; leukemias, including acute lymphoblastic leukemia in adults, acute lymphoblastic leukemia in infants, acute myeloid leukemia in adults, acute myeloid leukemia in infants, chronic lymphocytic leukemia, chronic myelogenous leukemia and hairy cell leukemia; multiple myeloma, including malignant plasma cells; lymphomas, including AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma in adults, lymphoma; Petition 870190036514, dated 04 / 16 / 2019, page 24 / 81 15 / 31 Hodgkin's lymphoma in infants, Hodgkin's lymphoma during pregnancy, mycosis fungoides, non-Hodgkin's lymphoma in adults, non-Hodgkin's lymphoma in infants, non-Hodgkin's lymphoma during pregnancy, lymphoma of the nervous system, Sézary syndrome and Waldenstrom macroglobulinemia; musculoskeletal cancers, including Ewing's sarcoma, osteosarcoma and malignant fibrous histiocytoma of bone, rhabdomyosarcoma and soft tissue sarcoma in infants; neurological cancers, including brain tumor in adults, brain tumor in infants, astrocytoma, brainstem glioma, atypical rhabdoid / teratoid tumor of the central nervous system, embryonal tumors of the central nervous system, craniopharyngioma, ependymoma, neuroblastoma, primary central nervous system (CNS) lymphoma; Respiratory / thoracic cancers, including non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, thymoma, and thymic carcinoma;and skin cancers, including Kaposi's sarcoma, melanoma, and squamous cell carcinoma.

[0026] As used herein, the term sample refers to any sample suitable for the processes provided by the present invention. The sample may be any sample that includes circulating tumor cells suitable for detection. Sample sources include whole blood, bone marrow, pleural fluid, peritoneal fluid, central spinal fluid, urine, saliva, and bronchial washings. In one aspect, the sample is a blood sample, including, for example, whole blood or any fraction or component thereof. A blood sample, suitable for Petition 870190036514, dated 04 / 16 / 2019, p. 25 / 81 16 / 31 For use with the present invention, it can be extracted from any known source that includes blood cells or their components, such as veins, arteries, peripheral tissues, umbilical cord and the like. For example, a sample can be obtained and processed using well-known and routine clinical processes (e.g., procedures for extracting and processing whole blood). In one embodiment, an exemplary sample may include peripheral blood extracted from an individual with cancer.

[0027] As used herein, the term identify or identification refers to visualizing the existence of a CTC.

[0028] As used herein, the term isolate or isolation refers to physically separating CTCs from other cell types found in an individual's sample.

[0029] As used herein, the term contact or contacting refers to causing an individual, tissue, organ or cells to come into contact with a PLE analogue of the present invention. As used herein, the contact may be made ex vivo or in vitro, i.e., in a test tube, on cells or tissues of living organisms, for example, humans. A patient or individual, used equivalently herein, refers to a mammal, preferably a human being.

[0030] As used herein, the term alkyl refers to a branched or straight-chain alkyl group comprising a saturated hydrocarbon group of 1 to 24 carbon atoms (C1-C24), unless otherwise indicated. The alkyl group may be cyclic or acyclic. Petition 870190036514, dated 04 / 16 / 2019, p. 26 / 81 17 / 31

[0031] As used herein, the term amine refers to a functional group comprising a nitrogen atom with a single pair of electrons.

[0032] As used herein, the term azide refers to a functional group comprising three consecutive nitrogen atoms.

[0033] As used herein, the term alkyne refers to a functional group comprising two carbon atoms that have triple bonds between them.

[0034] As used herein, the term carboxyl refers to a functional group comprising a C(O)O structure.

[0035] As used herein, the term hydroxyl refers to a functional group comprising an OH.

[0036] As used here, n is an integer from 16 to 30.

[0037] As used herein, Y is selected from the group comprising H, -OH, -OR, -C(O)OH, and OC(O)R.

[0038] As used herein, R refers to an alkyl group.

[0039] As used herein, the term R refers to H, CH3, C2H5 and C3H7.

[0040] As used herein, X is a luminescent molecule or a magnetic sphere bonded to a ligand.

[0041] The flow cytometry useful in the present invention includes, but is not limited to, fluorescence-activated cell separation (FACS) and multicolor flow cytometry. Petition 870190036514, dated 04 / 16 / 2019, page 27 / 81 18 / 31

[0042] Magnetic spheres useful in the present invention include, but are not limited to, nanomagnetic spheres having a size in the nanometer range and which are sometimes referred to as magnetic nanoparticles, for example, 50 nm MACS® spheres (MACS is a registered trademark and is available from Miltenyi Biotec GmbH), micromagnetic spheres with a size in the micrometer range, for example, 1 μm to 3 μm Dynabeads® spheres (Dynabeads is a registered trademark and is available from Invitrogen Dynal AS Corp), paramagnetic spheres and superparamagnetic spheres.

[0043] Luminescent molecules useful in the present invention include fluorophores.

[0044] Fluorophores include, but are not limited to, Alexa Fluor® compounds (Alex Fluor is a registered trademark of Molecular Probes, Inc.), including Brilliant Violet™ compounds 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750 and 790 (Brilliant Violet is available from BioLegend®), including Brilliant Ultra-Violet™ compounds 420, 510, 605, 650, 711 and 786 (Brilliant Ultra-Violet is available from BD Biosciences, Inc.), including those with frequencies of 395 and 737 nm, Dylight® compounds (Dylight is a registered trademark and available from Pierce Biotechnology, Inc.), including 350, 405, 488, 550, 594, 633, 650, 680, 755 and 800, Violetfluor® 450, Redfluor® 710, (Violetfluor and Redfluor are registered trademarks and are available from Tonbo Biotechnologies Corporation), allophycocyanin (APC), APC Alexa Fluor® 750, APC-Cy7, Petition 870190036514, dated 04 / 16 / 2019, p. 28 / 81 19 / 31 chlorophyll protein peridinin (PerCP), PerCP-Cy5, PerCPCy5.5, PerCP-Cy7, propidium iodide (PI), phycoerythrin (PE), PE-Cy5, PE-Cy5.5, PE-Cy7, PE-exas RED® (Texas Red is a registered trademark of Molecular Probes, Inc.), fluorescein (FITC), aminomethylcoumarin (AMCA), Marina Blue®, Cascade Blue® (Marina Blue and Cascade Blue are registered trademarks and available from Molecular Probes, Inc.), Cascade Yellow, Pacific Blue, Qdot® 605 (Qdot is a registered trademark of Life Technologies Corp), tetramethylrhodamine (TR1TC), Cy3, Cy5, Cy5.5, Texas Red® and compounds of the following structures: selected independently of H, CH3, C2H5 and C3H7.

[0045] The ligands useful in the present invention include, but are not limited to, a linkage, a biotin-streptavidin linker, an azetidinone linker, and an amine, azide, alkyne, carboxyl, and hydroxyl group linker and combinations thereof. Petition 870190036514, dated 04 / 16 / 2019, page 29 / 81 20 / 31

[0046] The following preferred embodiments are provided for illustrative purposes only and are not intended in any way to limit the invention. Preferred Options

[0047] In a preferred embodiment, the present invention is directed to a process for identifying a circulating prostate tumor cell, a process comprising: (i) placing a blood or blood serum sample from an individual in contact with a composition comprising a compound of: (ii) subjecting an individual's blood or blood serum sample to fluorescence microscopy or flow cytometry.

[0048] In a preferred embodiment, the present invention is directed to a process for isolating a circulating prostate tumor cell, a process comprising: Petition 870190036514, dated 04 / 16 / 2019, page 30 / 81 21 / 31 (i) placing a blood or blood serum sample from an individual in contact with a composition comprising a compound of: II © (CH2)IS—OPOCH2CH2NMe3formula (V) CLR1501 or (CHjhs-OPOCH2CH2NMe3formula (VI) CLR1502; and (ii) subjecting an individual's blood or blood serum sample to fluorescence-activated cell screening.

[0049] The following examples are provided for illustrative purposes only and are not intended in any way to limit the invention. EXAMPLES Example 1 - Synthesis of a conjugate of magnetic spheres - PLE

[0050] Firstly, both a PLE of formula I-IV and a magnetic sphere, as described herein, are linked to their own functional group. The functional groups are then linked by means of click chemistry. As an example, a PLE of formula I of the present invention can be linked to an azide functional group and a magnetic sphere can be linked to an alkyne functional group. The azide and alkyne functional groups can then be linked by means of click chemistry, such as azide-alkyne cycloaddition. Petition 870190036514, dated 04 / 16 / 2019, page 31 / 81 22 / 31 copper-catalyzed (CuAAC). In general, magnetic spheres bonded to a functional group are known as “functionalized magnetic spheres” and are available from various sources, such as Nanocs, Inc. I II ® (CH2)i8OPOCH2CH2NMe3 THE NaN3, THE II CLR1401 CuI, Ascorbato de Na EtOH-H2O, 80 °C, 95% (CH2)18OPOCH2CH2NMe3O Azida CLR1401 H2, Pd / C / =\ ii ® ----------- H2N—4 / (CH2)18OPOCH2CH2NMe3MeOH, 87%1' ^ AzidaCLR1401 Síntese de azida CLR1401

[0051] 18-(p-iodophenyl)octadecyl phosphocholine (4.01 g, 6.3 mmol), sodium azide (818 mg, 12.6 mmol), and sodium ascorbate (140 mg, 0.71 mmol) were dissolved in a mixture of degassed ethanol (28 mL) and water (12 mL) in the reaction vessel. Copper(I) iodide (120 mg, 0.63 mmol) and N,N'-dimethylethylenediamine (0.1 mL, 0.94 mmol) were added to the reaction mixture. The reaction vessel was tightly closed, and the mixture was stirred at 80°C for 45 minutes. The reaction mixture was cooled to room temperature, water (60 mL) was added, and the mixture was stirred for 30 minutes in the open air. The mixture was transferred to a separatory funnel, chloroform (80 mL) and methanol (52 mL) were added, and extraction was performed by shaking. The chloroform layer was removed, and the extraction was repeated (2 x 80 mL of chloroform). Combined chloroform extracts were washed with 0.01 N HCl, dried over Na2SO4, filtered, and evaporated to dryness.The residue was dissolved in chloroform (4 mL) and acetone (170 mL) was added slowly with stirring. The mixture was stirred. Petition 870190036514, dated 04 / 16 / 2019, page 32 / 81 23 / 31 for 30 minutes and filtered. The product was rinsed in the filter with acetone and dried under high vacuum to yield 3.31 g (95%) of 18-(p-iodophenyl)octadecyl phosphocholine. Bonding of a PLE-azide to an alkyne-functionalized magnetic sphere / =\OCu(I) / ==\O® X = H + N3—4 (CH2)18OPOCH2CH2NMe3----- O^ ON=No Magnetic spheres, alcine function Azide CLR1401

[0052] Azide CLR1401 is linked to an alkyne-functionalized magnetic bead via “click” chemistry. An example of CuAAC is shown above. CuAAC is described in Himo F., et al., Copper(I)-catalyzed synthesis of azoles. DFT study predicts unprecedented reactivity and intermediates, J Am Chem Soc, January 12, 2005, 127(1), 210-216, which is incorporated by reference in its entirety. Briefly, the alkyne-functionalized magnetic bead and azide CLR1401 are mixed in a 1:1 ratio of water and tert-butyl alcohol in the presence of a copper oxide (Cu(I)) catalyst for 6 to 12 hours. Optionally, sodium ascorbate is added to the mixture. The final magnetic-PLE bead can then be isolated from the solution using filtration or simple extraction. N. XN 'N XN3 + Magnetic spheres, azide function H O ® (CH2) 18OPOCH2CH2NMe3 THE CLR1401 acetylene Cu(I) O ® (CH2)18OPOCH2CH2NMe3 THE Connecting a PLE-acetylene to an azide-functionalized magnetic sphere

[0053] Acetylene CLR1401 is bonded to an azide-functionalized magnetic sphere via chemistry Petition 870190036514, dated 04 / 16 / 2019, p. 33 / 81 24 / 31 click. The same CuAAC reaction used for binding azide CLR1401 to an alkyne-functionalized magnetic sphere can be used co ..11 / =\11 X- NH2+ HO-C—(CH2)18OPOCH2CH2NMe3 Magnetic nanoparticles, ftjnçãoamma carboxjCLR1401 Agent of , , coupling ito above. coupling agent HO / =\ O ® XNC—(CH2)18OPOCH2CH2NMe3O Binding of a PLE-carboxy to a magnetically functionalized amine nanoparticle

[0054] Carboxi CLR1401 is linked to an amine-functionalized magnetic nanoparticle via an amide linkage. The amide linkage can be achieved using any suitable coupling reagent for amide linkage formation, such as reagents used for peptide synthesis including, but not limited to, (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium) hexafluorophosphate (HBTU), (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5b]pyridinium) 3-oxide hexafluorophosphate (HATU), COMU® (COMU is available from Sigma Aldrich Co., LLC is a registered trademark of Luxembourg. Biotechnologies Ltd.) and propanephosphonic acid anhydride (PPAA or T3P®, T3P is available and is a registered trademark of Euticals SPA). X-NH2 + Magnetic nanoparticles, amine function IIw(CH2)i8OPOCH2CH2NMe3 Carboxi-AZD CLR1401 OO OΘY_NA_ / ''-N-1LHC^(CH2)i8OPOCH2CH2NMe3XH HO Binding of a carboxy-AZD PLE to an amine-functionalized magnetic nanoparticle

[0055] Alternatively to the amide linkage described above, amine-functionalized magnetic nanoparticles can be linked to a carboxy-PLE via a Petition 870190036514, dated 04 / 16 / 2019, p. 34 / 81 25 / 31 azetidinone ligand (AZD). As an example, carboxy CLR1401AZD can be linked to an amine-functionalized magnetic nanoparticle via an AZD ligand. AZD linkage is described in Roberts LR et al., Kappa Agonist CovX-Bodies, Bioorg Med Chem Lett, June 15, 2012, 22(12), 4173-4178 and Sato S. et al., Chemically Programmed Antibodies AS HIV-1 Attachment Inhibitors, ACS Med Chem Lett, May 9, 2013, 4(5), 460-465. Example 2 - Identification and counting of circulating tumor cells of lung, thyroid, breast, cervical, squamous cell carcinoma, and colorectal cancer in patients using a fluorescent PLE analog. Processes

[0056] Whole blood was collected in Cell Save® collection tubes or ethylenediaminetetraacetic acid (EDTA) collection tubes from seven patients with lung cancer (patients 101 and 103), thyroid cancer (patient 102), breast cancer (patient 106), cervical cancer (patient 104), squamous cell carcinoma (patient 107), and colorectal cancer (patient 108) before (collection 1) and after (collection 2) therapy, when available. Mononuclear cells were isolated from whole blood using Ficoll-Paque density gradient. Cells from each patient were then incubated with an Fc blocker. The cells were then stained with fluorescently labeled antibodies for CD45, CD14, CD34, EpCAM, and pancytokeratin (CK), and a fluorescent PLE analog (CLR1501) for 30 minutes. The cells were then analyzed by flow cytometry to indicate the number of cells in each whole blood sample that were positive. Petition 870190036514, dated 04 / 16 / 2019, page 35 / 81 26 / 31 and / or negative for a particular marker. According to the current definition, CTCs were identified as live cells that were CD45-, CD14-, CD34-, CK+, and EpCAM+ (“by definition”). Due to previous experience with CD14+ monocytes / macrophages (Figure 1), CD14+ cells were removed from the analyses due to high CLR1501 uptake. Normal CD34+ epithelial cells were also removed from the analyses. In comparison, remaining CD45+ and CD34+ cells in the same patient will not utilize CLR1501 to a great extent. The results of this analysis are explained below and summarized in Tables 1 and 2.

[0057] The first row of Tables 1 and 2 shows the number of cells from each patient's blood sample that met the definition of a CTC: CD45-, CD14-, CD34-, CK+, and EpCAM+, and contained a nucleus. The second row shows the number of cells from each patient's blood sample that were CD45-, CD14-, CD34-, CK+, and contained a nucleus. The third row shows the number of cells from each patient's blood sample that were CLR 1501+, CD45-, CD14-, CD34-, CK+, and contained a nucleus. The fourth row shows the number of cells from each patient's blood sample that were CD45-, CD14-, CD34-, and EpCAM+, and contained a nucleus. The fifth line shows the number of cells in each patient's blood sample that were CLR 1501+, CD45-, CD14-, CD34-, and EpCAM+, and contained one nucleus. The sixth line shows the number of cells in each patient's blood sample that were CLR 1501+, CD45-, CD14-, CD34-, CK-, and EpCAM-, and contained one nucleus. Petition 870190036514, dated 04 / 16 / 2019, page 36 / 81 27 / 31 Table 1. Identification and Enumeration of Circulating Tumor Cells in Blood Samples from Patients with Various Types of Cancer, Collected in Cell Save® Collection Tubes. Cell Save® (number) Lung Cancer Lung Cancer Thyroid Cancer Thyroid Cancer Lung Cancer Cervical Cancer 101 (1) 101 (2) 102 (1) 102 (2) 103 104 (1) CTCs (by definition) 0 10 29,980 22,246 6,380 944 CK+ 4.7 M 10 1.0 M 3.5 M 3.3 M 6.8 M CK+ / CLR 1501+ 4.7 M 10 1.0 M 3.3 M 3.3 M 6.8 M EpCAM+ 20 41 38,594 24,674 10,610 1,087 EpCAM+ / CLR 1501+ 20 31 37,355 24,919 11,139 998 CK- / EpCAM- / CLR1501+ 1.4M 121,696 6.7M 7.9M 5.3M 1.3M Cell Save® (number) Cervical Cancer Breast Cancer Carcinoma Colorectal Cancer Colorectal Cancer 104 (2) 106 107 108 (1) 108 (2) CTCs (by definition) 26 30 1,034 5 30 CK+ 789 60 54,661 5 15,423 CK+ / CLR 1501+ 789 60 54,391 5 15,418 EpCAM+ 1069 105 6,968 40 222 EpCAM+ / CLR 1501+ 942 75 6,686 15 109 CK- / EpCAM- / CLR1501+ 36.4 M 278 611,622 1,062 1.1 M M denotes millions Table 2. Identification and Enumeration of Circulating Tumor Cells in Blood Samples from Patients with Various Types of Cancer, Collected in EDTA Collection Tubes Petition 870190036514, dated 04 / 16 / 2019, page 37 / 81 28 / 31 EDTA (number) Lung Cancer Lung Cancer Thyroid Cancer Thyroid Cancer Lung Cancer 101 (1) 101 (2) 102 (1) 102 (2) 103 CTCs (by definition) 26 0 730 8,808 192 CK+ 1.8 M 0 46,155 16,797 13,290 CK+ / CLR 1501+ 1.8 M 0 46,132 16,755 13,012 EpCAM+ 178 0 2,353 10,236 1,517 EpCAM+ / CLR 1501+ 153 0 2,720 10,375 1,896 CK- / EpCAM- / CLR1501+ 1.8 M 30,222 1.5 M 10.9 M 2.5 M EDTA (number) Cervical Cancer Breast Cancer Carcinoma Colorectal Cancer Colorectal Cancer 104 106 107 108 (1) 108 (2) CTCs (by definition) 22,965 12 1,896 0 76 CK+ 9.6 M 98 13,805 76 968 CK+ / CLR 1501+ 9.6 M 86 13,788 76 917 EpCAM+ 24,722 17 8,088 662 433 EpCAM+ / CLR 1501+ 23,493 17 9,469 382 229 CK- / EpCAM- / CLR1501+ 4.0 M 19,585 147,641 29,584 753,387 M denotes millions Results

[0058] All seven samples contained detectable CTCs (by definition) that showed positive CLR1501 uptake (compare line 1 with lines 3, 5, and 6 in Tables 1 and 2). An illustrative flow cytometry image representing high CLR1501 uptake in CD14+ cells from blood samples of patient 8 is shown in Figure 1. In Figure 1, the upper left quadrant indicates CD14 Petition 870190036514, dated 04 / 16 / 2019, pp. 38 / 81 29 / 31 / CLR1501+, the upper right quadrant indicates CD14+ / CLR1501+, the lower right quadrant indicates CD14+ / CLR1501-, and the lower left quadrant indicates CD14 / CLR1501-. In panel A, CD45- and CD34-negative cells (i.e., possible circulating tumor cells), isolated from blood drawn from patient 108, are shown stained with all markers except Brilliant Violet™785 CD14. CD14-positive cells are shown on the x-axis, and CLR1501-positive cells are shown on the y-axis. Panel A was used as a control to set the gates for Brilliant Violet™785 CD14-positive cells. In panel B, CD45- and CD34-negative cells, isolated from blood drawn from patient 108, are stained with all markers, including Brilliant Violet™785 CD14. As shown, 99.7% of the cells that are positive for CD14+ are also positive for CLR1501. Compare Figure 1, Panel B to Panel A.

[0059] Furthermore, CLR 1501 was able to identify ~99%-100% CK+ cells (compare row 2 and row 3 of Table 1 or 2) and ~35%-100% EpCAM+ cells (compare row 4 or row 5 of Table 1 or 2) in all cancer types, regardless of which blood collection tube was used. Surprisingly, there was a large number of cells that were CLR 1501+ but CK-, EpCAM-, CD45-, CD14-, CD34-, and contained a nucleus (row 6, Tables 1-2). The cells indicated in row 6 of Tables 1 and 2 are not blood cell types, but may be other tumor cells that may have decreased or absent expression of EpCAM and CK. Many cancers are reported to express markers Petition 870190036514, dated 04 / 16 / 2019, pp. 39 / 81 30 / 31 alternative tumor cells have heterogeneous expression of EpCAM and / or CK, or may be undergoing epithelial-mesenchymal transition (EMT) and thus losing expression of epithelial markers, Yu (2013), Eichelberg (2013) and Spizzo (2011). Thus, CLR1501 is able to identify circulating tumor cells that would otherwise not be detected in current single and multi-marker assays.

[0060] Patients 101, 102, 104, and 108 had blood samples collected before and after therapy (collections 1 and 2, respectively). Patient 101 had a very good partial response to therapy; however, between collections 1 and 2, this patient's CTC count, by definition, increased from 0 to 10, indicating cancer progression (Table 1). If only CK was used as a tumor cell marker, patient 101's tumor cell count decreased from 4.7 million to 10 (Table 1, compare collection 1 with collection 2). If only EpCAM was used as a tumor cell marker, patient 101's tumor cell count increased from 20 to 41 (Table 1, compare count 1 with count 2).However, the total tumor cell count of patient 101 (CK+ and EpCAM+ numbers) decreased between collections 1 and 2, indicating that separate markers may represent a more accurate measure of clinical response, and that patient 101's tumor cells reflect heterogeneous CK and EpCAM expression. Furthermore, patient 101's count for all CLR 1501+ cells decreased dramatically between collections 1 and 2. This trend is also seen in the EDTA blood collection tubes for patient 101 (Table 2). Thus, CLR 1501. Petition 870190036514, dated 04 / 16 / 2019, pp. 40 / 81 31 / 31 alone may be a more accurate measure of clinical response compared to current assays, negating the need for EpCAM and CK identification.

[0061] In general, the fluorescent PLE analog CLR1501 has been successfully used to identify CTCs from patients with lung, thyroid, breast, cervical, squamous cell carcinoma, and colorectal cancer. Petition 870190036514, dated 04 / 16 / 2019, pp. 41 / 81

Claims

1 / 6 X——(CH2)n -OPOCH2CH2N(CH3)3 where: n is an integer from 16 to 30; X is a luminescent molecule or one linked by means of a ligand; and (ii) subject the blood sample CLAIMS 1.Method for identifying one or more circulating tumor cells (CTCs) characterized in that it comprises: (i) placing a blood or blood serum sample from an individual in contact with a composition comprising a compound of formula (II), (II) , and a magnetic sphere or blood serum from the individual under fluorescence microscopy or flow cytometry, wherein one or more circulating tumor cells are selected from the group consisting of a breast cancer cell, a lung cancer cell, a thyroid cancer cell, a cervical cancer cell, a squamous cell carcinoma cell, a prostate cancer cell, a pancreatic cancer cell, a colorectal cancer cell, a cancer stem cell, a leukemia cell and a lymphoma cell.

2. Method according to claim 1, characterized in that X is a luminescent molecule.

3. Method according to claim 2, characterized in that the luminescent molecule is a fluorophore. Petition 870260050292, dated 05 / 26 / 2026, page 12 / 25 2 / 6 4. Method according to the claim characterized in that the fluorophore is selected from the group consisting of: wherein each R is selected independently from CH3, C2H5 and C3H7.

5. Method according to the claim characterized in that the fluorophore is selected from the group consisting of: V f' n according to is a fact that 6. Claim method characterized by a magnetic sphere bonded by means of a ligand.

7. Method according to claim 6 characterized in that the magnetic sphere is selected from the group consisting of nanomagnetic spheres, micromagnetic spheres, paramagnetic spheres and superparamagnetic spheres.

8. Method according to claim 1, characterized in that the ligand is selected from the group consisting of a biotin-streptavidin ligand, an azetidinone ligand and a ligand of -amine, -azide, alkyne, -carboxyl and -hydroxyl groups and combinations thereof.

9. Method for isolating one or more circulating tumor cells (CTCs) characterized in that it comprises the steps of: (i) placing a blood or blood serum sample from an individual in contact with a composition comprising a compound of formula (II), wherein: n is an integer from 16 to 30; and X is a luminescent molecule or a magnetic sphere; and (ii) subjecting the individual's blood or blood serum sample to flow cytometry or a magnetic field, wherein the individual's blood or blood serum sample is subjected to flow cytometry when X is a luminescent molecule; wherein the individual's blood or blood serum sample is subjected to a magnetic field when X is a magnetic sphere linked by means of a ligand; and Petition 870260050292, dated 05 / 26 / 2026, p.14 / 25 4 / 6 in which one or more CTCs is selected from the group consisting of a breast cancer cell, a lung cancer cell, a thyroid cancer cell, a cervical cancer cell, a squamous cell carcinoma cell, a prostate cancer cell, a pancreatic cancer cell, a colorectal cancer cell, a cancer stem cell, a leukemia cell, and a lymphoma cell.

10. Method, according to which X 11. Method, characterized in accordance with the fact that it is a fluorophore.

12. Method, characterized by the fact that the group consists of: with claim 9, is a luminescent molecule; with claim 10, the luminescent molecule is a group consisting of: with claim 11, the fluorophore is selected from the group in which each R is selected independently from among H, CH3, C2H5 and C3H7. Petition 870260050292, dated 05 / 26 / 2026, page 15 / 25 5 / 6 13. Method according to claim 12, characterized in that the fluorophore is selected from the group consisting of: characterized in that X is a magnetic sphere linked by means of a ligand.

15. Method according to claim 14, characterized in that the magnetic sphere is selected from the group consisting of nanomagnetic spheres, micromagnetic spheres, paramagnetic spheres and superparamagnetic spheres.

16. Method according to claim 14, characterized in that the ligand is selected from the group consisting of a biotin-streptavidin ligand, an azetidinone ligand and a ligand of -amine, -azide, alkyne, -carboxyl and -hydroxyl groups and combinations thereof.

17. Method according to claim 9, characterized in that it further comprises isolating one or more isolated CTCs.

18. Method, according to claim 17, characterized in that it further comprises using one or more isolated CTCs in a technology selected from the group consisting of protein isolation, RNA isolation, DNA isolation, gene translocation analysis, gene amplification analysis and fluorescent in situ hybridization. Petition 870260050292, dated 05 / 26 / 2026, p. 16 / 25 6 / 6 19. A method according to any one of claims 1 or 9, characterized in that the leukemia cell is an acute lymphoblastic leukemia cell, an acute myeloid leukemia cell, a chronic lymphocytic leukemia cell, a chronic myeloid leukemia cell, or a hairy cell leukemia cell.

20. Method, according to any one of claims 1 or 9, characterized in that the leukemia cell is a cutaneous T-cell lymphoma cell, a Hodgkin lymphoma cell, or a non-Hodgkin lymphoma cell. Petition 870260050292, dated 05 / 26 / 2026, p. 17 / 25

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