diagnostic and prognostic methods for cancers that express alpha folate receptor

专利摘要:
ALPHA FOLATE RECEPTOR AS A DIAGNOSTIC AND PROGNOSTIC MARKER FOR CANCERS EXPRESSING ALPHA FOLATE RECEPTOR The present invention relates to methods and kits to assess whether a subject is suffering from cancer that expresses FRa, methods and kits to predict the progression of ovarian cancer in a subject suffering from a cancer that expresses FRa, methods and kits to assess the level of risk that a subject will develop a cancer that expresses FRa, and methods of stratifying a subject with a cancer that expresses FRa in therapy groups cancer. The methods involve determining the level of alpha folate receptor (FRa) that is not bound to a cell in a sample derived from the subject and comparing this level with the level of FRa in a control sample.
公开号:BR112013011072B1
申请号:R112013011072-4
申请日:2011-11-04
公开日:2021-02-02
发明作者:Daniel J. O'shannessy；Luigi Grasso；Shanhong Wan；Qimin Chao；Elizabeth Brooke Somers
申请人:Eisai Inc.；
IPC主号:

专利说明:

Related Requests
[001] This application claims the benefit of the filing date of US provisional application No. 61 / 410,497, filed on November 5, 2010, and US provisional application No. 61 / 508,444, filed on July 15, 2011, the contents of which in the whole they are hereby incorporated by reference. Background of the Invention
[002] In humans, the high affinity receptor for folate comes in three isoforms: alpha, beta and gamma. Alpha and beta forms are typically attached to cell membranes by a glycosyl phosphatidylinositol (GPI) anchor. They recycle between extracellular and endocytic compartments and are capable of transporting folate in the cell. Soluble forms of FRα can be derived by the action of proteases or phospholipase on folate receptors anchored in the membrane.
[003] Alpha folate receptor (also referred to as FRα, FR-alpha, FOLR-1 or FOLR1) is expressed in a variety of epithelial tissues, including those of the choroid plexus, lung, thyroid, kidney, uterus, breast, fallopian, epididymis and salivary glands. Weitman, SD et al. Cancer Res 52: 3396-3401 (1992); Weitman SD et al, Cancer Res 52: 6708-6711. Overexpression of FRα has been observed in several cancers, including lung cancer (for example, bronchialalveolar carcinomas, carcinoid tumors and non-small cell lung cancers, such as adenocarcinomas); mesothelioma; Ovary cancer; kidney cancer; brain cancer (for example, anaplastic ependymoma, cerebellar juvenile pilocytic astrocytoma, and brain metastases); cervical cancer; nasopharyngeal cancer; mesodermally derived tumor; squamous cell carcinoma of the head and neck; endometrial cancer; endometrioid adenocarcinomas of the ovary, serous cystadenocarcinomas, breast cancer; bladder cancer; pancreatic cancer; bone cancer (eg, high-grade osteosarcoma); pituitary cancer (for example, pituitary adenomas); colorectal cancer and medullary thyroid cancer. See, for example, U.S. Patent No. 7,754,698; U.S. patent application No. 2005/0232919; WO 2009/132081; Bueno R et al. J of Thoracic and Cardiovascular Surgery, 121 (2): 225-233 (2001); Elkanat H & Ratnam M. Frontiers in Bioscience, 11, 506-519 (2006); Fisher R.E. J Nucl Med, 49: 899-906 (2008); Franklin, WA et al. Int J Cancer, Suppl 8: 89-95 (1994); Hartman L.C. et al. Int J Cancer 121: 938-942 (2007); Iwakiri S et al. Annals of Surgical Oncology, 15 (3): 889-899; Parker N. et al. Analytical Biochemistry, 338: 284-293 (2005); Weitman, SD et al. Cancer Res 52: 3396-3401 (1992); Saba N.F. et al. Head Neck, 31 (4): 475-481 (2009); Yang R et al. Clin Cancer Res 13: 2557-2567 (2007). In some types of cancers (for example, squamous cell carcinoma of the head and neck), a higher level of FRα expression is associated with a worsening prognosis, while in other types of cancers (for example, non-cell lung cancers) small), a higher level of FRα expression is associated with an improvement prognosis. See, for example, Iwakiri S et al. Annals of Surgical Oncology, 15 (3): 889-899; Saba N.F. et al. Head Neck, 31 (4): 475-481 (2009).
[004] Early cancer detection increases survival rates and quality of life. To increase the likelihood of early detection and treatment, there is an urgent need for non-invasive methods to diagnose cancer, determine the level of risk of developing cancer, and predict the progression of cancer. The present invention satisfies these needs for cancers that express FRα. Summary of the Invention
[005] The present invention relates to methods of assessing whether a subject is suffering from cancers that express FRα, such as lung or ovarian cancer, methods of assessing the progression of cancers that express FRα, such as lung or ovarian cancer, in a subject suffering from cancers that express FRα, methods of stratifying a subject with cancer that expresses FRα in at least one of the four cancer therapy groups, methods of evaluating the effectiveness of the MORAb-003 treatment of ovarian cancer or cancer of lung and kits to assess whether a subject is suffering from cancers that express FRα, such as lung or ovarian cancer, or to assess the progression of cancers that express FRα, such as lung or ovarian cancer, in a subject. Methods of Assessing if a Subject Is Suffering from a Cancer That Expresses FRα
[006] In a first aspect, the present invention concerns a method of evaluating whether a subject is suffering from a cancer that expresses FRα, determining the level of alpha folate receptor (FRα) that is not bound to a cell, in a sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell with the level of FRα in a control sample, in which a difference between the level of FRα in the sample derived from the subject and the level of FRα in the sample control is an indication that the subject is suffering from a cancer that expresses FRα; wherein the level of FRα in the sample derived from the subject is assessed by placing the sample in contact with an antibody that binds FRα. In a particular embodiment, the sample is either urine, serum, plasma or ascites.
[007] In another aspect, the present invention relates to a method of assessing whether a subject is suffering from a cancer that expresses FRα, by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a urine sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the subject's derived urine sample with the level of FRα in a control sample, in which a difference between the level of FRα in the derived urine sample of the subject and the level of FRα in the control sample is an indication that the subject is suffering from a cancer that expresses FRα. In a further aspect, the present invention relates to a method of assessing whether a subject is suffering from a cancer that expresses FRα, by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a serum sample. derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the subject's derived serum sample with the level of FRα in a control sample, in which a difference between the level of FRα in the derived serum sample of the subject and the level of FRα in the control sample is an indication that the subject is suffering from a cancer that expresses FRα.
[008] In various modalities of the previous aspects of the invention, the cancer that expresses FRα is selected from the group consisting of lung cancer, mesothelioma, ovarian cancer, kidney cancer, brain cancer, cervical cancer, nasopharyngeal cancer, carcinoma of squamous cell of the head and neck, cancer of the brain, breast cancer, bladder cancer, pancreatic cancer, bone cancer, pituitary cancer, colorectal cancer and medullary thyroid cancer. In a particular embodiment, the cancer that expresses FRα is ovarian cancer. In another embodiment, cancer that expresses FRα is non-small cell lung cancer, such as an adenocarcinoma.
[009] In another aspect, the present invention relates to methods of assessing whether a subject is suffering from ovarian cancer by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a urine sample. derived from the subject, in which the presence of FRα that is not bound to a cell in the urine sample at a concentration greater than about 9,100 pg / mL is an indication that the subject is suffering from ovarian cancer.
[0010] In several aspects of the previous aspects of the invention, the presence of FRα in the urine sample at a concentration greater than about 9,500 pg / mL, about 10,000 pg / mL, about 11,000 pg / mL, about 12,000 pg / ml, about 13,000 pg / ml, about 14,000 pg / ml, about 15,000 pg / ml, about 16,000 pg / ml, about 17,000 pg / ml, about 18,000 pg / ml, about 19,000 pg / mL, or about 20,000 pg / mL, is an indication that the subject is suffering from ovarian cancer.
[0011] In several respects, the level of FRα is determined by placing the sample in contact with an antibody that binds FRα. For example, the antibody is selected from the group consisting of: (a) an antibody that binds to the same epitope as the MORAb-003 antibody; (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRLH1 SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3; (c) the MOV18 antibody; (d) an antibody that binds to the same epitope as the MOV18 antibody; (e) antibody 548908; (f) an antibody that binds to the same epitope as the 548908 antibody; (g) the 6D398 antibody; (h) an antibody that binds to the same epitope as the 6D398 antibody; (i) an antibody that binds to the same epitope as the 26B3 antibody; (j) an antibody comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as CDRL2 and SEQ ID NO: 53 (QHHYAFPWT) as CDRL3; (k) the 26B3 antibody; (l) an antibody that binds to the same epitope as the 19D4 antibody; (m) an antibody comprising SEQ ID NO: 39 (HPYMH) as CDRH1, SEQ ID NO: 40 (RIDPANGNTKYDPKFQG) as CDRH2, SEQ ID NO: 41 (EEVADYTMDY) as CDRH3, SEQ ID NO: 35 (RASESVDTYGNNFIH) as CDRL1 SEQ ID NO: 36 (LASNLES) as CDRL2 and SEQ ID NO: 37 (QQNNGDPWT) as CDRL3; (n) the 19D4 antibody; (o) an antibody that binds to the same epitope as the 9F3 antibody; (p) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3; (q) the 9F3 antibody; (r) an antibody that binds to the same epitope as the 24F12 antibody; (s) an antibody comprising SEQ ID NO: 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 (ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3; (t) the 24F12 antibody; (u) an antibody comprising a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16); (v) an antibody comprising a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21); (w) an antibody comprising the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); (x) an antibody comprising the LK26HuVH SLF heavy chain variable region (SEQ ID NO: 19) and the LK26HuVKPW variable region light chain, Y (SEQ ID NO: 16); and (y) an antibody comprising the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0012] In a particular embodiment, the antibody binds to the same epitope as the MORAb-003 antibody. In another embodiment, the antibody includes SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRL1, SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3. In another embodiment, the antibody is the MOV18 antibody. In yet another embodiment, the antibody binds on the same epitope as the MOV18 antibody. In an additional embodiment, the antibody comprises a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16). Alternatively, or in combination, the antibody includes a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21). In certain embodiments, the antibody includes (i) the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); the heavy chain variable region LK26HuVH SLF (SEQ ID NO: 19) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); or the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0013] In a particular modality, the level of FRα in the sample derived from said subject is evaluated by placing the sample in contact with a pair of antibodies selected from the group consisting of (a) MOV18 antibody immobilized on a solid support and MORAB- antibody 003 marked; (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody; (c) antibody 26B3 immobilized on a solid support and labeled antibody 19D4; and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody.
[0014] In certain embodiments, the antibody is selected from the group consisting of a murine antibody, a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, Fab'2, ScFv, SMIP, affibody, avimer , versabody, nanobody, and an antibody from the domain. Alternatively, or in combination, the antibody is labeled, for example, with a marker selected from the group consisting of a radio marker, a biotin marker, a chromophore marker, a fluorophore marker, or an enzyme marker.
[0015] In certain modalities, the level of FRα is determined using a technique selected from the group consisting of western blot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, solution phase assay, electrochemical chemiluminescent immunoassay (ECLIA ) and ELISA assay.
[0016] In several modalities of the previous aspects of the invention, the control sample is a standardized level of control of FRα in a healthy subject.
[0017] In certain embodiments, the sample is treated with guanidine before determining the level of FRα in the sample. Alternatively, or in combination, the sample is diluted before determining the level of FRα in the sample. Alternatively, or in combination, the sample is centrifuged, swirled, or both, before determining the level of FRα in the sample.
[0018] In yet another aspect, the present invention relates to a method of assessing whether a subject is suffering from ovarian cancer by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the sample with the level of FRα in a control sample, where a difference between the levels of FRα in the sample derived from the subject and in the control sample is an indication that the subject is suffering from ovarian cancer; where the level of FRα in the sample derived from the subject is assessed by placing the sample in contact with (a) MOV18 antibody immobilized on a solid support and labeled MORAB-003 antibody, (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody , (c) 26B3 antibody immobilized on a solid support and labeled 19D4 antibody, and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody. For example, the sample can be urine, serum, plasma or ascites. Methods of Assessing the Progression of a Cancer Expressing FRα in a Subject
[0019] In an additional aspect, the present invention relates to a method of evaluating the progression of a cancer that expresses FRα in a subject suffering from a cancer that expresses FRα, determining the level of alpha folate receptor (FRα) that does not it is attached to a cell, in a sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell with the level of FRα in a control sample, in which an increase in the level of FRα in the sample derived from the subject compared with the level of FRα in the sample control is an indication that the cancer will progress quickly; and in which a decrease in the level of FRα in the sample derived from the subject compared to the level of FRα in the control sample is an indication that the cancer will progress slowly or regress, thereby assessing the progression of the cancer that expresses FRα in the subject; wherein the level of FRα that is not bound to a cell in the sample derived from the subject is assessed by placing the sample in contact with an antibody that binds FRα. In a particular embodiment, the sample is urine, serum, plasma or ascites.
[0020] In another aspect, the present invention relates to a method of evaluating the progression of a cancer that expresses FRα in a subject suffering from a cancer that expresses FRα, determining the level of alpha folate receptor (FRα) that does not it is attached to a cell in a urine sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the urine sample derived from the subject with the level of FRα in a control sample, in which an increase in the level of FRα in the urine sample derived from subject compared to the level of FRα in the control sample is an indication that the cancer will progress rapidly; and that a decrease in the level of FRα in the urine sample derived from the subject compared to the level of FRα in the control sample is an indication that the cancer will progress slowly or regress, thereby assessing the progression of the cancer that expresses FRα in the subject .
[0021] In an additional aspect, the present invention relates to methods of evaluating the progression of a cancer that expresses FRα in a subject suffering from a cancer that expresses FRα, determining the level of alpha folate receptor (FRα) that is not bound to a cell in a serum sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the subject-derived serum sample with the level of FRα in the control sample, in which an increase in the level of FRα in the subject-derived serum sample compared to the level of FRα in the control sample is an indication that the cancer will progress rapidly; and that a decrease in the level of FRα in the serum sample derived from the subject compared to the level of FRα in the control sample is an indication that the cancer will progress slowly or regress, thereby assessing the progression of the cancer that expresses FRα on the subject.
[0022] In various modalities of the previous aspects of the invention, cancer that expresses FRα is selected from the group consisting of lung cancer, mesothelioma, ovarian cancer, kidney cancer, brain cancer, cervical cancer, nasopharyngeal cancer, carcinoma of squamous cell of the head and neck, cancer of the brain, breast cancer, bladder cancer, pancreatic cancer, bone cancer, pituitary cancer, colorectal cancer and medullary thyroid cancer. In a particular embodiment, the cancer that expresses FRα is ovarian cancer. In another embodiment, cancer that expresses FRα is non-small cell lung cancer, such as an adenocarcinoma.
[0023] In another aspect, the present invention relates to methods of assessing whether a subject is suffering from ovarian cancer by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a urine sample. derived from the subject, in which the presence of FRα that is not bound to a cell in the urine sample at a concentration greater than about 9,100 pg / mL is an indication that the subject is suffering from ovarian cancer.
[0024] In various aspects of the foregoing aspects of the invention, the presence of FRα in the urine sample at a concentration greater than about 9,500 pg / mL, about 10,000 pg / mL, about 11,000 pg / mL, about 12,000 pg / ml, about 13,000 pg / ml, about 14,000 pg / ml, about 15,000 pg / ml, about 16,000 pg / ml, about 17,000 pg / ml, about 18,000 pg / ml, about 19,000 pg / mL, or about 20,000 pg / mL, is an indication that the subject is suffering from ovarian cancer.
[0025] In several respects, the level of FRα is determined by placing the sample in contact with an antibody that binds FRα. For example, the antibody is selected from the group consisting of: (a) an antibody that binds to the same epitope as the MORAb-003 antibody; (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRLH1 SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3; (c) the MOV18 antibody; (d) an antibody that binds to the same epitope as the MOV18 antibody; (e) antibody 548908; (f) an antibody that binds to the same epitope as the 548908 antibody; (g) the 6D398 antibody; (h) an antibody that binds to the same epitope as the 6D398 antibody; (i) an antibody that binds to the same epitope as the 26B3 antibody; (j) an antibody comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as CDRL2 and SEQ ID NO: 53 (QHHYAFPWT) as CDRL3; (k) the 26B3 antibody; (l) an antibody that binds to the same epitope as the 19D4 antibody; (m) an antibody comprising SEQ ID NO: 39 (HPYMH) as CDRH1, SEQ ID NO: 40 (RIDPANGNTKYDPKFQG) as CDRH2, SEQ ID NO: 41 (EEVADYTMDY) as CDRH3, SEQ ID NO: 35 (RASESVDTYGNNFIH) as CDRL1 SEQ ID NO: 36 (LASNLES) as CDRL2 and SEQ ID NO: 37 (QQNNGDPWT) as CDRL3; (n) the 19D4 antibody; (o) an antibody that binds to the same epitope as the 9F3 antibody; (p) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3; (q) the 9F3 antibody; (r) an antibody that binds to the same epitope as the 24F12 antibody; (s) an antibody comprising SEQ ID NO: 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 (ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3; (t) the 24F12 antibody; (u) an antibody comprising a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16); (v) an antibody comprising a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21); (w) an antibody comprising the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); (x) an antibody comprising the LK26HuVH SLF heavy chain variable region (SEQ ID NO: 19) and the LK26HuVKPW variable region light chain, Y (SEQ ID NO: 16); and (y) an antibody comprising the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0026] In a particular embodiment, the antibody binds to the same epitope as the MORAb-003 antibody. In another embodiment, the antibody includes SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRL1, SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3. In another embodiment, the antibody is the MOV18 antibody. In yet another embodiment, the antibody binds on the same epitope as the MOV18 antibody. In an additional embodiment, the antibody comprises a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16). Alternatively, or in combination, the antibody includes a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21). In certain embodiments, the antibody includes (i) the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); the heavy chain variable region LK26HuVH SLF (SEQ ID NO: 19) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); or the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0027] In a particular embodiment, the level of FRα in the sample derived from said subject is evaluated by placing the sample in contact with a pair of antibodies selected from the group consisting of (a) MOV18 antibody immobilized on a solid support and MORAB- antibody. 003 marked; (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody; (c) antibody 26B3 immobilized on a solid support and labeled antibody 19D4; and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody.
[0028] In certain embodiments, the antibody is selected from the group consisting of a murine antibody, a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, Fab'2, ScFv, SMIP, affibody, avimer , versabody, nanobody, and an antibody from the domain. Alternatively, or in combination, the antibody is labeled, for example, with a marker selected from the group consisting of a radio marker, a biotin marker, a chromophore marker, a fluorophore marker, or an enzyme marker.
[0029] In certain modalities, the level of FRα is determined using a technique selected from the group consisting of western blot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, solution phase assay, electrochemical chemiluminescent immunoassay (ECLIA ) and ELISA assay.
[0030] In several modalities of the previous aspects of the invention, the control sample is a standardized level of control of FRα in a healthy subject. In another modality, the control sample is a sample previously obtained from the subject.
[0031] In certain embodiments, the sample is treated with guanidine before determining the level of FRα in the sample. Alternatively, or in combination, the sample is diluted before determining the level of FRα in the sample. Alternatively, or in combination, the sample is centrifuged, swirled, or both, before determining the level of FRα in the sample.
[0032] In a further aspect, the present invention relates to methods of assessing the progression of ovarian cancer in a subject suffering from ovarian cancer, by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the sample with the level of FRα in a control sample, in which an increase in the level of FRα in the sample derived from the subject compared with the level of FRα in the control sample it is an indication that ovarian cancer will progress rapidly; and in which a decrease in the level of FRα in the sample derived from the subject compared to the level of FRα in the control sample is an indication that ovarian cancer will progress slowly or regress, thereby assessing the progression of ovarian cancer in the subject; where the level of FRα in the sample derived from the subject is assessed by placing the sample in contact with (a) MOV18 antibody immobilized on a solid support and labeled MORAB-003 antibody, (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody , (c) 26B3 antibody immobilized on a solid support and labeled 19D4 antibody, and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody. For example, the sample can be urine, serum, plasma or ascites. Methods of Stratifying a Cancer That Expresses FRα in Cancer Therapy Groups
[0033] In a further aspect, the present invention relates to a method of stratifying a subject suffering from cancer that expresses FRα in at least one of the four cancer therapy groups by determining the level of alpha folate receptor (FRα) that it is not bound to a cell, in a sample derived from the subject; and stratifying the subject into at least one of the four cancer therapy groups based on the level of alpha folate receptor (FRα) that is not bound to a cell; wherein the level of FRα that is not bound to a cell in the sample derived from the subject is assessed by placing the sample in contact with an antibody that binds FRα. For example, the sample is selected from the group consisting of urine, serum, plasma or ascites.
[0034] In yet another aspect, the present invention relates to a method of stratifying a subject suffering from cancer that expresses FRα in at least one of the four cancer therapy groups by determining the level of alpha folate receptor (FRα) that it is not bound to a cell in a urine sample derived from the subject; and stratifying the subject into at least one of the four cancer therapy groups based on the level of alpha folate receptor (FRα) that is not bound to a cell in the sample. In a further aspect, the present invention relates to methods of stratifying a subject suffering from cancer that expresses FRα in at least one of the four cancer therapy groups by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a serum sample derived from the subject; and stratifying the subject into at least one of the four cancer therapy groups based on the level of alpha folate receptor (FRα) that is not bound to a cell in the serum sample.
[0035] In various modalities of the previous aspects of the invention, cancer that expresses FRα is selected from the group consisting of lung cancer, mesothelioma, ovarian cancer, kidney cancer, brain cancer, cervical cancer, nasopharyngeal cancer, carcinoma of squamous cell of the head and neck, cancer of the brain, breast cancer, bladder cancer, pancreatic cancer, bone cancer, pituitary cancer, colorectal cancer and medullary thyroid cancer. In a particular embodiment, the cancer that expresses FRα is ovarian cancer. In another embodiment, cancer that expresses FRα is non-small cell lung cancer, such as an adenocarcinoma.
[0036] In another aspect, the present invention relates to methods of assessing whether a subject is suffering from ovarian cancer by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a urine sample. derived from the subject, in which the presence of FRα that is not bound to a cell in the urine sample at a concentration greater than about 9,100 pg / mL is an indication that the subject is suffering from ovarian cancer.
[0037] In various aspects of the foregoing aspects of the invention, the presence of FRα in the urine sample at a concentration greater than about 9,500 pg / mL, about 10,000 pg / mL, about 11,000 pg / mL, about 12,000 pg / ml, about 13,000 pg / ml, about 14,000 pg / ml, about 15,000 pg / ml, about 16,000 pg / ml, about 17,000 pg / ml, about 18,000 pg / ml, about 19,000 pg / mL, or about 20,000 pg / mL, is an indication that the subject is suffering from ovarian cancer.
[0038] In several respects, the level of FRα is determined by placing the sample in contact with an antibody that binds FRα. For example, the antibody is selected from the group consisting of: (a) an antibody that binds to the same epitope as the MORAb-003 antibody; (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRLH1 SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3; (c) the MOV18 antibody; (d) an antibody that binds to the same epitope as the MOV18 antibody; (e) antibody 548908; (f) an antibody that binds to the same epitope as the 548908 antibody; (g) the 6D398 antibody; (h) an antibody that binds to the same epitope as the 6D398 antibody; (i) an antibody that binds to the same epitope as the 26B3 antibody; (j) an antibody comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as CDRL2 and SEQ ID NO: 53 (QHHYAFPWT) as CDRL3; (k) the 26B3 antibody; (l) an antibody that binds to the same epitope as the 19D4 antibody; (m) an antibody comprising SEQ ID NO: 39 (HPYMH) as CDRH1, SEQ ID NO: 40 (RIDPANGNTKYDPKFQG) as CDRH2, SEQ ID NO: 41 (EEVADYTMDY) as CDRH3, SEQ ID NO: 35 (RASESVDTYGNNFIH) as CDRL1 SEQ ID NO: 36 (LASNLES) as CDRL2 and SEQ ID NO: 37 (QQNNGDPWT) as CDRL3; (n) the 19D4 antibody; (o) an antibody that binds to the same epitope as the 9F3 antibody; (p) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3; (q) the 9F3 antibody; (r) an antibody that binds to the same epitope as the 24F12 antibody; (s) an antibody comprising SEQ ID NO: 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 (ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3; (t) the 24F12 antibody; (u) an antibody comprising a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16); (v) an antibody comprising a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21); (w) an antibody comprising the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); (x) an antibody comprising the LK26HuVH SLF heavy chain variable region (SEQ ID NO: 19) and the LK26HuVKPW variable region light chain, Y (SEQ ID NO: 16); and (y) an antibody comprising the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0039] In a particular embodiment, the antibody binds to the same epitope as the MORAb-003 antibody. In another embodiment, the antibody includes SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRL1, SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3. In another embodiment, the antibody is the MOV18 antibody. In yet another embodiment, the antibody binds on the same epitope as the MOV18 antibody. In an additional embodiment, the antibody comprises a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16). Alternatively, or in combination, the antibody includes a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21). In certain embodiments, the antibody includes (i) the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); the heavy chain variable region LK26HuVH SLF (SEQ ID NO: 19) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); or the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0040] In a particular embodiment, the level of FRα in the sample derived from said subject is evaluated by placing the sample in contact with a pair of antibodies selected from the group consisting of (a) MOV18 antibody immobilized on a solid support and MORAB- antibody. 003 marked; (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody; (c) antibody 26B3 immobilized on a solid support and labeled antibody 19D4; and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody.
[0041] In certain embodiments, the antibody is selected from the group consisting of a murine antibody, a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, Fab'2, ScFv, SMIP, affibody, avimer , versabody, nanobody, and an antibody from the domain. Alternatively, or in combination, the antibody is labeled, for example, with a marker selected from the group consisting of a radio marker, a biotin marker, a chromophore marker, a fluorophore marker, or an enzyme marker.
[0042] In certain modalities, the level of FRα is determined using a technique selected from the group consisting of western blot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, solution phase assay, electrochemical chemiluminescent immunoassay (ECLIA ) and ELISA assay.
[0043] In various modalities of the previous aspects of the invention, the control sample is a standardized level of control of FRα in a healthy subject.
[0044] In certain embodiments, the sample is treated with guanidine before determining the level of FRα in the sample. Alternatively, or in combination, the sample is diluted before determining the level of FRα in the sample. Alternatively, or in combination, the sample is centrifuged, swirled, or both, before determining the level of FRα in the sample.
[0045] In a particular modality, the subject is stratified into Stage I, Stage II, Stage III or Stage IV ovarian cancer.
[0046] In a further aspect, the present invention relates to a method of stratifying a subject with ovarian cancer into at least one of the four cancer therapy groups by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a sample derived from the subject; and stratifying the subject into at least one of the four cancer therapy groups based on the level of alpha folate receptor (FRα) that is not bound to a cell in the sample; where the level of FRα in the sample derived from the subject is assessed by placing the sample in contact with (a) MOV18 antibody immobilized on a solid support and labeled MORAB-003 antibody, (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody , (c) 26B3 antibody immobilized on a solid support and labeled 19D4 antibody, and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody. For example, the sample can be urine, serum, plasma or ascites. Methods of Monitoring the Effectiveness of MORAb-003 Treatment for Ovarian Cancer or Lung Cancer
[0047] In one aspect, the present invention relates to a method of monitoring the effectiveness of MORAb-003 in the treatment of ovarian cancer or lung cancer in a subject suffering from ovarian cancer or lung cancer, by determining the level of alpha folate receptor (FRα) that is not bound to a cell, in a sample derived from the subject, in which the subject was previously administered with MORAb-003; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell with the level of FRα in a control sample, in which an increase in the level of FRα in the sample derived from the subject compared with the level of FRα in the sample control is an indication that treatment with MORAb-003 is not efficient; and that a decrease in the level of FRα in the sample derived from the subject compared to the level of FRα in the control sample is an indication that treatment with MORAb-003 is efficient. In particular modalities, the level of FRα that is not bound to a cell in the sample derived from the subject is assessed by placing the sample in contact with an antibody that binds FRα. For example, the sample can be urine, serum, plasma or ascites.
[0048] In a further aspect, the present invention relates to a method of monitoring the effectiveness of treatment with MORAb-003 for ovarian cancer or lung cancer in a subject suffering from ovarian cancer or lung cancer, by determining the level an alpha folate receptor (FRα) that is not bound to a cell in a subject-derived urine sample, in which the subject was previously administered with MORAb-003; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the urine sample derived from the subject with the level of FRα in a control sample, in which an increase in the level of FRα in the urine sample derived from subject compared to the level of FRα in the control sample is an indication that treatment with MORAb-003 is not efficient; and that a decrease in the level of FRα in the urine sample derived from the subject compared to the level of FRα in the control sample is an indication that treatment with MO-RAb-003 is efficient.
[0049] In yet another aspect, the present invention relates to a method of monitoring the effectiveness of treatment with MORAb-003 for ovarian cancer or lung cancer in a subject suffering from ovarian cancer or lung cancer, determining the level of alpha folate receptor (FRα) that is not bound to a cell in a serum sample derived from the subject, where the subject was previously administered with MORAb-003; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the serum sample derived from the subject with the level of FRα in a control sample, in which an increase in the level of FRα in the serum sample derived from subject compared to the level of FRα in the control sample is an indication that treatment with MORAb-003 is not efficient; and that a decrease in the level of FRα in the serum sample derived from the subject compared to the level of FRα in the control sample is an indication that treatment with MORAb-003 is efficient.
[0050] In several respects, the level of FRα is determined by placing the sample in contact with an antibody that binds FRα. For example, the antibody is selected from the group consisting of: (a) an antibody that binds to the same epitope as the MORAb-003 antibody; (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRLH1 SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3; (c) the MOV18 antibody; (d) an antibody that binds to the same epitope as the MOV18 antibody; (e) antibody 548908; (f) an antibody that binds to the same epitope as the 548908 antibody; (g) the 6D398 antibody; (h) an antibody that binds to the same epitope as the 6D398 antibody; (i) an antibody that binds to the same epitope as the 26B3 antibody; (j) an antibody comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as CDRL2 and SEQ ID NO: 53 (QHHYAFPWT) as CDRL3; (k) the 26B3 antibody; (l) an antibody that binds to the same epitope as the 19D4 antibody; (m) an antibody comprising SEQ ID NO: 39 (HPYMH) as CDRH1, SEQ ID NO: 40 (RIDPANGNTKYDPKFQG) as CDRH2, SEQ ID NO: 41 (EEVADYTMDY) as CDRH3, SEQ ID NO: 35 (RASESVDTYGNNFIH) as CDRL1 SEQ ID NO: 36 (LASNLES) as CDRL2 and SEQ ID NO: 37 (QQNNGDPWT) as CDRL3; (n) the 19D4 antibody; (o) an antibody that binds to the same epitope as the 9F3 antibody; (p) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3; (q) the 9F3 antibody; (r) an antibody that binds to the same epitope as the 24F12 antibody; (s) an antibody comprising SEQ ID NO: 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 (ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3; (t) the 24F12 antibody; (u) an antibody comprising a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16); (v) an antibody comprising a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21); (w) an antibody comprising the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); (x) an antibody comprising the LK26HuVH SLF heavy chain variable region (SEQ ID NO: 19) and the LK26HuVKPW variable region light chain, Y (SEQ ID NO: 16); and (y) an antibody comprising the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0051] In a particular embodiment, the antibody binds to the same epitope as the MORAb-003 antibody. In another embodiment, the antibody includes SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRL1, SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3. In another embodiment, the antibody is the MOV18 antibody. In yet another embodiment, the antibody binds on the same epitope as the MOV18 antibody. In an additional embodiment, the antibody comprises a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16). Alternatively, or in combination, the antibody includes a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21). In certain embodiments, the antibody includes (i) the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); the heavy chain variable region LK26HuVH SLF (SEQ ID NO: 19) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); or the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0052] In a particular embodiment, the level of FRα in the sample derived from said subject is evaluated by placing the sample in contact with a pair of antibodies selected from the group consisting of (a) MOV18 antibody immobilized on a solid support and MORAB- antibody. 003 marked; (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody; (c) antibody 26B3 immobilized on a solid support and labeled antibody 19D4; and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody.
[0053] In certain embodiments, the antibody is selected from the group consisting of a murine antibody, a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, Fab'2, ScFv, SMIP, affibody, avimer , versabody, nanobody, and an antibody from the domain. Alternatively, or in combination, the antibody is labeled, for example, with a marker selected from the group consisting of a radio marker, a biotin marker, a chromophore marker, a fluorophore marker, or an enzyme marker.
[0054] In certain modalities, the level of FRα is determined using a technique selected from the group consisting of western blot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, solution phase assay, electrochemiluminescent immunoassay (ECLIA ) and ELISA assay.
[0055] In several modalities of the previous aspects of the invention, the control sample is a standardized level of control of FRα in a healthy subject. In another modality, the control sample is a sample previously obtained from the subject.
[0056] In certain embodiments, the sample is treated with guanidine before determining the level of FRα in the sample. Alternatively, or in combination, the sample is diluted before determining the level of FRα in the sample. Alternatively, or in combination, the sample is centrifuged, swirled, or both, before determining the level of FRα in the sample. Methods of Predicting whether a Subject Will Respond to Treatment with MORAb-003
[0057] In one aspect, the present invention relates to a method for predicting whether a subject suffering from a cancer that expresses FRα, such as ovarian cancer or lung cancer, will respond to treatment with MORAb-003, determining the level of alpha folate receptor (FRα) that is not bound to a cell in a sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the sample derived from the subject with the level of FRα in a control sample, in which a difference between the level of FRα in the sample derived from the subject and the FRα level in the control sample is an indication that the subject will respond to treatment with MORAb-003.
[0058] In one aspect, the present invention relates to a method for predicting whether a subject suffering from a cancer that expresses FRα, such as ovarian cancer or lung cancer, will respond to treatment with MORAb-003, determining the level of alpha folate receptor (FRα) that is not bound to a cell in a urine sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the subject's derived urine sample with the level of FRα in a control sample, in which a difference between the level of FRα in the derived urine sample of the subject and the level of FRα in the control sample is an indication that the subject will respond to treatment with MORAb-003.
[0059] In an additional aspect, the present invention relates to a method for predicting whether a subject suffering from a cancer that expresses FRα, such as ovarian cancer or lung cancer, will respond to treatment with MORAb-003, determining the level an alpha folate receptor (FRα) that is not bound to a cell in a serum sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the subject's derived serum sample with the level of FRα in a control sample, in which a difference between the level of FRα in the derived serum sample of the subject and the level of FRα in the control sample is an indication that the subject will respond to treatment with MORAb-003.
[0060] In additional modalities, cancer that expresses FRα is selected from the group consisting of lung cancer, mesothelioma, ovarian cancer, kidney cancer, brain cancer, cervical cancer, nasopharyngeal cancer, squamous cell carcinoma of the head and neck, endometrial cancer, breast cancer, bladder cancer, pancreatic cancer, bone cancer, pituitary cancer, colorectal cancer and medullary thyroid cancer. In a particular embodiment, the cancer that expresses FRα is ovarian cancer. In another embodiment, cancer that expresses lung FRα is non-small cell lung cancer, such as adenocarcinoma.
[0061] In a further aspect, the present invention relates to methods for predicting whether a subject suffering from ovarian cancer will respond to treatment with MORAb-003, by determining the level of alpha folate receptor (FRα) that is not linked to a cell in a urine sample derived from the subject, in which the presence of FRα that is not bound to a cell in the urine sample at a concentration greater than about 9,100 pg / mL is an indication that the subject will respond to treatment with MORAb -003.
[0062] In various embodiments of the foregoing aspects of the invention, the presence of FRα in the urine sample at a concentration greater than about 9,500 pg / mL, about 10,000 pg / mL, about 11,000 pg / mL, about 12,000 pg / ml, about 13,000 pg / ml, about 14,000 pg / ml, about 15,000 pg / ml, about 16,000 pg / ml, about 17,000 pg / ml, about 18,000 pg / ml, about 19,000 pg / mL, or about 20,000 pg / mL, is an indication that the subject is suffering from ovarian cancer.
[0063] In various embodiments of the foregoing aspects of the invention, the level of FRα is determined by placing the sample in contact with an antibody that binds FRα. For example, the antibody is selected from the group consisting of: (a) an antibody that binds to the same epitope as the MORAb-003 antibody; (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRLH1 SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3; (c) the MOV18 antibody; (d) an antibody that binds to the same epitope as the MOV18 antibody; (e) antibody 548908; (f) an antibody that binds to the same epitope as the 548908 antibody; (g) the 6D398 antibody; (h) an antibody that binds to the same epitope as the 6D398 antibody; (i) an antibody that binds to the same epitope as the 26B3 antibody; (j) an antibody comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as CDRL2 and SEQ ID NO: 53 (QHHYAFPWT) as CDRL3; (k) the 26B3 antibody; (l) an antibody that binds to the same epitope as the 19D4 antibody; (m) an antibody comprising SEQ ID NO: 39 (HPYMH) as CDRH1, SEQ ID NO: 40 (RIDPANGNTKYDPKFQG) as CDRH2, SEQ ID NO: 41 (EEVADYTMDY) as CDRH3, SEQ ID NO: 35 (RASESVDTYGNNFIH) as CDRL1 SEQ ID NO: 36 (LASNLES) as CDRL2 and SEQ ID NO: 37 (QQNNGDPWT) as CDRL3; (n) the 19D4 antibody; (o) an antibody that binds to the same epitope as the 9F3 antibody; (p) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3; (q) the 9F3 antibody; (r) an antibody that binds to the same epitope as the 24F12 antibody; (s) an antibody comprising SEQ ID NO: 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 (ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3; (t) the 24F12 antibody; (u) an antibody comprising a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16); (v) an antibody comprising a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21); (w) an antibody comprising the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); (x) an antibody comprising the LK26HuVH SLF heavy chain variable region (SEQ ID NO: 19) and the LK26HuVKPW variable region light chain, Y (SEQ ID NO: 16); and (y) an antibody comprising the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0064] In a particular embodiment, the antibody binds to the same epitope as the MORAb-003 antibody. In another embodiment, the antibody includes SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRL1, SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3. In another embodiment, the antibody is the MOV18 antibody. In yet another embodiment, the antibody binds on the same epitope as the MOV18 antibody. In an additional embodiment, the antibody comprises a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16). Alternatively, or in combination, the antibody includes a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21). In certain embodiments, the antibody includes (i) the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); the heavy chain variable region LK26HuVH SLF (SEQ ID NO: 19) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); or the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0065] In a particular modality, the level of FRα in the sample derived from said subject is evaluated by placing the sample in contact with a pair of antibodies selected from the group consisting of (a) MOV18 antibody immobilized on a solid support and MORAB- antibody. 003 marked; (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody; (c) antibody 26B3 immobilized on a solid support and labeled antibody 19D4; and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody.
[0066] In certain embodiments, the antibody is selected from the group consisting of a murine antibody, a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, Fab'2, ScFv, SMIP, affibody, avimer , versabody, nanobody, and an antibody from the domain. Alternatively, or in combination, the antibody is labeled, for example, with a marker selected from the group consisting of a radio marker, a biotin marker, a chromophore marker, a fluorophore marker, or an enzyme marker.
[0067] In certain modalities, the level of FRα is determined using a technique selected from the group consisting of western blot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, solution phase assay, electrochemiluminescent immunoassay (ECLIA ) and ELISA assay.
[0068] In various modalities of the previous aspects of the invention, the control sample is a standardized level of control of FRα in a healthy subject.
[0069] In certain embodiments, the sample is treated with guanidine before determining the level of FRα in the sample. Alternatively, or in combination, the sample is diluted before determining the level of FRα in the sample. Alternatively, or in combination, the sample is centrifuged, swirled, or both, before determining the level of FRα in the sample.
[0070] In a further aspect, the present invention relates to a method for predicting whether a subject suffering from a cancer that expresses FRα, such as ovarian cancer or lung cancer, will respond to treatment with MORAb-003, determining the level an alpha folate receptor (FRα) that is not bound to a cell in a sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the sample with the level of FRα in a control sample, where a difference between the levels of FRα in the sample derived from the subject and in the control sample is an indication that the subject will respond to treatment with MORAb-003; where the level of FRα in the sample derived from the subject is assessed by placing the sample in contact with (a) MOV18 antibody immobilized on a solid support and labeled MORAB-003 antibody, (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody , (c) 26B3 antibody immobilized on a solid support and labeled 19D4 antibody, and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody. For example, the sample can be urine, serum, plasma or ascites.
[0071] In various embodiments of the foregoing aspects of the invention, MO-RAb-003 for treatment is (a) an antibody comprising the heavy chain amino acid sequence shown in SEQ ID NO: 7 and the amino acid sequence light chain acids shown in SEQ ID NO: 8; (b) an antibody that binds to the same epitope as the MORAb-003 antibody; or (c) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as , SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3. Methods of Treating a Subject With Ovarian Cancer or Lung Cancer
[0072] In another aspect, the present invention relates to methods of treating a subject with ovarian cancer or lung cancer by determining the level of alpha folate receptor (FRα) that is not bound to a cell, in a derived sample said subject (e.g., urine, serum, plasma or ascites); and comparing the level of alpha folate receptor (FRα) that is not bound to a cell with the level of FRα in a control sample, in which a difference between the level of FRα in the sample derived from said subject and the level of FRα in the control sample is an indication that the subject is suffering from ovarian cancer or lung cancer; and administering a therapeutically effective amount of MORAb-003 to said subject.
[0073] In another aspect, the present invention relates to methods of treating a subject with ovarian cancer or lung cancer by determining the level of alpha folate receptor (FRα) that is not bound to a cell, in a sample of urine derived from said subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell with the level of FRα in a control sample, in which a difference between the level of FRα in the urine sample derived from said subject and the level of FRα in the control sample is an indication that the subject is suffering from ovarian cancer or lung cancer; and administering a therapeutically effective amount of MORAb-003 to said subject.
[0074] In another aspect, the present invention relates to methods of treating a subject with ovarian cancer or lung cancer by determining the level of alpha folate receptor (FRα) that is not bound to a cell, in a sample of serum derived from said subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell with the level of FRα in a control sample, in which a difference between the level of FRα in the serum sample derived from said subject and the level of FRα in the control sample is an indication that the subject is suffering from ovarian cancer or lung cancer; and administering a therapeutically effective amount of MORAb-003 to said subject.
[0075] In a further aspect, the present invention relates to methods for treating a subject suffering from ovarian cancer by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a subject-derived urine sample. , in which the presence of FRα that is not bound to a cell in the urine sample at a concentration greater than about 9,100 pg / mL is an indication that the subject will respond to treatment with MORAb-003; and administering a therapeutically effective amount of MORAb-003 to said subject.
[0076] In particular modalities, the level of FRα that is not bound to a cell in the sample derived from said subject is evaluated by placing the sample in contact with an antibody that binds FRα.
[0077] In various embodiments of the foregoing aspects of the invention, the presence of FRα in the urine sample at a concentration greater than about 9,500 pg / mL, about 10,000 pg / mL, about 11,000 pg / mL, about 12,000 pg / ml, about 13,000 pg / ml, about 14,000 pg / ml, about 15,000 pg / ml, about 16,000 pg / ml, about 17,000 pg / ml, about 18,000 pg / ml, about 19,000 pg / mL, or about 20,000 pg / mL, is an indication that the subject is suffering from ovarian cancer.
[0078] In various embodiments of the foregoing aspects of the invention, the level of FRα is determined by placing the sample in contact with an antibody that binds FRα. For example, the antibody is selected from the group consisting of: (a) an antibody that binds to the same epitope as the MORAb-003 antibody; (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRLH1 SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3; (c) the MOV18 antibody; (d) an antibody that binds to the same epitope as the MOV18 antibody; (e) antibody 548908; (f) an antibody that binds to the same epitope as the 548908 antibody; (g) the 6D398 antibody; (h) an antibody that binds to the same epitope as the 6D398 antibody; (i) an antibody that binds to the same epitope as the 26B3 antibody; (j) an antibody comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as CDRL2 and SEQ ID NO: 53 (QHHYAFPWT) as CDRL3; (k) the 26B3 antibody; (l) an antibody that binds to the same epitope as the 19D4 antibody; (m) an antibody comprising SEQ ID NO: 39 (HPYMH) as CDRH1, SEQ ID NO: 40 (RIDPANGNTKYDPKFQG) as CDRH2, SEQ ID NO: 41 (EEVADYTMDY) as CDRH3, SEQ ID NO: 35 (RASESVDTYGNNFIH) as CDRL1 SEQ ID NO: 36 (LASNLES) as CDRL2 and SEQ ID NO: 37 (QQNNGDPWT) as CDRL3; (n) the 19D4 antibody; (o) an antibody that binds to the same epitope as the 9F3 antibody; (p) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3; (q) the 9F3 antibody; (r) an antibody that binds to the same epitope as the 24F12 antibody; (s) an antibody comprising SEQ ID NO: 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 (ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3; (t) the 24F12 antibody; (u) an antibody comprising a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16); (v) an antibody comprising a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21); (w) an antibody comprising the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); (x) an antibody comprising the LK26HuVH SLF heavy chain variable region (SEQ ID NO: 19) and the LK26HuVKPW variable region light chain, Y (SEQ ID NO: 16); and (y) an antibody comprising the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0079] In a particular embodiment, the antibody binds to the same epitope as the MORAb-003 antibody. In another embodiment, the antibody includes SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRL1, SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3. In another embodiment, the antibody is the MOV18 antibody. In yet another embodiment, the antibody binds on the same epitope as the MOV18 antibody. In an additional embodiment, the antibody comprises a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16). Alternatively, or in combination, the antibody includes a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21). In certain embodiments, the antibody includes (i) the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); the heavy chain variable region LK26HuVH SLF (SEQ ID NO: 19) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); or the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0080] In a particular embodiment, the level of FRα in the sample derived from said subject is evaluated by placing the sample in contact with a pair of antibodies selected from the group consisting of (a) MOV18 antibody immobilized on a solid support and MORAB- antibody. 003 marked; (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody; (c) antibody 26B3 immobilized on a solid support and labeled antibody 19D4; and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody.
[0081] In certain embodiments, the antibody is selected from the group consisting of a murine antibody, a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, Fab'2, ScFv, SMIP, affibody, avimer , versabody, nanobody, and an antibody from the domain. Alternatively, or in combination, the antibody is labeled, for example, with a marker selected from the group consisting of a radio marker, a biotin marker, a chromophore marker, a fluorophore marker, or an enzyme marker.
[0082] In certain modalities, the level of FRα is determined using a technique selected from the group consisting of western blot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, solution phase assay, electrochemical chemiluminescent immunoassay (ECLIA ) and ELISA assay.
[0083] In several modalities of the previous aspects of the invention, the control sample is a standardized level of control of FRα in a healthy subject.
[0084] In certain embodiments, the sample is treated with guanidine before determining the level of FRα in the sample. Alternatively, or in combination, the sample is diluted before determining the level of FRα in the sample. Alternatively, or in combination, the sample is centrifuged, swirled, or both, before determining the level of FRα in the sample.
[0085] In a further aspect, the present invention relates to a method for treating a subject suffering from ovarian cancer or lung cancer, by determining the level of alpha folate receptor (FRα) that is not bound to a cell in a sample derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the sample with the level of FRα in a control sample, in which a difference between the levels of FRα in the sample derived from the subject and in the control sample is an indication that the subject will respond to treatment with MORAb-003; where the level of FRα in the sample derived from the subject is assessed by placing the sample in contact with (a) MOV18 antibody immobilized on a solid support and labeled MORAB-003 antibody, (b) 9F3 antibody immobilized on a solid support and labeled 24F12 antibody , (c) 26B3 antibody immobilized on a solid support and labeled 19D4 antibody, and (d) 9F3 antibody immobilized on a solid support and labeled 26B3 antibody. For example, the sample can be urine, serum, plasma or ascites.
[0086] In various embodiments of the foregoing aspects of the invention, MO-RAb-003 for treatment is (a) an antibody comprising the heavy chain amino acid sequence shown in SEQ ID NO: 7 and the amino acid sequence light chain acids shown in SEQ ID NO: 8; (b) an antibody that binds to the same epitope as the MORAb-003 antibody; or (c) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as , SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3. Cases of the Invention
[0087] In one aspect, the present invention relates to a kit to assess whether a subject is suffering from a cancer that expresses FRα or to evaluate the progression of a cancer that expresses FRα in a subject, the kit including means for determining the level of alpha folate receptor (FRα) that is not bound to a cell in a sample derived from the subject; and instructions for using the kit to assess whether the subject is suffering from a cancer that expresses FRα or to assess the progression of a cancer that expresses FRα. For example, cancer that expresses FRα is selected from the group consisting of lung cancer, mesothelioma, ovarian cancer, kidney cancer, brain cancer, cervical cancer, nasopharyngeal cancer, squamous cell carcinoma of the head and neck, endometrial cancer, breast cancer, bladder cancer, pancreatic cancer, bone cancer, pituitary cancer, colorectal cancer and medullary thyroid cancer. In a particular embodiment, the cancer that expresses FRα is ovarian cancer. In yet another modality, cancer that expresses FRα is non-small cell lung cancer, for example, adenocarcinoma. In an additional embodiment, the sample is either urine, serum, plasma or ascites.
[0088] In an additional embodiment, the medium includes a folate-binding agent (FRα) alpha receptor, for example, an antibody. In an additional embodiment, the antibody is selected from the group consisting of: (a) an antibody that binds to the same epitope as the MORAb-003 antibody; (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRLH1 SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3; (c) the MOV18 antibody; (d) an antibody that binds to the same epitope as the MOV18 antibody; (e) antibody 548908; (f) an antibody that binds to the same epitope as the 548908 antibody; (g) the 6D398 antibody; (h) an antibody that binds to the same epitope as the 6D398 antibody; (i) an antibody that binds to the same epitope as the 26B3 antibody; (j) an antibody comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as CDRL2 and SEQ ID NO: 53 (QHHYAFPWT) as CDRL3; (k) the 26B3 antibody; (l) an antibody that binds to the same epitope as the 19D4 antibody; (m) an antibody comprising SEQ ID NO: 39 (HPYMH) as CDRH1, SEQ ID NO: 40 (RIDPANGNTKYDPKFQG) as CDRH2, SEQ ID NO: 41 (EEVADYTMDY) as CDRH3, SEQ ID NO: 35 (RASESVDTYGNNFIH) as CDRL1 SEQ ID NO: 36 (LASNLES) as CDRL2 and SEQ ID NO: 37 (QQNNGDPWT) as CDRL3; (n) the 19D4 antibody; (o) an antibody that binds to the same epitope as the 9F3 antibody; (p) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3; (q) the 9F3 antibody; (r) an antibody that binds to the same epitope as the 24F12 antibody; (s) an antibody comprising SEQ ID NO: 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 (ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3; (t) the 24F12 antibody; (u) an antibody comprising a variable region light chain selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16); (v) an antibody comprising a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21); (w) an antibody comprising the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); (x) an antibody comprising the LK26HuVH SLF heavy chain variable region (SEQ ID NO: 19) and the LK26HuVKPW variable region light chain, Y (SEQ ID NO: 16); and (y) an antibody comprising the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0089] In certain embodiments, the antibody is labeled including, but not limited to, a radio marker, a biotin marker, a chromophore marker, a fluorophore marker, or an enzyme marker.
[0090] In yet another modality, the kit includes a means to obtain a sample from the subject.
[0091] The present invention is further illustrated by the following detailed description and drawings. Brief Description of Drawings
[0092] Figure 1 is a schematic representation of an electrochemiluminescence method immunoassay (ECLIA) to evaluate FRα in urine as described in the examples. MOV18 antibody attached to solid supports binds FRα in urine. FRα was subsequently detected by binding to Ru-labeled MORAb-003 antibody.
[0093] Figure 2 shows the distribution of FRα levels in the urine of subjects with ovarian cancer and normal control subjects measured by ECLIA (see Example 1).
[0094] Figure 3 represents the detection of FRα in the urine of patients with ovarian cancer (faded band in lane 1, clear band in lane 2) using immunoblotting (see Example 5).
[0095] Figure 4 shows the distribution of FRα levels in the urine of subjects with ovarian cancer and normal control subjects measured by ECLIA after the urine was treated with guanidine, as described in example 7.
[0096] Figure 5 represents a ROC curve showing the sensitivity and specificity of the ECLIA measurement of FRα levels in urine after urine has been treated with guanidine, as described in example 7. The area under the curve (AUC) measures the accuracy of the test in separating ovarian cancer from control subjects. A cutoff value (above which the test results were considered abnormal) of 9,100 pg / mL was used.
[0097] Figure 6 shows the distribution of FRα levels in ovarian cancer (OC) and normal control subjects after correction for creatinine levels. There is a statistically significant difference between patients with ovarian cancer and controls at corrected creatinine levels of FRα (p = 0.007) (see Example 8).
[0098] Figure 7 represents a ROC analysis of creatinine-corrected FRα levels determined using the electrochemiluminescence assay (ECLIA) of urine samples treated with guanidine (see Example 8).
[0099] Figure 8 is a schematic representation of the enzyme immunoassay (EIA) method used to assess the level of FRα (ie, FRα) in the samples, as described in example 9. MOV-18 served as the antibody capture, which binds FRα of biological fluids. FRα was detected by binding to the bio-tinylated MORAb-003, which was detected using avidin conjugated to horseradish peroxidase (avidin-HRP).
[00100] Figure 9 represents results obtained for the measurement of FRα in serum using one- and two-step incubation procedures, as described in example 9.
[00101] Figure 10 is a schematic representation of the three different combinations of capture antibodies and detectors that were used with the enzyme immunoassay (EIA) method to assess the level of FRα in human plasma, as described in the example 11.
[00102] Figure 11 shows the plasma concentrations of FRα (pg / mL) for individual subjects determined using EIA with three combinations of capture antibodies and detectors, as described in example 11.
[00103] Figure 12 represents the relationship between OD values and FRα concentrations (see Example 11).
[00104] Figure 13 shows the distribution of FRα concentrations in plasma in subjects with ovarian cancer and normal control subjects determined using EIA (see Example 12).
[00105] Figure 14 represents the correlation between FRαs plasma concentrations determined using EIA and ECLIA (see Example 12).
[00106] Figure 15 shows correlations between ECLIA measurements of FRα levels in serum and urine. The correlation for patients with lung cancer was r = 0.24 (upper panel) and the correlation for patients with ovarian cancer was r = -0.76 (lower panel) (see Example 13).
[00107] Figure 16 shows the correlation of FRα levels in serum versus plasma for assays conducted using pair 1 (see Example 16).
[00108] Figure 17 shows the correlation of FRα levels in serum versus plasma for assays conducted using pair 2 (see Example 16).
[00109] Figure 18 shows the correlation of FRα levels in serum for assays conducted using pair 1 versus pair 2 (see Example 16).
[00110] Figure 19 shows the correlation of FRα levels in plasma for assays conducted using pair 1 versus pair 2 (see Example 16).
[00111] Figure 20 shows the intraday correlation of FRα levels in serum for assays conducted using pair 2 (Example 16). Detailed Description of the Invention
[00112] The present invention is based, at least in part, on the surprising discovery that alpha folate receptor (FRα), not bound to a cell, is observed at high levels in body fluids, for example, urine or serum , from a subject with a cancer that expresses FRα such as lung or ovarian cancer, compared to a control sample. In addition, the present invention is based, at least in part, on the identification of an immunological assay that exhibits the sensitivity needed to assess levels of FRα in samples, where previous attempts at evaluation have repeatedly failed. As a result, the present invention relates to methods for diagnosing a cancer that expresses FRα by assessing levels of a FRα not bound to a cell in samples derived from the subject. Certainly, the present invention overcomes the challenges observed during previous attempts to develop an FRα-based diagnostic assay for cancers that express FRα, such as ovarian cancer, by providing an immunological assay capable of accurately assessing levels of FRα not bound to a cell in samples.
[00113] In this way, methods and kits are provided to assess whether a subject has a cancer that expresses FRα or is at risk of developing it and, additionally, to assess the progression of cancer that expresses FRα. In several modalities, the methods involve comparing levels of FRα not bound to a cell in the samples, for example, urine and serum, in relation to the control levels, in assessing the presence, degree or risk of developing ovarian cancer in the subject. In particular embodiments, the methods involve the use of the MO-RAb-003 antibody, antibodies that bind the same epitope as the MORAb-003 antibody or antibodies with SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG ) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRL1, SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3, in the evaluation of the levels of FRα not bound to a cell in a sample, for example, urine or serum. Alternatively, or in addition, the MOV18 antibody or an antibody that binds to the same epitope as the MOV18 antibody, the 548908 antibody, an antibody that binds to the same epitope as the 548908 antibody, the 6D398 antibody or an antibody that binds to the same epitope as the antibody 548908 can be used according to the methods of the present invention. Various aspects of the invention are described in more detail in the following subsections: I. Definitions
[00114] As used herein, each of the following terms has the meaning associated with it in this section.
[00115] The articles "one" and "one" are used here to refer to one or more than one (ie q at least one) of the grammatical purpose of the article. For example, "an element" means an element or more than an element.
[00116] As used herein, the term "subject" refers to human and non-human animals, including veterinary subjects. The terms "non-human animal" include all vertebrates, for example, mammals and non-mammals, such as non-human primates, mice, rabbits, goats, dog, cat, horse, cow, chickens, amphibians and reptiles. In a preferred embodiment, the subject is a human.
[00117] The terms "cancer" or "tumor" are well known in the art and refer to the presence, for example, in a subject, of characteristics having cells typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone in a subject, or they may be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" includes pre-malignant as well as malignant cancers.
[00118] As used here, a “cancer that expresses FRα” includes any type of cancer characterized in which the cancer cells express FRα. In particular modalities, cancer that expresses FRα includes cancerous conditions characterized in that the cancer cells are able to secrete, harbor, export or release FRα in such a way that high levels of FRα are detectable in a biological sample of the subject. Cancers that express FRα include, but are not limited to, lung cancer (for example, bronchialalveolar carcinomas, carcinoid tumors, and non-small cell lung cancers, such as adenocarcinomas); mesothelioma; Ovary cancer; kidney cancer; brain cancer (for example, anaplastic ependymoma and cerebellar juvenile pilocytic astrocytoma); cervical cancer; nasopharyngeal cancer; mesodermally derived tumor; squamous cell carcinoma of the head and neck; endometrial cancer; endometrioid adenocarcinomas of the ovary, serous cystadenocarcinomas, breast cancer; bladder cancer; pancreatic cancer; bone cancer (eg, high-grade osteosarcoma); pituitary cancer (for example, pituitary adenoma). See, for example, U.S. Patent No. 7,754,698; U.S. patent application No. 2005/0232919; WO 2009/132081; Bueno R et al. J of Thoracic and Cardiovascular Surgery, 121 (2): 225233 (2001); Elkanat H & Ratnam M. Frontiers in Bioscience, 11, 506-519 (2006); Franklin, WA et al. Int J Cancer, Suppl 8: 89-95 (1994); Hartman L.C. et al. Int J Cancer 121: 938-942 (2007); Iwakiri S et al. Annals of Surgical Oncology, 15 (3): 889-899; Weitman, SD et al. Cancer Res 52: 3396-3401 (1992); Saba N.F. et al. Head Neck, 31 (4): 475-481 (2009); Yang R et al. Clin Cancer Res 13: 2557-2567 (2007). In a particular embodiment, the cancer that expresses FRα is ovarian cancer. In another embodiment, cancer that expresses FRα is lung cancer just like non-small cell lung cancer. In other modalities, the cancer that expresses FRα is colorectal cancer and medullary thyroid cancer.
[00119] As used herein, a subject who is "suffering from a cancer that expresses FRα" is one who is clinically diagnosed with a cancer like this by a qualified physician (for example, by the methods of the present invention), or one who shows one or more signs or symptoms (e.g., high levels of FRα in biological fluids) from a cancer like that and is subsequently clinically diagnosed with a cancer like that by a qualified physician (for example, by the methods of the present invention). A non-human subject that serves as an animal model of cancer that expresses FRα may also fall within the scope of the terms a subject “suffering from cancer that expresses FRα”.
[00120] The terms "ovarian cancer" refer to the disease recognized by the technique and each includes epithelial ovarian cancer (EOC;> 90% ovarian cancer in western countries), germ cell tumors (circa 2- 3% of ovarian cancer), and stromal ovarian cancer. Ovarian cancer is stratified in different groups based on the differentiation of the tumor tissue. In grade I, the tumor tissue is well differentiated. In grade II, the tumor tissue is moderately well differentiated. In grade III, the tumor tissue is poorly differentiated. This grade correlates with a less favorable prognosis than grades I and II.
[00121] Ovarian cancer is stratified at different stages based on the spread of the cancer. Stage I is generally confined within the capsule involving one (stage IA) or both (stage IB) ovaries, although in some stage I cancers (ie stage IC), malignant cells can be detected in ascites, in washing fluid peritoneal, or on the surface of the ovaries. Stage II involves tumor extension or metastasis from one or both ovaries to other pelvic structures. In stage IIA, the tumor extends or has been metastasized in the uterus, in the fallopian tubes, or both. Stage IIB involves extending the tumor to the pelvis. Stage IIC is stage IIA or IIB in which malignant cells can be detected in ascites, in peritoneal lavage fluid, or on the surface of the ovaries. In stage III, the tumor comprises at least one malignant extension to the small intestine or omentum, extrapelvic peritoneal implants of microscopic (stage IIIA) or macroscopic size (<2 cm in diameter, stage IIIB;> 2 cm in size) have been formed. diameter, stage IIIC), or was metastasized to a retroperitoneal or inguinal lymph node (an alternate indicator of stage IIIC). In stage IV, distant (ie non-peritoneal) metastases from the tumor can be detected.
[00122] The durations of the various stages of ovarian cancer are not known at present, but are believed to be at least about a year each (Richart et al., 1969, Am. J. Obstet. Gynecol. 105: 386 ). The prognosis declines with greater stage designation. For example, 5-year survival rates for human subjects diagnosed with stage I, II, III, and IV ovarian cancer are 80%, 57%, 25%, and 8%, respectively.
[00123] Each of the previous types, groups and stages of ovarian cancer are encompassed by the terms "ovarian cancer" in the manner used here.
[00124] As used herein, the terms "lung cancer" refer to a disease in lung tissues involving uncontrolled cell growth, which in some cases leads to metastasis. Lung cancer is the most common cause of cancer-related death in men and women. Most primary lung cancers are lung carcinomas, derived from epithelial cells. The main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). In a particular embodiment, the cancer that expresses FRα is a non-small cell lung cancer.
[00125] Small cell lung cancer or small cell lung carcinoma (SCLC) is a malignant lung cancer, in which the cancer cells have a flat shape and a scarce cytoplasm; therefore, SCLC is sometimes called “lymphocytoid carcinoma”. SCLC is generally more metastatic than NSCLC and is sometimes seen in combination with squamous cell carcinoma.
[00126] As used herein, the terms "non-small cell lung cancer," also known as non-small cell lung cancer (NSCLC), refer to epithelial lung cancer other than small cell lung cancer (SCLC). There are three main subtypes: adenocarcinoma, squamous cell lung carcinoma, and large cell lung carcinoma. Other less common types of non-small cell lung cancer include pleomorphic carcinoid tumors, salivary gland carcinoma, and unclassified carcinoma. Adenocarcinomas represent approximately 40% of lung cancers, and are the most common type of lung cancer in people who have never smoked. Squamous cell carcinomas account for about 25% of lung cancers. Squamous cell lung cancer is more common in men than in women and is even more highly correlated with a history of tobacco smokers who are other types of lung cancer. There are at least four variants (papillary, small cell, clear cell, and basaloid) of squamous cell lung carcinoma. Large cell lung carcinomas are a heterogeneous group of malignant neoplasms that originate from transformed epithelial cells in the lung. Large cell lung carcinomas are carcinomas that do not have mild microscopic features of small cell carcinoma, squamous cell carcinoma, or adenocarcinoma.
[00127] Different stage systems are used for SCLC and NSCLC. SCLC is categorized as a limited disease confined to the ipsilateral hemithorax or as an extensive disease with metastasis beyond the ipsilateral hemithorax.
[00128] NSCLC can be categorized using the tumor-nodule-metastasis (TNM) system. See Spira J & Ettinger, D.S. Multidisciplinary management of lung cancer, N Engl J Med, 350: 382- (2004) (then, Spira); Greene FL, Page DL, Fleming ID, Fritz AG, Balch CM, Haller DG, et al (eds). AJCC Cancer Staging Manual. 6th edition. New York: Springer-Verlag, 2002: 167-77 (hereinafter Greene); Sobin LH, Wittekind CH (eds). International Union Against Cancer. TNM classi fication of maligant tumors. 6th edition. New York: Wiley-Liss (2002) (then Sobin). In addition, NSCLC is typically treated according to the stage of cancer determined by the following classification scheme (see http://www.cancer.gov/cancertopics/pdq/treatment/non-small-cell- lung / Patient / page2 # Keypoint10).
[00129] In the hidden (hidden) stage, cancerous cells are seen in sputum (mucus expelled from the lungs), but no tumor in the lung can be observed by imaging or bronchoscopy, or the tumor is too small to be verified.
[00130] In stage 0 (carcinoma in situ), abnormal cells are observed in the lining of the airways. These abnormal cells can become cancer and spread to normal tissue nearby. Stage 0 is also called carcinoma in situ.
[00131] Stage I, in which cancer was formed, is divided into Stages IA and IB.
[00132] In the LlA stage, the tumor is only in the lung and is 3 centimeters or less.
[00133] In stage IB, the cancer did not spread in the lymph nodes and one or more of the following is true: (i) The tumor is larger than 3 centimeters but not larger than 5 centimeters; (ii) the cancer has spread in the main bronchi and is at least 2 centimeters below where the trachea joins the bronchi; (iii) the cancer has spread to the innermost layer of the membrane that covers the lung; (iv) part of the lung has collapsed or developed pneumonitis (inflammation of the lung) in the area where the trachea joins the bronchi.
[00134] In stage IIA, the cancer spread in certain lymph nodes on the same side of the breast as the primary tumor; the cancer is (a) 5 cm or less than, (b) spread in the main bronchi, and / or (c) spread in the innermost layer of the lung lining. The cancer did not spread to the lymph nodes; the cancer is (d) larger than 5 cm, but not larger than 7 cm, (e) spread in the main bronchi, and / or (f) spread in the innermost layer of the lung lining. Part of the lung may have collapsed or become inflamed. Stage IIA is divided into two sections, depending on the size of the tumor, where the tumor is found, and whether there is cancer in the lymph nodes. In the first section, the cancer spread to the lymph nodes on the same side of the tumor's chest. The lymph nodes with cancer are inside the lung or close to the bronchi. Also, one or more of the following is true: (i) the tumor is no larger than 5 centimeters, (ii) the cancer has spread in the main bronchi and is at least 2 centimeters below where the trachea joins the bronchi, (iii) the cancer spread in the innermost layer of the membrane that covers the lung, (iv) part of the lung collapsed or developed pneumonitis (inflammation of the lung) in the area where the trachea joins the bronchi. In the second section, the cancer has not spread to the lymph nodes and one or more of the following is true: (i) the tumor is larger than 5 centimeters, but not larger than 7 centimeters, (ii) the cancer has spread in the main bronchi and is at least 2 centimeters below where the trachea joins the bronchi, (iii) the cancer has spread to the innermost layer of the membrane covering the lung, (iv) part of the lung has collapsed or developed pneumonitis (inflammation of the lung) in the area where the trachea joins the bronchi .
[00135] In stage IIB, the cancer spread in certain lymph nodes on the same side of the breast as the primary tumor; the cancer is (a) larger than 5 cm but not larger than 7 cm, (b) spread in the main bronchi, and / or (c) spread in the innermost layer of the lung lining. Part of the lung may have collapsed or become inflamed. Alternatively, (d) the cancer is larger than 7 cm; (e) spread in the main bronchi, (f) in the diaphragm, (g) in the chest wall or the lining of the chest wall; and / or (h) spread on the membrane around the heart. there may be one or more separate tumors in the same lung lobe; cancer may have spread to the nerve that controls the diaphragm; the whole lung may have collapsed or become inflamed. Stage IIB is divided into two sections depending on the size of the tumor, where the tumor is found, and whether there is cancer in the lymph nodes. In the first section, the cancer spread in the vicinity of the lymph nodes on the same side of the tumor's chest. The lymph nodes with cancer are inside the lung or close to the bronchi. Also, one or more of the following is true: (i) the tumor is larger than 5 centimeters, but not larger than 7 centimeters, (ii) the cancer has spread in the main bronchi and is at least 2 centimeters below where the trachea joins the bronchi , (iii) the cancer has spread to the innermost layer of the membrane covering the lung, (iv) part of the lung has collapsed or developed pneumonitis (inflammation of the lung) in the area where the trachea joins the bronchi. In the second section, the cancer has not spread in the lymph nodes and one or more of the following is true: (i) the tumor is larger than 7 centimeters, (ii) the cancer has spread in the main bronchi (and is less than 2 centimeters below where the trachea joins the bronchi), on the chest wall, on the diaphragm, or on the nerve that controls the diaphragm, (iii) the cancer has spread on the membrane around the heart or lining of the chest wall, (iv) the whole lung has collapsed or developed pneumonitis (inflammation of the lung), (v) there are one or more separate tumors in the same lobe of the lung.
[00136] Stage IIIA is divided into three sections depending on the size of the tumor, where the tumor is found, and which lymph nodes have cancer (if any). In the first section of Stage IIIA, the cancer spread to the lymph nodes on the same side of the tumor's chest. Lymph nodes with cancer are close to the sternum (breast bone) or where the bronchi enter the lung. Also, the tumor can be of any size; part of the lung (where the trachea joins the bronchi) or the whole lung may have collapsed or developed pneumonitis (inflammation of the lung); there may be one or more separate tumors in the same lung lobe; and the cancer may have spread in any of the following: (i) main bronchi, but not the area where the trachea joins the bronchi, (ii) chest wall, (iii) diaphragm and the nerve that controls it, (iv ) membrane around the lung or lining of the chest wall, (iv) membrane around the heart. In the second section of Stage IIIA, the cancer spread to the lymph nodes on the same side of the tumor's chest. The lymph nodes with cancer are inside the lung or close to the bronchi. Also, the tumor can be of any size; the whole lung may have collapsed or developed pneumonitis (inflammation of the lung); there may be one or more separate tumors in any of the lobes of the lung with cancer; and the cancer may have spread in any of the following: (i) main bronchi, but not the area where the trachea joins the bronchi, (ii) chest wall, (iii) diaphragm and the nerve that controls it, (iv) membrane around the lung or lining of the chest wall, (v) heart or the membrane around it, (vi) main blood vessels entering or leaving the heart, (vi) trachea, (vii) esophagus, (viii) nerve that controls the larynx (voice box), (ix) sternum (breast bone) or clavicle, (x) carina (where the trachea joins the bronchus). In the third section of Stage IIIA, the cancer did not spread to the lymph nodes and the tumor could be any size, and the cancer spread to any of the following: (i) heart, (ii) major blood vessels entering and leaving the heart, (iii) trachea, (iv) esophagus, (v) nerve that controls the larynx (voice box), (vi) sternum (chest bone) or backbone, (vi) carina (where the trachea joins the bronchus).
[00137] Stage IIIB is divided into two sections depending on the size of the tumor, where the tumor is found, and which lymph nodes have cancer. In the first section, the cancer spread in the lymph nodes above the collarbone or in the lymph nodes on the opposite side of the tumor's chest; the tumor can be of any size; part of the lung (where the trachea joins the bronchi) or the whole lung may have collapsed or developed pneumonitis (inflammation of the lung); there may be one or more separate tumors in any of the lobes of the lung with cancer; and the cancer may have spread in any of the following: (i) major bronchi, (ii) chest wall, (iii) diaphragm and the nerve that controls it, (iv) membrane around the lung or lining of the chest wall, (iv) heart or the membrane around it, (v) main blood vessels entering and leaving the heart, (vi) trachea, (vii) esophagus, (viii) nerve that controls the larynx (voice box), (ix ) sternum (breast bone) or backbone, (x) carina (where the trachea joins the bronchus). In the second section of Stage IIIB, the cancer spread to the lymph nodes on the same side of the tumor's chest; lymph nodes with cancer are close to the sternum (breast bone) or where the bronchi enter the lung; the tumor can be of any size; there may be separate tumors in different lobes of the same lung; and cancer spread in any of the following: (i) heart, (ii) major blood vessels entering and leaving the heart, (iii) trachea, (iv) esophagus, (v) nerve that controls the larynx (voice box ), (vi) sternum (breast bone) or backbone, (vii) carina (where the trachea joins the bronchus).
[00138] In stage IV, the tumor may have any size and the cancer may have spread to the lymph nodes. One or more of the following is true: there are one or more tumors in both lungs; cancer is found in the fluid around the lungs or the heart; cancer has spread to other parts of the body, such as the brain, liver, adrenal glands, kidneys, or bone.
[00139] Thus, in various modalities of the previous invention, lung cancer can be stratified in any of the previous stages (for example, hidden, stage 0, stage IA, stage IB, stage IIA, stage IIB, stage IIIA, stage IIIB or stage IV) based on the assessment of FRα levels not bound to a cell, such as a normal or cancerous cell, in an example sample (by urine or serum) from a subject.
[00140] As used herein, the terms "alpha folate receptor" (also referred to as FRα, FR-alpha, FOLR-1 or FOLR1) refer to the alpha isoform of the high affinity receptor for folate. Membrane-bound FRα is attached to the cell surface by recycling the glycosyl phosphatidylinositol (GPI) anchor between extracellular and endocytic compartments and is capable of transporting folate in the cells. FRα is expressed in a variety of epithelial tissues including those of the female reproductive tract, placenta, breast, tubular proximal kidney, choroid plexus, lung and salivary glands. Soluble forms of FRα can be derived by the action of proteases or phospholipase on folate receptors anchored in the membrane.
[00141] The consensus nucleotide and amino acid sequences for human FRα are presented here as SEQ ID NOs: 24 and 25, respectively. Variants, for example, naturally occurring allelic variants or sequences containing at least one amino acid substitution, are encompassed by the terms as used herein.
[00142] As used herein, the terms "not bound to a cell" refer to FRα which is not attached to the cell membrane of a cell, such as a cancer cell. In a particular embodiment, FRα not bound to a cell is unbound to any cell and is freely floating or solubilized in biological fluids, for example, urine or serum. For example, FRα can be housed, secreted or exported from normal or cancer cells, for example, from the surface of cancer cells, in biological fluids.
[00143] The "level" of alpha folate receptor not bound to a cell, as used herein, refers to the protein level of the alpha folate receptor determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, assays colorimetric, spectrophotometric assays, flow cytometer, immunodiffusion (single or double), solution phase assay, immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme linked immunosorbent assays (ELISAs), immunofluorescent assays, and electrochemical immunoassay (exemplified below), and the like. In a preferred embodiment, the level is determined using antibody-based techniques, as described in more detail here.
[00144] It is generally preferred both to immobilize an antibody and a specific binding protein for FRα not bound to a cell on a solid support for Western blots and immunofluorescence techniques. Suitable solid-phase carriers or vehicles include any support capable of binding an antigen or an antibody. Well-known supports or vehicles include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
[00145] Those skilled in the art will perceive many other vehicles suitable for binding antibody or antigen, and will be able to adapt such support for use with the present invention. For example, protein isolated from a sample of the subject (eg, urine or serum) can be run on a polyacrylamide gel electrophoresis and immobilized on a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of marker attached to the solid support can then be detected by conventional means. Means of detecting proteins using electrophoretic techniques are well known to those skilled in the art (see generally R. Escopes (1982) Protein Purification, Springer-Verlag, NY; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification , Academic Press, Inc., NY).
[00146] Other standard methods include immunoassay techniques that are well known to those skilled in the art and can be seen in Principles And Practice OD Immunoassay, 2nd Edition, Price and Newman, eds., MacMillan (1997) and Antibodies, a Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, Ch. 9 (1988), each of which is incorporated herein by reference in its entirety.
[00147] Antibodies used in immunoassays to determine the level of alpha folate receptor expression can be marked with a detectable marker. The term "labeled", with respect to the binding agent or antibody, is intended to encompass direct labeling of the binding agent or coupling antibody (i.e., physically binding) a detectable substance in the binding agent or antibody, as well as indirect labeling of the agent binding or antibody by reactivity with another reagent that is directly labeled. An example of indirect labeling includes detection of a primary antibody using a fluorescent-labeled secondary antibody. In one embodiment, the antibody is, for example, radiolabeled, chromophore labeled, labeled fluorophore, or labeled enzyme. In another embodiment, the antibody is a derivative of the antibody (for example, an antibody conjugated to a substrate or to the protein or linker of a protein-linker pair (for example, biotin-streptavidin), or a fragment of the antibody (for example, example, a single chain antibody, an isolated hypervariable domain) that specifically binds with FRα not bound to a cell.
[00148] In an embodiment of the invention, proteomic methods are used, for example, mass spectrometry. Mass spectrometry is an analytical technique that consists of ionizing chemical compounds to generate charged molecules (or their fragments) and measure their mass to charge ratios. In a typical mass spectrometry procedure, a sample is obtained from a subject, loaded into mass spectrometry, and its components (for example, FRα) are ionized by different methods (for example, impacting them with an electronic beam), resulting in the formation of charged particles (ions). The mass to charge ratio of the particles is then calculated from the movement of the ions as they pass through the electromagnetic fields.
[00149] For example, laser desorption / ionization flight time mass spectrometry associated with the matrix (MALDI-TOF MS) or surface-enhanced laser desorption / ionization flight time mass spectrometry (SELDI -TOF MS) which involves applying a sample, such as urine or serum, to a protein binding chip (Wright, GL, Jr., et al. (2002) Expert Rev Mol Diagn 2: 549; Li, J ., et al. (2002) Clin Chem 48: 1296; Laronga, C., et al. (2003) Dis Markers 19: 229; Petricoin, EF, et al. (2002) 359: 572; Adam, BL, et al. (2002) Cancer Res 62: 3609; Tolson, J., et al. (2004) Lab Invest 84: 845; Xiao, Z., et al. (2001) Cancer Res 61: 6029) can be used to determine the FRα level.
[00150] In addition, in vivo techniques for determining the level of FRα not bound to a cell include introducing into a subject a labeled antibody directed against FRα, which binds and transforms FRα into a detectable molecule. The presence, level, or location of unbound FRα detectable to a cell in a subject can be determined using standard imaging techniques.
[00151] The term "sample" as used herein refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. In preferred embodiments, the sample is a biological fluid containing FRα not bound to a cancer cell. Biological fluids are typically liquid at physiological temperatures and may include naturally occurring fluids present, removed, expressed or otherwise extracted from a subject or biological source. Certain biological fluids are derived from particular tissues, organs, or localized regions, and certain other biological fluids may be more globally or systematically located in a biological subject or source. Examples of biological fluids include blood, serum and serous fluids, plasma, lymph, urine, cerebrospinal fluid, saliva, eye fluids, cystic fluid, tear drops, feces, sputum, mucous secretions from secretory tissues and secretions from the vaginal organ, gynecological fluids, ascites fluids such as those associated with non-solid tumors, fluids from the pleura, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage and the like. In a particular embodiment, the sample is urine or serum. In another embodiment, the sample does not include ascites or is not a sample of ascites. In another embodiment, the sample does not include peritoneal fluid or is not peritoneal fluid.
[00152] In one embodiment, the sample is removed from the subject. In another modality, the sample is present in the subject. Biological fluids can also include liquid solutions in contact with a biological subject or source, for example, cell and organ culture medium including conditioned cell or organ medium, washing fluids and the like.
[00153] In some embodiments, only a portion of the sample is subjected to an assay to determine the level of FRα not bound to a cell, or several portions of the sample are subjected to several assays to determine the level of FRα not bound to a cell . Also, in many embodiments, the sample can be pretreated by physical or chemical means prior to testing. For example, in modalities discussed in more detail in the examples section, samples, for example, urine samples, were subjected to centrifugation, dilution and / or treatment with a solubilizing substance (for example, treatment with guanidine) before testing the samples for FRα not bound to a cell. Such techniques serve to increase the accuracy, reliability and reproducibility of the assays of the present invention.
[00154] The terms "control sample," as used herein, refer to any clinically relevant control sample, including, for example, a sample from a healthy subject not suffering from ovarian cancer, a sample from a subject with a less severe or slower progression of ovarian cancer than the subject being evaluated, a sample of a subject with some other type of cancer or disease, and the like. A control sample can include a sample derived from one or more subjects. A control sample can also be a sample taken at an early point in time for the subject being evaluated. For example, the control sample could be a sample taken from the subject to be evaluated before the onset of cancer that expresses FRα such as lung or ovarian cancer, at an early stage of the disease, or before administration of treatment or a treatment portion. The control sample can also be a sample of an animal model, or of a tissue or cell line derived from the animal model, of cancer that expresses FRα such as lung or ovarian cancer. The level of FRα not bound to a cell in a control sample consisting of a group of measurements can be determined based on any appropriate statistical measure, such as, for example, measures of central tendency including mean, median, or modal values.
[00155] The terms "level of control" refer to an accepted or predetermined level of FRα that is used to compare with the level of FRα in a sample derived from a subject. In one embodiment, the level of FRα control is based on the level of FRα not linked to a sample cell (s) from a subject (s) with low disease progression. In another embodiment, the level of control of FRα not bound to a cell is based on the level in a sample from a subject (s) with rapid disease progression. In another modality, the level of control of FRα is based on the level of FRα not bound to a cell in a sample (s) of a subject (s), unaffected, ie , not sick that is, a subject who does not have a cancer that expresses FRα such as lung or ovarian cancer. In yet another modality, the level of control of FRα is based on the level of FRα not bound to a cell in a sample of a subject (s) prior to the administration of an ovarian cancer therapy. In another embodiment, the level of control of FRα is based on the level of FRα not bound to a cell in a sample (s) of a subject (s) with a cancer that expresses FRα such as cancer of lung or ovary that is not in contact with a test compound. In another embodiment, the level of control of FRα is based on the level of FRα not bound to a cell in a sample (s) of a subject (s) not with a cancer that expresses FRα such as lung or ovarian cancer that is in contact with a test compound. In one embodiment, the level of control of FRα is based on the level of FRα not bound to a cell in a sample (s) of an animal model of a cancer that expresses FRα such as lung or ovarian cancer, a cell, or a cell line derived from the animal model of a cancer that expresses FRα such as lung or ovarian cancer.
[00156] In one embodiment, the control is a standardized control, such as, for example, a control that is predetermined using an average of the levels of FRα not bound to a cell in a population of subjects without any cancer that expresses FRα such as lung or ovarian cancer. In yet other embodiments of the invention, a level of control of FRα is based on the level of FRα not bound to a cell in a non-cancerous sample (s) derived from the subject with a cancer that expresses FRα such as lung or ovarian cancer. For example, when a laparotomy or other medical procedure reveals the presence of ovarian cancer in a portion of the ovaries, the level of control of FRα can be determined using the unaffected portion of the ovaries, and this level of control can be compared with FRα level in an affected portion of the ovaries. Similarly, when a biopsy or other medical procedure reveals the presence of lung cancer in a portion of the lungs, the level of control of FRα can be determined using the unaffected portion of the lungs, and this level of control can be compared with FRα level in an affected portion of the lungs.
[00157] As used herein, "a difference" between the level of alpha folate receptor not bound to a cell in a subject sample (ie, a test sample) and the level of alpha folate receptor not bound to one cell in a control sample largely refers to any clinically relevant and / or statistically significant difference in the level of alpha folate receptor in the two samples. In an exemplary modality, the difference is selected based on a cutoff value determined using a characteristic receiver operational analysis (ROC), an example of which is shown in example 6.
[00158] In other modalities, the difference must be greater than the detection limits of the method to determine the level of FRα not bound to a cell. It is preferred that the difference is at least greater than the standard error of the evaluation method, and preferably a difference of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 100, about 500, about 1,000 times or more than the standard error of the evaluation method. The difference can be assessed by any appropriate comparison, including any appropriate parametric or nonparametric statistics or descriptive comparison. For example, "an increase" in the level of FRα not bound to a cell can refer to a level in a test sample that is about two, and more preferably about three, about four, about five, about six, about seven, about eight, about nine, about ten or more times the level of FRα in the control sample. An increase can also refer to a level in a test sample that is preferably at least about 1.5, and more preferably about two, about three, about four, about five or more standard deviations above the level mean of FRα in the control sample. Similarly, "a decrease" in the level of FRα not bound to a cell can refer to a level in a test sample that is preferably at least about two, and more preferably about three, about four, about five, about six, about seven, about eight, about nine, about ten or more times less than the level of FRα in the control sample. A decrease can also refer to a level in a test sample that is preferably at least about 1.5, and more preferably about two, about three, about four, about five or more standard deviations below the mean level of FRα in the control sample.
[00159] As used herein, the terms "contacting the sample" with an FRα binding agent, for example, an anti-FRα antibody, includes exposing the sample, or any portion thereof with the agent or antibody, to such that at least a portion of the sample comes into contact with the agent or antibody. The sample or portion of it can be altered in some way, such as by subjecting it to physical or chemical treatments (for example, dilution or treatment with guanidine), before the act of putting it in contact with the agent or antibody.
[00160] The term "antibody", as used herein, comprises four polypeptide chains, two heavy chains (H) and two light chains (L), interconnected by disulfide bonds, as well as any mutant, variant, variant functional, or one of these (ie, antigen binding) that retain the essential epitope binding resources of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art, and include molecules such as Fab fragments, Fab 'fragments, F (ab') 2 fragments, Fd fragments, Fabc fragments, Sc antibodies (single chain antibodies), diabody, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and the like. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
[00161] Each heavy chain is comprised of a variable heavy chain region (abbreviated here as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated here as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of a domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interlaced with regions that are more conserved, called frame regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino to carboxy terminals in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody isotype as IgG, IgM, IgA, IgD and IgE, respectively.
[00162] The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The antibody constant regions can mediate the binding of immunoglobulin in host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (Clq) of the classic complement system.
[00163] The terms "antigen binding portion" of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (for example, FRα not bound to a cell). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the terms "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al. (1989) Nature 341, 544-546), which consists of a VH domain; (vii) a dAb consisting of a VH domain or a VL; and (viii) an isolated complementarity determining region (CDR) or (ix) a combination of two or more isolated CDRs that can optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded for separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be made as a single protein chain in which the regions VL and VH pair to form monovalent molecules (known as single-chain Fv (scFv); see for example, Bird et al. (1988) Science 242, 423-426; and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85, 5879-5883). Such single chain antibodies should also be encompassed in the terms "antigen binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are classified for utility in the same way since they are intact antibodies. Antigen binding moieties can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
[00164] The term "antibody", as used herein, includes polyclonal antibodies, monoclonal antibodies, murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies, and those that occur naturally or are produced recombinantly according to well-established methods. known in the art.
[00165] In one embodiment, an antibody for use in the methods of the invention is a bispecific antibody. A "bispecific antibody" is an artificial hybrid antibody with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or ligation of Fab 'fragments. See, for example, Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79, 315-321; Kostelny et al. (1992) J. Immunol. 148, 1547-1553.
[00166] In another embodiment, an antibody for use in the methods of the invention is a camelid antibody as described, for example, in PCT publication WO 94/04678, the contents of which are incorporated herein by reference.
[00167] A region of the camelid antibody that is the small single variable domain, identified as VHH can be obtained by genetic engineering to produce a small protein with high affinity for a target, resulting in a protein derived from the low molecular weight antibody, known as a “camelid nanocorp”. See U.S. Patent No. 5,759,808; see also Stijlemans et al., 2004 J. Biol. Chem. 279: 1256-1261; Dumoulin et al., 2003 Nature 424: 783-788; Pleschberger et al., 2003 Bioconjugate Chem. 14: 440-448; Cortez-Retamozo et al., 2002 Int. J. Cancer 89: 456-62; and Lauwereys et al., 1998 EMBO J. 17: 3512-3520. Engineered libraries modified by camelid antibodies and antibody fragments, for example, are from Ablynx, Ghent, Belgium. Thus, a feature of the present invention is a camelid nanocorp with a high affinity for FRα.
[00168] In other embodiments of the invention, an antibody for use in the methods of the invention is a body, a single chain body, or a di-body.
[00169] Bodies are bivalent, bispecific molecules in which VH and VL domains are expressed in a single polypeptide chain, connected by a linker that is too small to allow pairing between the two domains in the same chain. The VH and VL domains pair with complementary domains of another chain, thereby creating two antigen binding sites (see, for example, Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90: 6444-6448 ; Poljak et al., 1994 Structure 2: 1121-1123). Diabodies can be produced by expressing two polypeptide chains both with the VHA-VLB and VHB-VLA structure (VH-VL configuration), and VLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. Most of them can be expressed in bacteria-soluble form.
[00170] Single-chain (scDb) bodies are produced by connecting the two diabody-forming polypeptide chains with a linker of approximately 15 amino acid residues (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45 (3-4): 128-30; Wu et al., 1996 Immunotechnology, 2 (1): 21-36). scDb can be expressed in bacteria in active soluble monomeric form (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45 (34): 128-30; Wu et al., 1996 Immunotechnology, 2 (1): 21-36; Pluckthun and Pack, 1997 Immunotechnology, 3 (2): 83-105; Ridgway et al., 1996 Protein Eng., 9 (7): 617-21).
[00171] A diabody can be fused in the Fc to generate a "di-body" (see Lu et al., 2004 J. Biol. Chem., 279 (4): 2856-65).
[00172] FRα binding molecules that exhibit functional properties of antibodies, but derive their frame and antigen binding portions from other polypeptides (e.g., polypeptides other than those encoded for antibody genes or generated by recombination of antibody genes in live) can also be used in the methods of the present invention. The antigen binding domains (for example, FRα binding domains) of these binding molecules are generated through a targeted evolution process. See U.S. Patent No. 7,115,396. Molecules that have a general fold similar to that of an antibody variable domain (an "immunoglobulin-like" fold) are suitable scaffolding proteins. Suitable scaffold proteins to derive antigen binding molecules include fibronectin or a fibronectin dimer, tenascin, N-cadherin, E-cadherin, ICAM, titin, GCSF receptor, cytokine receptor, glucosidase inhibitor, chromoprotein antibiotic, adhesion molecule myelin membrane P0, CD8, CD4, CD2, MHC class I, T cell antigen receptor, CD1, C2 and I-set domains of VCAM-1, immunoglobulin I-set domain of myosin-binding protein C, immunoglobulin I-set domain of myosin H binding protein, telokine I-set immunoglobulin domain, NCAM, twitchin, neurogliana, growth hormone receptor, erythropoietin receptor, prolactin receptor, gamma interferon receptor, β -galactosidase / glucuronidase, β-glucuronidase, transglutaminase, T cell antigen receptor, superoxide dismutase, tissue factor domain, cytochrome F, green fluorescent protein, GroEL, and thaumatin.
[00173] "Specific binding" when used in the context of antibodies, or fragments of the antibody, represents binding through domains encoded for immunoglobulin genes or fragments of immunoglobulin genes with one or more epitopes of a protein of interest, but which do not recognizes and binds substantially and other molecules in a sample containing a mixed population of antigenic molecules. Typically, an antibody binds to a cognate antigen with a Kd of less than about 1x10-8 M, as measured by a surface plasmon resonance assay or a cell binding assay.
[00174] As used herein, a "binding agent" folate alpha receptor includes an antibody that binds FRα not bound to a cell as well as non-antibody binding agents. To generate non-antibody binding agents or binding molecules, a library of clones can be created in which sequences in regions of the scaffold protein that form antigen binding surfaces (for example, analogous regions in position and structure for CDRs of a fold of immunoglobulin (the variable domain of the antibody) are randomized. Library clones are tested for specific binding to the antigen of interest (for example, FRα) and for other functions (for example, inhibition of FRα biological activity). Selected clones can be used as the basis for randomization and additional selection to produce derivatives of greater affinity for the antigen.
High-affinity binding molecules are generated, for example, using the tenth module of fibronectin III (10Fn3) as the scaffold, described in U.S. Patent Nos. 6,818,418 and 7,115,396; Roberts and Szostak, 1997 Proc. Natl. Acad. Sci USA 94: 12297; U.S. Patent No. 6,261,804; U.S. Patent No. 6,258,558; and Szostak et al. WO98 / 31700, whose contents in full are incorporated herein by reference.
[00176] Non-antibody binding molecules can be produced as dimers or multimers to increase avidity for the antigen target. For example, the antigen binding domain is expressed as a fusion with a constant region (Fc) of an antibody that forms fr Fc-Fc dimers. See, for example, U.S. Patent No. 7,115,396, the contents of which are fully incorporated herein by reference.
[00177] An "antigen" is a molecule recognized by the immune system; the term originally comes from "antibody generator" and includes a molecule that specifically binds to an antibody. At the molecular level, an antigen is characterized by its ability to bind to the antigen binding site of an antibody. In the present invention, the antigen is FRα, just like FRα that is not bound to an FRα cell or a portion thereof.
[00178] As used herein, the term "epitope" refers to the molecular surface resources of an antigen, for example, FRα, capable of being bound by an antibody. Antigenic molecules, usually being "large" biological polymers, usually have several surface features that can act as points of interaction for specific antibodies. Any such distinct molecular resource constitutes an epitope. Most antigens, therefore, have the potential to be linked by several distinct antibodies, each of which is typically specific to a particular epitope. In one embodiment of the present invention, a binding agent, for example, antibody, binds to an epitope in FRα that is available in the form of the receptor that is not bound to a cell, but not in the form of the bound membrane of the receptor. For example, the antibody can bind to the same epitope on the FRα to which MORAB-003 binds.
[00179] As used here, the phrase "progression from a cancer that expresses FRα in a subject suffering from a cancer that expresses FRα" includes the progression of a cancer like that from a less serious state to a more serious one. This could include an increase in the number or severity of tumors, the degree of metastasis, the speed with which the cancer grows and spreads, and the like. For example, "the progression of ovarian cancer" includes the progression of a cancer like that from a less severe to a more serious state, such as progression from Stage I to Stage II, Stage II to Stage III, etc. . Alternatively, the phrase "progression of a cancer that expresses FRα in a subject suffering from a cancer that expresses FRα" can refer to the regression of a cancer that expresses FRα from a more serious state to a less severe state. For example, in one modality, “the progression of ovarian cancer” refers to the regression from Stage IV to Stage III, from Stage III to Stage II, etc. In other modalities, the “progression of a cancer that expresses FRα in a subject suffering from a cancer that expresses FRα” can refer to the survival rate determined from the onset of symptoms of cancer that expresses FRα, or to the survival rate from the time of cancer diagnosis that expresses FRα.
[00180] As used herein, the term "stratify" refers to the characterization of a cancer that expresses FRα, for example, ovarian or lung cancer, at an appropriate stage, for example, based on the degree of spread of the cancer , also accepts stratifications in technique. For example, stratification includes cancer characterization that expresses FRα in Stage I, Stage II, Stage III or Stage IV. In certain modalities, Stage I refers to cancers that are located in one part of the body. In certain modalities, Stages II and III refer to cancers that are locally advanced, where a distinction between stages is often specific to the particular cancer. Finally, Stage IV refers to cancers that have often been metastasized, or spread to another organ or the entire body.
[00181] As used here, the term "survival" refers to the continuation of the life of a subject who has been treated for cancer. In one embodiment, survival refers to the failure of a tumor to recur. As used herein, the term "reoccurrence" refers to the regrowth of a tumor or cancer cells in a subject to whom the primary treatment for the tumor was administered. The tumor may reoccur at the original site or in another part of the body. In one embodiment, a tumor that recurs is of the same type as the original tumor for which the subject was treated. For example, if a subject had ovarian tumor cancer, was treated and subsequently developed another ovarian tumor cancer, the tumor reoccurred. Furthermore, a cancer can reoccur in a different organ or tissue than the one where it originally occurred. II. Methods and Cases of the Invention
[00182] The present invention is based, at least in part, on the unexpected discovery that alpha folate receptor (FRα), not bound to a cell, is observed at high levels in body fluids, for example, urine or serum, of a subject with a cancer that expresses FRα compared to a control sample. In addition, the present invention is based, at least in part, on the identification of an immunological assay that exhibits the sensitivity needed to assess levels of FRα not bound to a cell in the samples, where previous attempts at evaluation have repeatedly failed. Certainly, the present invention overcomes the challenges observed during previous attempts to develop a FRα-based diagnostic assay for cancer that expresses FRα such as lung or ovarian cancer by providing an immunological assay capable of accurately assessing levels of FRα not linked to a cell in a sample, for example, urine or serum.
[00183] In this way, methods and kits are provided to assess whether a subject has or is at risk of developing a cancer that expresses FRα and, additionally, to assess the progression of a cancer that expresses FRα. In several modalities, the methods involve comparing levels of FRα not bound to a cell in the samples, for example, urine and serum, in relation to the control levels, in assessing the presence, degree or risk of developing ovarian cancer in the subject. A. Diagnostic Methods, Prognostic Methods, Risk Assessment Methods, and Stratification Methods
[00184] Specifically, the present invention relates to diagnostic methods to assess whether a subject is suffering from a cancer that expresses FRα, such as lung or ovarian cancer, prognostic methods to predict the progression of a cancer that expresses FRα such such as lung or ovarian cancer, and risk assessment methods to assess the level of risk that a subject will develop cancer that expresses FRα. In addition, the invention relates to stratification methods for stratifying a cancer that expresses FRα as a subject with lung or ovarian cancer in the cancer therapy groups. The various aspects and modalities of the invention discussed here are considered non-limiting and encompass all possible combinations of the specific modalities mentioned, which can apply to any of the methods and kits discussed here or claimed below.
[00185] The methods of the present invention can be practiced along with any other method used by those skilled in the art to diagnose a cancer that expresses FRα, predict the progression of a cancer that expresses FRα, or to assess the level of risk that a subject develop a cancer that expresses FRα.
[00186] In one aspect, the invention relates to a method of assessing whether a subject is suffering from a cancer that expresses FRα, determining the level of alpha folate receptor (FRα) that is not bound to a cell, in a sample (such as urine or serum) derived from the subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell with the level of FRα in a control sample, in which a difference between the level of FRα in the sample derived from the subject and the level of FRα in the sample control is an indication that the subject is suffering from a cancer that expresses FRα. In a particular embodiment, the level of FRα in the sample derived from the subject is assessed by placing the sample in contact with an antibody that binds FRα not bound to a cell and is selected from the group consisting of (a) an antibody that binds to the same epitope as the MORAb-003 antibody; and (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as , SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3. In one embodiment, the sample is selected from the group consisting of urine and serum.
[00187] In another aspect, the present invention relates to a method of assessing whether a subject is suffering from a cancer that expresses FRα such as lung or ovarian cancer, the method comprising determining the level of alpha folate receptor (FRα) not bound to a cell in a urine sample derived from the subject; and comparing the level of alpha folate receptor (FRα) in the subject-derived urine sample with the level of FRα in a control sample, in which a difference between the level of FRα in the subject-derived urine sample and the level of FRα in the control sample it is an indication that the subject is suffering from a cancer that expresses FRα.
[00188] In another aspect, the invention concerns a method of evaluating whether a subject is suffering from a cancer that expresses FRα, determining the level of alpha folate receptor (FRα) not bound to a cell in a serum sample derived from the subject; and comparing the level of alpha folate receptor (FRα) in the subject-derived serum sample with the level of FRα in a control sample, in which a difference between the level of FRα in the subject-derived serum sample and the level of FRα in the control sample it is an indication that the subject is suffering from a cancer that expresses FRα. In particular modalities, the subject was not treated with an agent, such as a steroid, that increases the levels of FRα in serum. In a specific embodiment, the cancer that expresses FRα is ovarian cancer and the subject has not been treated with an agent, such as a steroid, that increases the levels of FRα in serum.
[00189] In the methods and kits of the present invention, cancers that express FRα include characterized cancers in which the cancer cells express FRα. In particular modalities, FRα is released from cancer cells, for example, from the surface of the cancer cell, and into the subject's biological fluids. Cancers that express FRα include lung cancer (for example, bronchialveolar carcinomas, carcinoid tumors, and non-small cell lung cancers, such as adenocarcinomas); mesothelioma; Ovary cancer; kidney cancer; brain cancer (for example, anaplastic ependymoma and juvenile cerebellar pilocytic astrocytoma); cervical cancer; nasopharyngeal cancer; mesodermally derived tumor; squamous cell carcinoma of the head and neck; endometrial cancer; endometrioid adenocarcinomas of the ovary, serous cystadenocarcinomas, breast cancer; bladder cancer; pancreatic cancer; bone cancer (eg, high-grade osteosarcoma); and pituitary cancer (for example, pituitary adenoma). In a particular embodiment, the cancer that expresses FRα is ovarian cancer.
[00190] In certain embodiments of the methods and kits of the present invention, the cancer that expresses FRα is lung cancer. In more specific modalities, lung cancer is non-small cell lung cancer (NSCLC). In such an embodiment, the NSCLC is selected from the group consisting of adenocarcinoma, squamous cell lung carcinoma, large cell lung carcinoma, pleomorphic NSCLC, carcinoid tumors, salivary gland carcinoma, and unclassified carcinoma. In a preferred embodiment, the NSCLC is adenocarcinoma. In alternative modalities, lung cancer is small cell lung cancer (SCLC). In another embodiment, lung cancer is bronchialveolar carcinoma. In yet another modality, lung cancer is a carcinoid tumor of the lung.
[00191] The present invention also concerns methods for assessing whether a subject is suffering from ovarian cancer by determining the level of alpha folate receptor (FRα) not bound to a cell in a subject-derived urine sample, in which the presence of FRα in the urine sample at a concentration greater than about 3,000 au / mL is an indication that the subject is suffering from ovarian cancer. In particular embodiments, the presence of FRα in the urine sample at a concentration greater than about 4,000 au / mL, about 5,000 au / mL, about 6,000 au / mL, about 7,000 au / mL, about 8,000 au / mL, about 9,000 au / mL, about 10,000 au / mL, about 11,000 au / mL, about 12,000 au / mL, about 13,000 au / mL, about 14,000 au / mL, about 15,000 au / mL , about 16,000 au / mL, about 17,000 au / mL, about 18,000 au / mL, about 19,000 au / mL, about 20,000 au / mL, about 21,000 au / mL, about 22,000 au / mL, about 23,000 au / mL, about 24,000 au / mL, about 25,000 au / mL, about 26,000 au / mL, about 27,000 au / mL, about 28,000 au / mL, about 29,000 au / mL or about 30,000 au / mL is an indication that the subject is suffering from ovarian cancer.
[00192] In yet another aspect, the present invention relates to a method of assessing whether a subject is suffering from ovarian cancer, by determining the level of alpha folate receptor (FRα) in a urine sample derived from the subject, in that the presence of FRα in the urine sample at a concentration greater than about 9,100 pg / mL is an indication that the subject is suffering from ovarian cancer or that a concentration of less than about 9,100 pg / mL is an indication that the subject is not suffering from ovarian cancer. For example, the presence of FRα in the urine sample at a concentration greater than about 9,500 pg / mL, about 10,000 pg / mL, about 11,000 pg / mL, about 12,000 pg / mL, about 13,000 pg / mL , about 14,000 pg / mL, about 15,000 pg / mL, about 16,000 pg / mL, about 17,000 pg / mL, about 18,000 pg / mL, about 19,000 pg / mL, about 20,000 pg / mL, about 21,000 pg / mL, about 22,000 pg / mL, about 23,000 pg / mL, about 24,000 pg / mL, about 25,000 pg / mL, about 26,000 pg / mL, about 27,000 pg / mL , about 28,000 pg / mL, about 29,000 pg / mL, about 30,000 pg / mL, about 40,000 pg / mL, about 50,000 pg / mL, about 60,000 pg / mL, about 70,000 pg / mL, about 80,000 pg / mL, about 90,000 pg / mL, about 100,000 pg / mL or about 150,000 pg / mL is an indication that the subject is suffering from ovarian cancer.
[00193] In certain embodiments of the foregoing aspects of the invention, the levels of FRα not bound to a cell in a sample (e.g., a sample such as a urine or serum sample) derived from a subject are compared with the levels of FRα in a control sample, in which a difference between levels is an indication that the subject is suffering from a cancer that expresses FRα such as lung or ovarian cancer. In a particular modality, the difference constitutes an increase in the level of FRα not bound to a cell in the sample derived from the subject compared with the level of FRα in the control sample, in which this increase is indicative of the presence or growth of cancer that expresses FRα. Alternatively, the difference constitutes a decrease in the level of FRα, in which the decrease is indicative of the absence or regression of cancer that expresses FRα. As used herein, “a difference” between the level of alpha folate receptor not bound to a cell in a sample of a subject (ie, a test sample) and the level of alpha folate receptor in a control sample refers to widely to any clinically relevant change (including an increase or decrease) and / or statistically significant difference in the level of alpha folate receptor in the two samples. In an exemplary modality, the difference is selected based on a cutoff value determined using a characteristic receiver operational analysis (ROC), an example of which is shown in example 6. The ideal cutoff value may vary depending on the test methods and conditions employed. In other embodiments, the difference must be greater than the detection limits of the method to determine the level of FRα not bound to a cell. It is preferred that the difference is at least greater than the standard error of the evaluation method, and preferably a difference of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 100, about 500, about 1,000 times or more than the standard error of the evaluation method. The difference can be assessed by any appropriate comparison, including any appropriate parametric or nonparametric statistics or descriptive comparison. For example, "an increase" in the FRα level can refer to a level that exceeds a cutoff value determined using a ROC analysis. It can also refer to a level in a test sample that is two, and more preferably about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1,000% more than the level of FRα in the control sample. An increase can also refer to a level in a test sample that is preferably at least about 1.5, and more preferably about two, about three, about four, about five or more standard deviations above the mean level of FRα in the control sample. Similarly, "a decrease" in the level of FRα not bound to a cell can refer to a level in a test sample that does not exceed a cut-off value determined using a ROC analysis. It can also refer to a level in a test sample that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% less than the level of FRα in the control sample. A decrease can also refer to a level in a test sample that is preferably at least about 1.5, and more preferably about two, about three, about four, about five or more standard deviations below the mean level of FRα in the control sample.
[00194] Samples used in the methods and kits of the invention include any tissue, cell, biopsy, or body fluid that may contain detectable levels of FRα not bound to a cell. In one embodiment, a sample can be tissue, cell, whole blood, plasma, oral scraping, saliva, cerebrospinal fluid, prayers, or bronchoalveolar lavage. In some embodiments, the sample is a sample of tumor that expresses FRα or a sample of tissues or cells where cancer that expresses FRα can be observed. In preferred embodiments, the sample is a urine or serum sample.
[00195] Body samples can be obtained from a subject by a variety of techniques known in the art including, for example, using a biopsy or scraping or rubbing an area or using a needle to aspirate body fluids. Methods for collecting various body samples are well known in the art.
[00196] Samples suitable for detecting and quantifying the level of FRα protein can be fresh, frozen, or fixed according to methods known to those skilled in the art. Suitable tissue samples are preferably sectioned and placed on a microscope slide for further analysis. Solid samples, that is, tissue samples, can be solubilized and / or homogenized and subsequently analyzed as soluble extracts. Liquid samples can also be subjected to physical or chemical treatments. In some embodiments, urine samples are treated by centrifugation, vortexing, dilution and / or treatment with a solubilizing substance (such as, for example, treatment with guanidine).
[00197] In one embodiment, a newly obtained biopsy sample is frozen using, for example, liquid nitrogen or difluordichloromethane. The frozen sample is assembled for sectioning using, for example, OCT, and sectioned serially in a cryostat. Serial sections are collected on a glass microscope slide. For immunohistochemical staining the slides can be coated, for example, with chromium-alum, gelatin or poly-L-lysine to ensure that the sections stick to the slides. In another mode, samples are fixed and embedded before sectioning. For example, a tissue sample can be fixed, for example, in formalin, serially dehydrated and embedded, for example, in paraffin.
[00198] Once the sample is obtained, any method known in the art to be suitable for detecting and quantifying FRα not bound to a cell can be used (both at the nucleic acid and, preferably, at the protein level), as described in section (B) below. Exemplary methods are well known in the art and include, but are not limited to, western blots, northern blots, southern blots, immunohistochemistry, solution phase assay, ELISA, for example, amplified ELISA, immunoprecipitation, immunofluorescence, flow cytometer, immunocytochemistry , mass spectrometry analyzes, for example, MALDI-TOF and SELDI-TOF, nucleic acid hybridization techniques, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
[00199] In many embodiments, the level of FRα not bound to a cell in the sample (such as, for example, urine or serum) is assessed by placing the sample in contact with an antibody that binds FRα. Antibodies that bind FRα are known in the art and include (i) the murine monoclonal LK26 antibody (its heavy and light chains are presented here as SEQ ID NOs: 22 and 23), as described in European patent application No. 86104170,5 (Rettig) (whose contents in full are incorporated here by reference); (ii) the MORAB-003 antibody, as described in international publication No. WO2004 / 113388 and U.S. patent No. 5,646,253, the contents of which are fully incorporated herein by reference. Monoclonal antibodies MOV18 and MOv19 also bind different epitopes on the FRα molecule (previously known as gp38 / FBP). Miotti, S. et al. Int J Cancer, 38: 297-303 (1987). For example, the MOV18 antibody binds the epitope shown here as SEQ ID NO: 26 (TELLNVXMNAK * XKEKPXPX * KLXXQX) (note that at position 12, a tritophan or histidine residue is possible, and at position 21, an acid residue as - pártico or glutamic acid is possible), as prescribed in Coney et al. Cancer Res, 51: 6125-6132 (1991).
[00200] As used herein, the term "MORAb-003" refers to an antibody that specifically binds FRα and which comprises the mature heavy chain amino acid sequence shown in SEQ ID NO: 7 and the mature light chain sequence of SEQ ID NO: 8. The amino acid sequences of the corresponding preprotein for MORAb-003 are shown in SEQ ID NOs: 9 (heavy chain) and 10 (light chain). The MORAb-003 antibody comprises the following CDRs: SEQ ID NO: 1 as CDRH1, SEQ ID NO: 2 as CDRH2, SEQ ID NO: 3 as CDRH3, SEQ ID NO: 4 as CDRL1, SEQ ID NO: 5 as CDRL2, and SEQ ID NO: 6 as CDRL3. Cells producing the MORAb-003 antibody were deposited with the American Type Culture Collection (10801 University Blvd., Manassas, Virginia 20110-2209) on April 24, 2006 and were assigned accession number PTA-7552.
[00201] Other antibodies that bind FRα and for use in the methods of the present invention include 9F3.H9.H3.H3.B5.G2 (also referred to as 9F3), 19D4.B7 (also referred to as 19D4), 24F12.B1 (also referred to as 24F12), and 26B3.F2 (also referred to as 26B3). The amino acid sequences of these antibodies, their CDRs, and their heavy and light chain variable domains, as well as polynucleotide sequences that can encode them, are provided in table 33. In some embodiments, these antibodies are murine IgG, or their derivatives. In other embodiments, the antibodies are human, humanized, or chimeric. 9F3
[00202] In some embodiments, the antibody that binds FRα is an antibody or antigen binding fragment that includes an amino acid sequence of light chain CDR1 substantially the same, or identical to SEQ ID NO: 27. In some embodiments, the antibody that binds FRα includes an amino acid sequence of light chain CDR2 substantially the same, or identical to SEQ ID NO: 28. In some embodiments, the FRα-binding antibody includes a light chain CDR3 amino acid sequence substantially the same, or identical to SEQ ID NO 29. In some embodiments, the FRα-binding antibody includes a heavy chain CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO 31. In some embodiments, the antibody that binds FRα includes a heavy chain CDR2 amino acid sequence substantially the same, or identical to SEQ ID NO: 32. In some embodiments, the antibody that binds FRα includes a heavy chain CDR3 amino acid sequence substantially the same, or identical to SEQ ID NO: 33. The antibody that binds FRα can include a light chain with a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 27; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 28; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 29. The antibody that binds FRα can include a heavy chain with an amino acid sequence of CDR1 substantially the same, or identical to SEQ ID NO: 31; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 32; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 33. The antibody that binds FRα can include a light chain with a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 27; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 28; and a CDR3 amino acid sequence substantially the same, or identical to SEQ ID NO: 29, and also have a heavy chain with a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 31; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 32; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 33.
The antibody that binds FRα can include a light chain variable domain that includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 30. The antibody that binds FRα can include a heavy chain variable domain that includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 34. The antibody that binds FRα can include variable domains of a light chain and a heavy chain, where the variable domain of the light chain includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 30, and the variable domain of the heavy chain includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 34. In some embodiments, the antibody that binds FRα is antibody 9F3.H9.H3.H3.B5.G2 (9F3) or an antigen-binding fragment thereof, capable of binding both a native and unreduced form of FRα. In some embodiments, the antibody has a murine IgG constant region.
[00204] In some embodiments, the antibody that binds FRα is an antibody that is produced by antibody-producing cells deposited with the American Type Culture Collection (10801 University Blvd., Manassas, Virginia 20110-2209) on May 19, 2011 and were assigned with accession number PTA-11887. In some embodiments, the FRα-binding antibody comprises one or more of the light and heavy chain CDRs of the antibodies produced by the deposited antibody-producing cells. In some embodiments, antibody that binds FRα comprises the light and heavy chain variable regions of the antibodies produced by the deposited antibody-producing cells. 19D4
[00205] In some embodiments, the antibody that binds FRα is an antibody or antigen binding fragment that includes a light chain CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 35. In some embodiments, the antibody that binds FRα includes an amino acid sequence of light chain CDR2 substantially the same, or identical to SEQ ID NO: 36. In some embodiments, the antibody that binds FRα includes an amino acid sequence of light chain CDR3 substantially the same, or identical to SEQ ID NO: 37. In some embodiments, the FRα-binding antibody includes a heavy chain CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 39. In some embodiments, the antibody that binds FRα includes a heavy chain CDR2 amino acid sequence substantially the same, or identical to SEQ ID NO: 40. In some embodiments, the antibody that binds FRα includes a heavy chain CDR3 amino acid sequence substantially the same, or identical to SEQ ID NO: 41. The antibody that binds FRα can include a light chain having a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 35; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 36; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 37. The antibody that binds FRα can include a heavy chain with an amino acid sequence of CDR1 substantially the same, or identical to SEQ ID NO: 39; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 40; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 41. The antibody that binds FRα can include a light chain having a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 35; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 36; and a CDR3 amino acid sequence substantially the same, or identical to SEQ ID NO: 37, and also have a heavy chain with a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 39; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 40; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 41.
The antibody that binds FRα can include a light chain variable domain that includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 38. The antibody that binds FRα can include a heavy chain variable domain that includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 42. The antibody that binds FRα can include variable domains of a light chain and a heavy chain, where the variable domain of the light chain includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 38, and the variable domain of the heavy chain includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 42. In some embodiments, the antibody that binds FRα is the 19D4.B7 (19D4) antibody or an antigen binding fragment thereof, capable of binding both a native and unreduced form of FRα. In some embodiments, the antibody has a murine IgG constant region.
[00207] In some embodiments, the antibody that binds FRα is an antibody that is produced by antibody-producing cells deposited with the American Type Culture Collection (10801 University Blvd., Manassas, Virginia 20110-2209) on May 19, 2011 and were assigned with accession number PTA-11884. In some embodiments, the FRα-binding antibody comprises one or more of the light and heavy chain CDRs of the antibodies produced by the deposited antibody-producing cells. In some embodiments, antibody that binds FRα comprises the light and heavy chain variable regions of the antibodies produced by the deposited antibody-producing cells. 24F12
[00208] In some embodiments, the antibody that binds FRα is an antibody or antigen binding fragment that includes a light chain CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 43. In some embodiments, the antibody that binds FRα includes an amino acid sequence of light chain CDR2 substantially the same, or identical to SEQ ID NO: 44. In some embodiments, the antibody that binds FRα includes an amino acid sequence of light chain CDR3 substantially the same, or identical to SEQ ID NO: 45. In some embodiments, the antibody that binds FRα includes a heavy chain CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 47. In some embodiments, the FRα-binding antibody includes a heavy chain CDR2 amino acid sequence substantially the same, or identical to SEQ ID NO: 48. In some embodiments, the antibody that binds FRα includes a heavy chain CDR3 amino acid sequence substantially the same, or identical to SEQ ID NO: 49. The antibody that binds FRα can include a light chain having a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 43; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 44; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 45. The antibody that binds FRα can include a heavy chain with an amino acid sequence of CDR1 substantially the same, or identical to SEQ ID NO: 47; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 48; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 49. The antibody that binds FRα can include a light chain having a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 43; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 44; and a CDR3 amino acid sequence substantially the same, or identical to SEQ ID NO: 45, and also have a heavy chain with a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 47; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 48; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 49.
The antibody that binds FRα can include a light chain variable domain that includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 46. The antibody that binds FRα can include a heavy chain variable domain that includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 50. The antibody that binds FRα can include variable domains of a light chain and a heavy chain, where the variable domain of the light chain includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 46, and the variable domain of the heavy chain includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 50. In some embodiments, the antibody that binds FRα is antibody 24F12.B1 (24F12) or an antigen binding fragment thereof, capable of binding both a native and unreduced form of FRα. In some embodiments, the antibody has a murine IgG1 constant region.
[00210] In some embodiments, the antibody that binds FRα is an antibody that is produced by antibody-producing cells deposited with the American Type Culture Collection (10801 University Blvd., Manassas, Virginia 20110-2209) on May 19, 2011 and were assigned with accession number PTA-11886. In some embodiments, the FRα-binding antibody comprises one or more of the light and heavy chain CDRs of the antibodies produced by the deposited antibody-producing cells. In some embodiments, antibody that binds FRα comprises the light and heavy chain variable regions of the antibodies produced by the deposited antibody-producing cells. 26B3
[00211] In some embodiments, the antibody that binds FRα is an antibody or antigen binding fragment that includes a light chain CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 51. In some embodiments, the antibody that binds FRα includes an amino acid sequence of light chain CDR2 substantially the same, or identical to SEQ ID NO: 52. In some embodiments, the antibody that binds FRα includes an amino acid sequence of light chain CDR3 substantially the same, or identical to SEQ ID NO: 53. In some embodiments, the FRα-binding antibody includes a heavy chain CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 55. In some embodiments, the antibody that binds FRα includes a heavy chain CDR2 amino acid sequence substantially the same, or identical to SEQ ID NO: 56. In some embodiments, the antibody that binds FRα includes a heavy chain CDR3 amino acid sequence substantially the same, or identical to SEQ ID NO: 57. The antibody that binds FRα can include a light chain having a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 51; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 52; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 53. The antibody that binds FRα can include a heavy chain with an amino acid sequence of CDR1 substantially the same, or identical to SEQ ID NO: 55; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 56; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 57. The antibody that binds FRα can include a light chain having a CDR1 amino acid sequence substantially the same, or identical to SEQ ID NO: 51; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 52; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 53, and also have a heavy chain with an amino acid sequence of CDR1 substantially the same, or identical to SEQ ID NO: 55; an amino acid sequence of CDR2 substantially the same, or identical to SEQ ID NO: 56; and an amino acid sequence of CDR3 substantially the same, or identical to SEQ ID NO: 57. The antibody that binds FRα can include a light chain variable domain that includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 54. The antibody that binds FRα can include a heavy chain variable domain that includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 58. The antibody that binds FRα can include variable domains of a light chain and a heavy chain, where the variable domain of the light chain includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 54, and the variable domain of the heavy chain includes an amino acid sequence substantially the same, or identical to SEQ ID NO: 58. In some embodiments, the antibody that binds FRα is antibody 26B3.F2 (26B3) or an antigen binding fragment thereof, capable of binding both a native and unreduced form of FRα. In some embodiments, the antibody has a murine IgG1 constant region.
[00212] In some embodiments, the antibody that binds FRα is an antibody that is produced by antibody-producing cells deposited with the American Type Culture Collection (10801 University Blvd., Manassas, Virginia 20110-2209) on May 19, 2011 and were assigned with accession number PTA-11885. In some embodiments, the FRα-binding antibody comprises one or more of the light and heavy chain CDRs of the antibodies produced by the deposited antibody-producing cells. In some embodiments, antibody that binds FRα comprises the light and heavy chain variable regions of the antibodies produced by the deposited antibody-producing cells.
[00213] CDR antigen binding arrangements can be modified by engineering using antibody type proteins such as CDR scaffolding. Engineered modified antigen binding proteins are included in the scope of antibodies that bind FRα.
[00214] Other reagent antibodies that bind FRα are known in the art, and currently, multiple such reagent antibodies are commercially available (based on anti-FRαs antibody research at http://www.biocompare.com), as shown in table a follow.








[00215] In a preferred embodiment, the FRα-binding antibody comprises at least one of the following CDRs, as derived from the heavy and light chains of murine LK26: SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 ( MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRL1, SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPY3Y. See US Patent No. 5,646,253, the contents of which, since they relate to the anti-FRαs antibody that can be used in the present invention, are incorporated herein by reference. Additional mutations can be made in the frame regions as required by US Patent No. 5,646,253, the contents of which are hereby incorporated by reference.
[00216] In another preferred embodiment, the antibody includes a light chain variable region selected from the group consisting of LK26HuVK (SEQ ID NO: 13) LK26HuVKY (SEQ ID NO: 14), LK26HuVKPW (SEQ ID NO: 15), and LK26HuVKPW, Y (SEQ ID NO: 16); and a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21). See US Patent No. 5,646,253 and US Patent No. 6,124,106. In another embodiment, the antibody comprises the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16). In another embodiment, the antibody comprises the heavy chain variable region LK26HuVH SLF (SEQ ID NO: 19) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16). In an additional embodiment, the antibody comprises the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[00217] In some embodiments, samples may need to be modified in order to make FRα accessible for antibody binding. In a particular aspect of immunocytochemistry or immunohistochemistry methods, slides can be transferred to a pretreatment buffer and optionally heated to increase accessibility of the antigen. Heating the sample in the pre-treatment buffer quickly disrupts the lipid bilayer of the cells and makes the antigens (may be the case in fresh species, but not typically what occurs in fixed species) (ie, FRα protein) more antibody binding. The terms "pretreatment buffer" are used interchangeably here to refer to a buffer that is used to prepare cytology or histology samples for immunoblotting, particularly increasing accessibility of the FRα protein for antibody binding. The pretreatment buffer may comprise a specific pH salt solution, a polymer, a detergent, or a nonionic or anionic surfactant such as, for example, an ethoxylated anionic or nonionic surfactant, an alkanoate or an alkoxylate or even mixtures of these surfactants or even the use of a bile salt. The pre-treatment buffer can, for example, be a 0.1% to 1% solution of deoxycholic acid, sodium salt, or a solution of sodium laureth-13-carboxylate (for example, Sandopan LS) or e ethoxylated anion complex. In some embodiments, the pretreatment buffer can also be used as a blade storage buffer. In a particular embodiment, the sample, for example, the urine sample, is centrifuged, swirled, diluted and / or subjected to treatment with guanidine.
Any method for making the FRα protein more accessible to antibody binding can be used in the practice of the invention, including antigen recovery methods known in the art. See, for example, Bibbo, et al. (2002) Minutes. Cytol. 46: 25-29; Saqi, et al. (2003) Diagn. Cytopathol. 27: 365-370; Bibbo, et al. (2003) Anal. Quant. Cytol. Histol. 25: 8-11, whose contents in full are incorporated herein by reference.
[00219] After pretreatment to increase accessibility of the FRα protein, samples can be blocked using an appropriate blocking agent, for example, a peroxidase blocking reagent such as hydrogen peroxide. In some embodiments, samples can be blocked using a protein blocking reagent to prevent non-specific binding of the antibody. The protein blocking reagent can comprise, for example, purified casein. An antibody, particularly a monoclonal or polyclonal antibody, that specifically binds to FRα is then incubated with the sample.
[00220] Techniques for detecting antibody binding are well known in the art. Antibody binding to FRα can be detected through the use of chemical reagents that generate a detectable signal that corresponds to the level of antibody binding and, thus, to the level of expression of the FRα protein. In one of the immunohistochemistry or immunocytochemistry methods of the invention, binding of the antibody is detected through the use of a secondary antibody that is conjugated to a labeled polymer. Examples of labeled polymers include, but are not limited to, polymer-enzyme conjugates. The enzymes in these complexes are typically used to catalyze the deposition of a chromogen at the antibody-antigen binding site, thereby resulting in cell staining that corresponds to the level of expression of the biomarkers of interest. Enzymes include, but are not limited to, horseradish peroxidase (HRP) and alkaline phosphatase (AP).
[00221] In an immunohistochemistry or immunocytochemistry method of the invention, binding of the antibody to the FRα protein is detected through the use of an HRP-labeled polymer that is conjugated to a secondary antibody. Antibody binding can also be detected through the use of a species-specific probe reagent, which binds to monoclonal or polyclonal antibodies, and an HRP-conjugated polymer, which binds to the species-specific probe reagent. Slides are stained for antibody binding using any chromogen, for example, the 3,3-diaminobenzidine chromogen (DAB), and then stained with hematoxylin and, optionally, a staining agent such as ammonium hydroxide or TBS / Tween-20. Other suitable chromogens include, for example, 3-amino-9-ethylcarbazole (AEC). In some aspects of the invention, slides are reviewed microscopically by a cytotechnologist and / or a pathologist to assess cell staining, for example, fluorescent staining (i.e., expression of FRα). Alternatively, samples can be reviewed by means of automated microscopy or by people with the assistance of computer software that facilitates the identification of positive staining cells.
[00222] In a preferred embodiment of the invention, the antibody is labeled. For example, detection of antibody binding can be facilitated by coupling the anti-FRα antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbeliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 35S, 14C, or 3H. In a particular embodiment, the antibody is labeled with a radio marker, chromophore marker, fluorophore marker, or enzyme marker.
[00223] In an embodiment of the invention frozen samples are prepared as described above and subsequently stained with antibodies against FRα diluted to an appropriate concentration using, for example, Tris-buffered saline (TBS). Primary antibodies can be detected by incubating the slides in biotinylated anti-immunoglobulin. This signal can optionally be amplified and visualized using diaminobenzidine precipitation from the antigen. In addition, slides can be optionally stained, for example, with hematoxylin, to visualize the cells.
[00224] In another embodiment, fixed and embedded samples are stained with antibodies against FRα and stained as previously described for frozen sections. In addition, samples can optionally be treated with agents to amplify the signal in order to visualize antibody staining. For example, a deposition catalyzed by biotinyl tyramide peroxidase can be used, which in turn is reacted with streptavidin conjugated to peroxidase (Catalyzed Signal Amplification System (CSA), DAKO, Carpinteria, CA).
[00225] Those skilled in the art will realize that the concentration of a particular antibody used to practice the methods of the invention will vary depending on such factors as time to bind, level of specificity of the antibody to FRα, and method of sample preparation. In addition, when multiple antibodies are used, the required concentration can be affected by the order in which the antibodies are applied to the sample, for example, simultaneously as a cocktail or sequentially as individual antibody reagents. In addition, the detection chemical used to visualize the antibody binding in FRα must be optimized to produce the desired signal for noise ratio.
[00226] In an embodiment of the invention, proteomic methods, for example, mass spectrometry, are used to detect and quantify the FRα protein. For example, laser desorption / ionization flight time mass spectrometry associated with the matrix (MALDI-TOF MS) or surface-enhanced laser desorption / ionization flight time mass spectrometry (SELDI-TOF MS ) which involves applying a sample, such as serum, to a protein binding chip (Wright, GL, Jr., et al. (2002) Expert Rev Mol Diagn 2: 549; Li, J., et al. (2002) Clin Chem 48: 1296; Laronga, C., et al. (2003) Dis Markers 19: 229; Petricoin, EF, et al. (2002) 359: 572; Adam, BL, et al. (2002) Cancer Res 62: 3609; Tolson, J., et al. (2004) Lab Invest 84: 845; Xiao, Z., et al. (2001) Cancer Res 61: 6029) can be used to detect and quantify the protein of FRα. Mass spectrometric methods are described, for example, in U.S. Patent Nos. 5,622,824, 5,605,798 and 5,547,835, whose contents in full are incorporated here by reference.
[00227] The present invention is further considered, at least in part, in the identification of FRα as a biomarker of prognosis, that is, as a biomarker of progression and / or severity, of a cancer that expresses FRα such as ovarian cancer or non-small cell lung cancer. Thus, the present invention concerns methods of assessing the progression of a cancer that expresses FRα in a subject suffering from ovarian cancer by comparing the level of FRα in a sample derived from a subject with the level of FRα in a control sample, where a difference in the level of FRα in the sample (such as a urine or serum sample) derived from the subject compared to the control sample is an indication that the cancer will progress rapidly. Similarly, methods of assessing the level of risk that a subject will develop cancer that expresses FRα involves comparing the level of FRα in a sample derived from a subject with the level of FRα in a control sample, in which a difference in the level of FRα in the sample (such as urine or serum sample) derived from the subject compared to the control sample is an indication that the subject has a higher level of risk of developing a cancer that expresses FRα compared to normal risk in a healthy individual.
[00228] In one modality, the difference is an increase. In another mode, the difference is a decrease. In some types of cancers (for example, squamous cell carcinoma of the head and neck, ovarian cancer), a higher level of expression of FRα is associated with a worsening prognosis, while in other types of cancers (for example, cancers of non-small cell lung), a higher level of FRα expression is associated with an improvement prognosis. Thus, in a specific modality, the cancer that expresses FRα is ovarian cancer or squamous cell carcinoma of the head and neck and the difference is an increase. In another specific modality, cancer that expresses FRα is a non-small cell lung cancer, and the difference is a decrease.
[00229] In certain respects, the invention relates to methods of assessing the progression of a cancer that expresses FRα in a subject suffering from a cancer that expresses FRα by comparing the level of FRα in a sample derived from a subject with the level of FRα in a control sample, where an increase in the level of FRα in the sample (such as a urine or serum sample) derived from the subject compared to the control sample is an indication that the cancer will progress rapidly, or a decrease in the level of FRα in the sample derived from the subject compared to the level of FRα in the control sample is an indication that the cancer will progress slowly or regress. Similarly, methods of assessing the level of risk that a subject will develop cancer that expresses FRα involves comparing the level of FRα in a sample derived from a subject with the level of FRα in a control sample, in which an increase in the level of FRα in the sample (such as urine or serum sample) derived from the subject compared to the control sample is an indication that the subject has a higher level of risk of developing a cancer that expresses FRα compared to normal risk in a healthy individual, or a decrease in the level of FRα in the sample derived from the subject compared to the level of FRα in the control sample is an indication that the subject has a lower level of risk of developing a cancer that expresses FRα compared to a normal risk in a healthy individual.
Any clinically relevant or statistically significant increase or decrease, using any analytical method known in the art, can be used in the prognosis, risk assessment and other methods of the invention. In one embodiment, an increase in the level of FRα refers to a level that exceeds a cutoff value determined using a ROC analysis as exemplified in example 6. In another embodiment, a decrease in the level of FRα refers to a level in a test sample that does not exceed a cutoff value determined using a ROC analysis.
[00231] In other modalities, the increase or decrease must be greater than the detection limits of the method to determine the level of FRα. In additional embodiments, the increase or decrease should be at least greater than the standard error of the assessment method, and preferably a difference of at least about 2, about 3, about 4, about 5, about 6, about from 7, about 8, about 9, about 10, about 15, about 20, about 25, about 100, about 500, about 1,000 times or more than the standard error of the evaluation method. In some modalities, the increase or decrease is evaluated using parametric or non-parametric descriptive statistics, analysis of comparisons, regression, and the like.
[00232] In other modalities, the increase or decrease is a level in the sample derived from the subject that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30% , about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% , about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1,000% more or less than the level of FRα in the control sample. In alternative modalities, the increase or decrease is a level in the sample derived from the subject that is at least about 1.5, and more preferably about two, about three, about four, about five or more standard deviations above or below the mean level of FRα in the control sample. As used herein, the phrase "progression from cancer that expresses FRα in a subject suffering from cancer that expresses FRα" can refer to the progression of a cancer that expresses FRα from a less severe to a more severe cancer state. This could include an increase in the number or severity of tumors, the degree of metastasis, the rate at which the cancer grows and spreads, and the like. In certain modalities, progression is a progression from a less severe stage to a more serious stage, where the stage is evaluated according to a staging scheme known in the art. In one modality, in which the cancer that expresses FRα is ovarian cancer, the progression refers to a progression from Stage I to Stage II, from Stage II to Stage III, etc. In another modality, in which the cancer that expresses FRα is non-small cell lung cancer (NSCLC), the progression refers to a progression from Stage 0 to Stage IA, Stage IA to Stage IB, Stage IB to Stage IIA, Stage IIA for Stage IIB, Stage IIB for Stage IIC, etc. In another modality, in which the cancer that expresses FRα is non-small cell lung cancer (NSCLC), the progression refers to a progression from a less severe stage to a more severe stage determined in the TNM classification system. See Spira; Greene; Sobin.
[00233] Alternatively, the phrase "progression of a cancer that expresses FRα in a subject suffering from a cancer that expresses FRα" can refer to a regression of a cancer that expresses FRα from a more serious state to a less serious state, such as a decrease in the number or severity of tumors, the degree of metastasis, the speed with which the cancer grows and spreads, and the like. In certain modalities, progression is a progression from a more severe stage to a less severe stage, where the stage is evaluated according to a staging scheme known in the art. In one modality, in which the cancer that expresses FRα is ovarian cancer, the progression refers to a regression from Stage IV to Stage III, from Stage III to Stage II, etc. In another modality, in which the cancer that expresses FRα is non-small cell lung cancer (NSCLC), the progression refers to a progression from Stage IV to Stage IIIB, Stage IIIB to Stage IIIA, Stage IIIA to Stage IIB, etc. In another modality, in which the cancer that expresses FRα is non-small cell lung cancer (NSCLC), the progression refers to a progression from a more severe to a less severe stage determined in the TNM classification system. See Spira; Greene; Sobin.
[00234] In additional modalities, the level of FRα can be used to calculate the probability that a subject is suffering from a cancer that expresses FRα, the progression of a cancer that expresses FRα in a subject, the level of risk of developing a cancer that expresses FRα, the risk of recurrence of cancer in a subject being treated for a cancer that expresses FRα, the survival of a subject being treated for a cancer that expresses FRα, the effectiveness of a treatment regimen to treat a cancer that expresses FRα, and the like, using the methods of the invention, which may include regression analysis methods known to those skilled in the art. For example, suitable regression models include, but are not limited to, CART (for example, Hill, T, and Lewicki, P. (2006) “STATISTICS Methods and Applications” StatSoft, Tulsa, OK), Cox (for example, www. evidence- based-medicine.co.uk), exponential, normal and normal log (eg www.obgyn.cam.ac.uk/mrg/statsbook/stsurvan.html), logistical (eg www.en.wikipedia .org / wiki / Logistic regression), parametric, nonparametric, semi-parametric (e.g. www.socserv.mcmaster.ca/jfox/Books/Companion), linear (e.g. www.en.wikipedia.org/wiki/Linear_regression ), or additives (for example, www.en.wikipedia.org/wiki/Generalized_additive_model).
[00235] In one modality, a regression analysis includes the level of FRα. In additional modalities, a regression analysis may include additional clinical and / or molecular covariates. Such clinical covariates include, but are not limited to, the subject's age, tumor stage, tumor grade, tumor size, treatment regimen, for example, chemotherapy and / or radiation therapy, clinical outcomes (for example, relapse, specific survival disease, therapy failure), and / or clinical results depending on the time after diagnosis, time after the start of therapy, and / or time after the end of treatment. Molecular covariates may include, but are not limited to, additional molecular marker values. For example, in modalities where the cancer expressing FRα is ovarian cancer, such markers may include, for example, serum CA125 levels, serum DF3 levels, and / or plasma LPA levels.
[00236] In other respects, the invention relates to methods for monitoring the efficiency of a therapy or treatment regimen. For example, the present invention relates to methods for monitoring the effectiveness of treatment with MO-RAb-003 for ovarian cancer or lung cancer in a subject suffering from ovarian cancer or lung cancer. Specifically, the methods involve determining the level of alpha folate receptor (FRα) that is not bound to a cell, in a sample derived from said subject, in which said subject was previously administered with MORAb-003; and compare the level of alpha folate receptor (FRα) that is not bound to a cell with the level of FRα in a control sample, in which an increase or change in the level of FRα in the sample derived from said subject compared with the level of FRα in the control sample is an indication that treatment with MORAb-003 is not efficient; and that a decrease in the level of FRα in the sample derived from said subject compared to the level of FRα in the control sample is an indication that treatment with MORAb-003 is efficient.
[00237] For example, the control sample can be derived from a subject not subjected to the treatment regimen and a test sample can be derived from a subject subjected to at least a portion of the treatment regimen. Alternatively, the test sample and the control sample can be derived from the same subject. For example, the test sample can be a sample derived from a subject after administration of a therapeutic product, such as MORAb-003. The control sample can be a sample derived from a subject prior to the administration of the therapeutic product or at an early stage of regimen of the therapeutic product. Thus, a decrease in the level of FRα expression in the test sample, relative to the control sample, is an indication that therapy has slowed the progression of cancer that expresses FRα, for example, ovarian cancer. For cancers that express FRα in which a higher level of FRα is associated with a worsening prognosis, such as, for example, ovarian cancer or squamous cell carcinoma of the head and neck, a decrease in the level of FRα expression in the test sample , relative to the control sample, is an indication that therapy is effective in decreasing the progression of cancer that expresses FRα, or causing a regression of cancer, in the subject suffering from cancer that expresses FRα. In a preferred embodiment, the cancer that expresses FRα is ovarian cancer.
[00238] In various embodiments of this aspect of the invention, the sample can be urine, serum, plasma or ascites. In particular modalities, the sample is urine or serum. In addition, FRα can be determined by contacting the sample with an antibody that binds FRα, optionally using antibodies as described here and assay methods as described here.
[00239] In several embodiments, the MORAb-003 antibody treatment is (a) an antibody comprising the heavy chain amino acid sequence shown in SEQ ID NO: 7 and the light chain amino acid sequence shown in SEQ ID NO: 8; (b) an antibody that binds to the same epitope as the MORAb-003 antibody; or (c) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNN1LH) as , SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3 ..
[00240] In a particular modality, the cancer that expresses FRα is ovarian cancer. In other modalities, the cancer that expresses FRα is lung cancer. In more specific modalities, lung cancer is non-small cell lung cancer (NSCLC). In such an embodiment, the NSCLC is selected from the group consisting of adenocarcinoma, squamous cell lung carcinoma, large cell lung carcinoma, pleomorphic NSCLC, carcinoid tumors, salivary gland carcinoma, and unclassified carcinoma. In a preferred embodiment, the NSCLC is adenocarcinoma. In alternative modalities, lung cancer is small cell lung cancer (SCLC). In another embodiment, lung cancer is bronchialveolar carcinoma. In yet another modality, lung cancer is a carcinoid tumor of the lung.
[00241] In another aspect, the invention relates to methods of stratifying a subject with a cancer that expresses FRα in cancer therapy groups based on the level of FRα determined in a sample. In a preferred embodiment, the method involves stratifying a subject with a cancer that expresses FRα in at least one of the four cancer therapy groups. In other modalities, the method involves stratifying a subject with a cancer that expresses FRα into at least one of about two, about three, about four, about five, about six, about seven, about eight, about of nine, or about ten cancer therapy groups.
[00242] According to the present invention, FRα levels can be associated with the severity, that is, the stage, of cancer that expresses FRα. For example, ovarian cancer is stratified at different stages based on the severity of the cancer, presented here. Accordingly, the present invention relates to methods for stratifying Stage I ovarian cancer, for example, Stage IA, Stage 1B or Stage IC ovarian cancer; Stage II, for example, Stage IIA, Stage IIB or Stage IIC; Stage III, for example, Stage IIIA, Stage IIIB or Stage IIIC; or Stage IV.
[00243] SCLS or NSCLC can be stratified at different stages based on the severity of the cancer, presented here. Thus, the present invention relates to methods for stratifying lung cancer, for example, SCLS or NSCLC, in the hidden (hidden) stage; stage 0; Stage I, for example, stages IA and IB; Stage II, for example, lung cancer in Stage IIA and IIB; Stage III, for example, Stage IIIA and IIIB; or Stage IV.
[00244] In yet another aspect, the present invention is considered, at least in part, in the observation that FRα can serve as a predictor of biomarkers for the treatment of cancers that express FRα. Specifically, the methods of the present invention provide for assessing whether a subject will respond to treatment, for example, with MORAb-003, and, if and when treatment begins, for example, with MORAb-003, by assessing the levels of FRα in a subject.
[00245] In one aspect, the present invention relates to a method for predicting whether a subject suffering from a cancer that expresses FRα, for example, ovarian or lung cancer, will respond to treatment with MORAb-003, determining the level of alpha folate receptor (FRα) that is not bound to a cell in a sample derived from said subject; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in the sample derived from said subject with the level of FRα in a control sample, in which a difference between the level of FRα in the sample derived from said subject and the level of FRα in the control sample is an indication that the subject will respond to treatment with MORAb-003.
[00246] In certain modalities, the degree of difference between the levels of FRα not bound to a cancer cell in the test sample compared to the control sample is indicative that the subject will respond to treatment with MORAb-003. For example, a difference of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 100, about 500, about 1,000 times or more than the standard error of the assessment method is indicative that the subject will respond to treatment with MORAb-003. Alternatively, or in combination, a difference of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50% , about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% , about 600%, about 700%, about 800%, about 900% or about 1,000% is indicative that the subject will respond to treatment with MORAb-003. Alternatively, or in combination, a difference of at least about 1.5, and more preferably about two, about three, about four, about five or more standard deviations is indicative that the subject will respond to treatment with MORAb -003.
[00247] In several embodiments, treatment with MORAb-003 antibody is (a) an antibody that binds to the same epitope as the MORAb-003 antibody; or (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as , SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3.
[00248] In several modalities, the sample is urine, plasma, serum or ascites. In particular modalities, the sample is urine or serum. In additional modalities, cancer that expresses FRα is selected from the group consisting of lung cancer, mesothelioma, ovarian cancer, kidney cancer, brain cancer, cervical cancer, nasopharyngeal cancer, squamous cell carcinoma of the head and neck, endometrial cancer , breast cancer, bladder cancer, pancreatic cancer, bone cancer, pituitary cancer, colorectal cancer and medullary thyroid cancer. In a particular embodiment, the cancer that expresses FRα is ovarian cancer. In another embodiment, cancer that expresses FRα is non-small cell lung cancer, such as adenocarcinoma. B. Assays Based on the Anti-FRα Antibody to Detect Cancers Expressing FRα
[00249] There are a variety of assay formats known to those skilled in the art to use an antibody to detect a polypeptide in a sample, including, but not limited to, linked to the enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunofluorimetry, immunoprecipitation, solution phase assay, equilibrium dialysis, immunodiffusion and other techniques. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Weir, D.M., Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston. For example, the assay can be performed in a Western blot format, in which a preparation of the protein from the biological sample is subjected to gel electrophoresis, transferred to a suitable membrane and reacted naturally with the antibody. The presence of the antibody on the membrane can then be detected using a suitable detection reagent, well known in the art and described below.
[00250] In another embodiment, the assay involves the use of an antibody immobilized on a solid support to bind to the target FRα polypeptide and remove it from the rest of the sample. The bound FRα polypeptide can then be detected using a second antibody reactive with a distinct FRα polypeptide antigenic determinant, for example, a reagent containing a detectable reporting fraction. As a non-limiting example, according to this modality the immobilized antibody and the second antibody that recognizes distinct antigenic determinants can be any of the two monoclonal antibodies described here selected from MORAb-003, MOV18, 548908, 6D398 or variants of these as described here. Alternatively, a competitive assay can be used, in which FRα is labeled with a detectable reporting fraction and naturally bound to the immobilized anti-FRα antibody after incubation of the immobilized antibody with the sample. The extent to which the components of the sample inhibit the binding of the labeled polypeptide to the antibody is indicative of the reactivity of the sample with the immobilized antibody, and as a result, indicative of the level of FRα in the sample.
[00251] The solid support can be any material known to those skilled in the art to which the antibody can be attached, such as a test well in a microtiter plate, a nitrocellulose filter or another suitable membrane. Alternatively, the support can be a sphere or disc, such as glass, fiberglass, latex or a plastic such as polystyrene or poly (vinylidene chloride). The antibody can be immobilized on the solid support using a variety of techniques known to those skilled in the art, which are generally described in the patent and scientific literature.
[00252] In certain preferred embodiments, the assay for detecting FRα in a sample is a two antibody sandwich assay. This assay can be performed first by contacting a specific FRα antibody (for example, MORAb-003, MOV18, 548908, 6D398 or variants of these as described here) that has been immobilized on a solid support, commonly the well of a microtiter plate. , with the biological sample, in such a way that a naturally occurring molecule soluble in the sample and with an antigenic determinant that is reactive with the antibody is naturally bound to the immobilized antibody (for example, an incubation time of 30 minutes at room temperature is generally sufficient) to form an antigen-antibody complex or an immune complex. The unbound constituents of the sample are then removed from the immobilized immune complexes. Then, a second antibody specific for FRα is added, where the antigen combining site of the second antibody does not competitively inhibit the binding of the antigen combining site of the first antibody immobilized on FRα (for example, MORAb-003, MOV18, 548908 , 6D398 or variants thereof as described here, which is not the same as that of the monoclonal antibody immobilized on the solid support). The second antibody can be detectably labeled as provided here, in such a way that it can be directly detected. Alternatively, the second antibody can be indirectly detected using a detectably labeled secondary anti-antibody (or "second stage"), or using a specific detection reagent as provided here. The method of the invention in question is not limited to any particular detection procedure, as one familiar with immunoassays will realize that there are numerous reagents and configurations for immunologically detecting a particular antigen (e.g., FRα) in a two-antibody sandwich immunoassay.
[00253] In certain preferred embodiments of the invention using the two antibody sandwich assay described above, the first, immobilized antibody specific to FRα is a polyclonal antibody and the second antibody specific to FRα is a polyclonal antibody. In certain other embodiments of the invention, the first, immobilized antibody specific to FRα is a monoclonal antibody and the second antibody specific to FRα is a polyclonal antibody. In certain other embodiments of the invention the first, immobilized antibody specific to FRα is a polyclonal antibody and the second antibody specific to FRα is a monoclonal antibody. In certain other embodiments of the invention, the first, immobilized antibody specific to FRα is a monoclonal antibody and the second antibody specific to FRα is a monoclonal antibody. For example, in these modalities it should be noted that monoclonal antibodies MORAb-003, MOV18, 548908, 6D398 or variants thereof as described here, as provided here, recognize distinct and non-competitive antigenic determinants (eg, epitopes) in the FRαs, de in such a way that any paired combination of these monoclonal antibodies can be employed. In other preferred embodiments of the invention, the first, immobilized antibody specific to FRα and / or the second antibody specific to FRα can be any of the types of antibodies known in the art and referred to here, for example, by way of illustration and not limitation, Fab fragments, F (ab ') 2s fragment, immunoglobulin V region fusion proteins or single chain antibodies. Those skilled in the art will appreciate that the present invention encompasses the use of other forms of antibody, fragments, derivatives and the like in the methods disclosed and claimed herein.
[00254] In certain particularly preferred embodiments, the second antibody may contain a detectable reporting fraction or marker such as an enzyme, dye, radionuclide, luminescent group, fluorescent or biotin group, or the like. The amount of the second antibody that remains bound on the solid support is then determined using an appropriate method for the specific detectable reporting fraction or marker. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Antibody-enzyme conjugates can be prepared using a variety of coupling techniques (for review see, for example, Scouten, W. H., Methods in Enzymology 135: 30-65, 1987). Spectroscopic methods can be used to detect dyes (including, for example, colorimetric products from enzymatic reactions), luminescent groups and fluorescent groups. Biotin can be detected using avidin or strepavidin, coupled with a different reporting group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporting groups can generally be detected by adding substrate (usually for a specific period of time), followed by spectroscopic, spectrophotometric or other analysis of the reaction products. Standards and standard additions can be used to determine the level of mesothelin polypeptide in a sample, using well-known techniques.
[00255] A method of classifying the presence of a cancer that expresses FRα according to the present invention can be further improved by detecting more than one tumor-associated marker in a biological sample from a subject. Thus, in certain embodiments, the present invention relates to a classification method that, in addition to detecting the reactivity of FRα not bound to a cell, also includes detecting at least one additional soluble marker of a malignant condition using established methods known in the art. technique and provided here. As noted earlier, there are currently numerous soluble tumor-associated antigens that are detectable in easily obtained biological fluid samples. C. Cases of the Invention
[00256] The invention also concerns kits to assess whether a subject is suffering from a cancer that expresses FRα, to assess the progression of a cancer that expresses FRα, to assess the level of risk that a subject will develop a cancer that expresses FRα, or to monitor the effectiveness of a therapy or treatment regimen for a cancer that expresses FRα. These kits include means to determine the level of expression of FRα and instructions for using the kit to assess the progression of a cancer that expresses FRα, to assess the level of risk that a subject will develop cancer that expresses FRα, or to monitor the efficiency of a therapy or treatment regimen for a cancer that expresses FRα.
[00257] The kits of the invention can optionally comprise additional components used to carry out the methods of the invention. As an example, the kits may comprise means for obtaining a sample from a subject, a control sample, for example, a sample from a subject with cancer progressing slowly and / or a subject without cancer, one or more sample compartments, and instructional material describing the performance of a method of the invention and specific tissue controls / standards.
[00258] Means for determining the level of FRα include known methods in the art for assessing protein levels, as discussed above, and preferred specific modalities, for example, using the MORAb-003 antibody, as discussed here. Thus, for example, in one embodiment, the level of FRα is assessed by contacting a sample derived from a subject (such as urine or serum) with an alpha receptor for folate binding agent (FRα). In a preferred embodiment, the binding agent is an antibody. Many of the types of antibodies that bind FRα are discussed earlier in the methods of the invention and can also be used in the kits of the invention.
[00259] The means for determining the level of FRα may additionally include, for example, buffers or other reagents for use in an assay to determine the level of FRα. The instructions can be, for example, printed instructions for performing the assay and / or instructions for assessing the level of expression of FRα.
[00260] The kits of the inventions may also include means for isolating a sample from a subject. Such means can comprise one or more items of equipment or reagents that can be used to obtain a fluid or tissue from a subject. Means for obtaining a sample from a subject may also comprise means for isolating blood components, such as serum, from a blood sample. Preferably, the kit is designed for use with a human subject. III. Classification Tests
[00261] In additional embodiments, the invention also relates to methods (also referred to here as "classification assays") to identify modulators, that is, candidate or test compounds or agents (for example, proteins, peptides, peptidomimetics, peptides, small molecules or other medications), which modulate the growth, progression and / or aggressiveness of cancer, for example, a cancer that expresses FRα, or a cancer cell, for example, an ovarian cancer cell, monitoring and comparing levels of FRα in a sample. Such assays typically comprise a test compound, or a combination of test compounds, whose activity against cancer or a cancer cell must be evaluated. Compounds identified by means of assays such as those described here can be used, for example, to modulate, for example, inhibit, alleviate, treat, or prevent aggressiveness of a cancer that expresses FRα or a cancer cell, for example, a cancer cell. Ovary cancer. By monitoring the level of FRα in a sample, it can be determined whether the cancer that expresses FRα is progressing or regressing and whether the test compound has the desired effect. For example, in modalities where cancer that expresses FRα is a cancer for which higher levels of FRα are associated with a prognosis of worsening, a decrease in the level of FRα after administration of the test compound (s) ) would be indicative of the effectiveness of the test compound. In contrast, an increase in the level of FRα after administration of the test compound (s) would indicate that the test compound is not effective in the treatment of ovarian cancer. Conversely, in modalities where cancer that expresses FRα is a cancer for which higher levels of FRα are associated with an improvement prognosis, an increase in the level of FRα after administration of the test compound (s) ) would be indicative of the effectiveness of the test compound. In contrast, a decrease in the level of FRα after administration of the test compound (s) would indicate that the test compound is not effective in treating ovarian cancer.
[00262] The test compounds used in the classification assays of the present invention can be obtained from any available source, including systematic libraries of natural and / or synthetic compounds. Test compounds can also be obtained by any of the numerous approaches to combinatorial library methods known in the art, including biological libraries; peptide libraries (molecule libraries with peptide functionalities, but with an unprecedented non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, for example, Zuckermann et al., 1994, J. Chem. 37: 2678-85); libraries of solid phase or phase in parallel spatially addressable solution; synthetic library methods requiring devolution; the 'one-sphere one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptide library approaches are limited to peptide libraries, while the other four approaches are applicable to the peptide, non-peptide oligomer or small molecule libraries of the compounds (Lam, 1997, Anticancer Drug Des. 12: 145).
[00263] Examples of methods for the synthesis of molecular libraries can be observed in the art, for example, in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994). J. Med. Chem. 37: 2678; Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; and in Gallop et al. (1994) J. Med. Chem. 37: 1233.
[00264] Libraries of the compounds can be presented in solution (for example, Houghten, 1992, Biotechniques 13: 412-421), or in spheres (Lam, 1991, Nature 354: 82-84), chips (Fodor, 1993, Nature 364: 555-556), bacteria and / or spores, (Ladner, USP 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci USA 89: 1865-1869) or in the phage (Scott and Smith, 1990, Science 249: 386-390; Devlin, 1990, Science 249: 404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. 87: 6378-6382; Felici, 1991, J. Mol. Biol. 222: 301 -310; Ladner, supra.).
[00265] The present invention is further illustrated by the following examples which should not be interpreted as additionally limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the figures, are expressly incorporated here by the reference in its entirety. EXAMPLES EXAMPLE 1. DETERMINATION OF FRα LEVELS IN URINE SAMPLES OF HUMAN SUBJECTS WITH AND WITHOUT Ovarian Cancer MEASURED BY ELECTROCHEMIOLUMINESCENT IMMUNOENSATION (ECLIA). Materials and methods
[00266] Urine samples were obtained from human subjects, including subjects suffering from ovarian cancer and normal control subjects not suffering from ovarian cancer. FRα levels in urine samples were determined using an electrochemical chemiluminescent immunoassay (ECLIA) according to the following procedure (see Namba et al. (1999) Analytical Science 15: 1087-1093): i. Antibody coating for microspheres
[00267] The monoclonal anti-folate alpha receptor antibody was coated on the surface of microspheres (Dynabeads M-450 Epoxy, Dynal). Thirty six milligrams of microspheres were mixed with 1.2 mL of MOV18 antibody (0.36 mg / mL, Enzo Life Science) in 0.15 mol / L of phosphate buffered saline pH 7.8 (PBS), followed by mixing soft for 16 hours at room temperature. The microspheres were then washed 5 times with 50 mM HEPES buffer containing 0.1% normal rabbit serum (NRS), 150 mmol / L NaCl, 0.01% Tween 20 pH 7.5 (wash buffer ). The coated microspheres were then suspended in 1.2 ml of 50 mM HEPES buffer containing 20% NRS, 150 mmol / L NaCl and 0.01% Tween 20 pH 7.5 (reaction buffer) to block the surface unbound, followed by gentle mixing for 3.5 hours at room temperature. Finally, the microspheres were washed 5 times with wash buffer and resuspended with 1.2 ml of 50 mM HEPES buffer containing 10% NRS, 150 mmol / L NaCl, 10 mmol / L EDTA-2Na and 0.01% Tween 20 pH 7.5 (reaction buffer) so that the microsphere concentration was 30 mg / mL. The microspheres were stored at 4 ° C until use. ii. Labeling of the Antibody with Ruthenium-Chelate-NHS (Ru)
[00268] One milliliter of MORAB-003 (1 mg / mL) in PBS was mixed with 14 μL of Ru (10 mg / mL), initial molar ratio of antibody to Ru was 1:20, followed by stirring for 30 minutes at room temperature in the dark. The reaction was terminated by adding 25 μL of 2 mol / L of glycine solution followed by incubation for 20 minutes. The labeled antibody was purified by gel filtration using Sephadex G-25 (GE Healthcare) eluted with PBS. The first yellow eluted fraction was collected and the concentration of antibody and Ru was determined using the Pierce BCA protein assay kit (Thermo Scientific) and absorption at 455 nm respectively. The final molar ratio was calculated using the formula: final molar ratio = [(absorption at 455) /13,700] / [Ab (mg / mL / 150,000)]. The labeled antibody was stored at 4 ° C until use. iii. One Step Immunoassay
[00269] The antibody coated microspheres were placed on the reagent table of the Picolumi 8220 (Sanko, Tokyo, Japan) after adjusting the concentration of the beads to 1.5 mg / mL (functional solution) in reaction buffer. The Ru-labeled antibody was placed on the reagent table of the Picolumi 8220 after adjusting the antibody concentration to 2 μg / mL (functional solution) in reaction buffer.
[00270] Ten microliters of urine (diluted 1:51 in reaction buffer) or standard FRα (prepared in reaction buffer) and 100 μL of reaction buffer were dispensed into a reaction tube (Sanko, Tokyo, Japan) and placed on the Picolumi 8220.
[00271] The following steps were automatically performed by Picolumi 8220. Thirty five microliters of beads (functional solution) and 180 μL of Ru-labeled antibody (functional solution) were dispensed. After 26 minutes of incubation at 30 +/- 2 ° C, the beads were washed and suspended with 300 μL of electrolyte solution (Sanko, Tokyo, Japan). The washed spheres were subsequently transferred to the electrode and the electrochemiluminescence emission (ECL) was measured. All ECL measurements were performed in duplicate. Results of
[00272] Table 1 represents the levels of FRα urine in individual subjects and female control subjects not suffering from ovarian cancer. Table 1: FRα levels in the urine of female control subjects with normal and ovarian cancer


[00273] Figure 2 represents the distribution of FRα levels in urine in subjects with ovarian cancer and in normal female control subjects, as shown in Table 1.
[00274] Table 2 summarizes the number of subjects (n), average values, standard deviation (SD), maximum (Max.) And minimum (Min.) For the levels of FRα in the group with ovarian cancer and in the control group normal female. Table 2: Summary of measurement of urine FRα
Discussion
[00275] A high level of FRα was detected in the urine of subjects with ovarian cancer. In addition, FRα levels differed significantly between the control group of females with ovarian cancer and normal (p = 0.03, one side). EXAMPLE 2. DILUTION LINEARITY - DETERMINATION OF FRα LEVELS IN SERIALLY DILUTED URINE SAMPLES MEASURED BY ELECTROCHEMIOLUMINESCENT IMMUNOENSATION (ECLIA)
[00276] Dilution linearity is a measure of the accuracy of an assay. Du- the urine samples were serially diluted by a factor of 10 and 100. The levels of FRα of each sample were measured shown in example 1 and compared to assess the percentage error. The percentage error was calculated as follows: [[(FRα in diluted sample) * (dilution factor)] - (F Rα in undiluted sample)] * 100 (FRα in undiluted sample)
[00277] The results are shown in Table 3: Table 3: Urine Dilution Linearity

[00278] The previous results demonstrate dilution linearity in the assessment of FRα levels in human urine samples and that, within acceptable errors, urine can be diluted to a factor of at least 100 while retaining accurate levels of FRα. In this way, dilution of urine samples can be considered before determining the levels of FRα. EXAMPLE 3: CENTRIFUGATION OF URINE SAMPLES - ADDRESSING REPRODUCIBILITY
[00279] The reproducibility of the ECLIA assay for a particular urine sample was also tested. For example, as reflected in table 4, ECLIA assays from the same sample resulted in varying results. Table 4. Reproducibility without sample centrifugation

[00280] The presence of insoluble material (precipitated) in urine samples was hypothesized to be responsible for the variability seen in measurements of FRα levels. As a result, centrifugation of samples in order to remove urine sediment, before measuring FRα levels, was considered an option to increase the accuracy and reproducibility of the assay.
[00281] Table 5 represents the results obtained when three samples were centrifuged before the performance of the ECLIA assay. Tabe a 5. Reproducibility with sample centrifugation

[00282] As previously presented, the results indicate that centrifugation provided more consistent measurements of FRα concentration.
[00283] In addition, two samples were subjected to (i) centrifugation (at 2,000 xg for 2 minutes) and the supernatant removed for FRα measurement (represented as “A” sample below in table 6) and (ii) centrifugation followed by turbulence (represented as sample “B” below in table 6), before the measurement of FRα levels by the ECLIA test presented in example 1. The results are reflected in table 6 below. Table 6: Effect of centrifugation on FRα levels in urine
Difference (%) was determined as follows: [(FRα level in “B”) - (FRα level in “A”)] * 100 (FRα level in “A”)
[00284] As shown in table 6, the FRα levels determined by the ECLIA assay vary depending on whether urine has been clarified by centrifugation to remove precipitates or whether the urine has been swirled to suspend or disperse sediment. Thus, in certain modalities, centrifugation or turbulence of urine samples can be performed before determining levels of FRα. EXAMPLE 4. DETERMINATION OF FRA LEVELS IN CENTRIFUGAL URINE SAMPLES OF HUMAN SUBJECTS WITH AND WITHOUT OVARY CANCER MEASURED BY ELECTROCCHEMIUMIUM INCENTIVE IMMUNOSENESS (ECLiA).
[00285] Based on the results of Example 3, the assay to assess FRα levels in subjects has been modified to introduce a centrifugation step. FRα levels were determined in the same samples used in example 1, including the group of subjects with ovarian cancer and the group of normal female control subjects. Materials and methods
[00286] The methodology used was as described in example 1, except that the urine samples were centrifuged for 10,000 x g for 1 minute and the resulting supernatant was subsequently diluted by 1:51 in reaction buffer. Results of
[00287] Table 7 represents the levels of FRα in centrifuged and non-centrifuged urine samples from subjects suffering from ovarian cancer and healthy female control subjects. Table 7: FRα1 level of urine in a control group with normal and ovarian cancer


[00288] According to the results presented in table 7, centrifugation resulted in a decrease in the measurement of FRα levels in some samples, as previously demonstrated in table 6. Example 5: Detection of FRα in Urine Sediment by Immunoblotting
[00289] Based on the results shown in examples 3 and 4, the presence or absence of FRα in urine / precipitate sediment was assessed using western blotting. Materials and methods
[00290] Urine samples from 2 ovarian cancer patients for whom FRα concentrations were measured at 18.747 pg / mL and 145.564 pg / mL, respectively (See table 10 above), underwent the following procedures. Control samples consisted of 10 μg HeLa cell lysate, 20 μg liver tissue lysate, and 20 μg ovarian cancer tissue lysate. • 900 μL of urine was centrifuged for 2 minutes at 10,000 g • supernatant was removed • the remaining precipitate was dissolved in 15 μL of PAGE sample buffer (containing 292 mM LDS) and subsequently boiled at 70 ° C for 10 minutes. • The entire sample (approx. 20 μL) was loaded onto the NuPAGE bis-tris gel (Invitrogen) • After electrophoresis, proteins were transferred to PVDF membrane • 1% skimmed milk / 0.05% Tween 20 / PBS were added for blocking • The membrane was washed with 0.05% Tween 20 / PBS • 0.5 mL of monoclonal antibody 548908 (R&D Systems) at 2 μg / mL was added and incubated for 60 minutes at room temperature • The membrane was washed with 0.05% Tween 20 / PBS • 10 mL of anti-mouse IgG-HRP (DAKO p0447, 1: 2000) was added and incubated naturally for 60 minutes • The membrane was washed with 0.05% Tween 20 / PBS • Pierce ECL substrate was added to the membrane • The membrane was removed from the substrates and then imaged using the LAS-3000 system (FUJIFILM) Results of
[00291] The resulting immunoblot is shown in figure 3. In this figure, lanes 15 correspond to FRα detected from the following sources: (1) urine from a patient with ovarian cancer with a measured FRα level of 18,747 pg / mL (2) urine of an ovarian cancer patient with a measured FRα level of 145,564 pg / mL (3) HeLa cell lysate: 10 μg (4) liver tissue lysate: 20 μg (5) ovarian cancer tissue lysate: 20 μg
[00292] Lane 6 in the western blot represents molecular weight markers and demonstrates that the range observed in lanes 1, 2, 3 and 5 runs at the expected molecular weight for FRα.
[00293] Lanes 3 and 5 are positive control samples and lane 4 is a negative control sample. The weak strip in lane 1 and the light strip in lane 2 demonstrated that FRα can be detected in the urine sediment of patients with ovarian cancer by western blotting. EXAMPLE 6. DETERMINATION OF FRα LEVELS IN NORMAL HUMAN URINE SAMPLES TREATED WITH GUANIDINE BY ELECTROCHEMIOLUMINESCENT IMMUNOENSATION (ECLIA).
[00294] Based on the results of example 4 in which centrifugation resulted in lower levels of FRα, and the results of example 5 where the urine sediment obtained from the centrifugation was shown to contain immunoreactive FRα, methods were sought to solubilize the sediments of urine to obtain more quantitative and accurate measurements of FRα.
[00295] In this regard, treatment of normal female urine samples with guanidine was attempted before assessing FRα levels.
[00296] The methodology used was as described in example 1, except that the urine samples were mixed in a 1: 1 ratio with either 6 M guanidine in buffer (PBS) or in buffer alone. Subsequently, the urine samples were diluted 1:51 in reaction buffer.
[00297] The results of this test are shown in Table 8. Table 8: FRα level of normal urine from treatment with or without guanidine

[00298] The results of this experiment indicated that guanidine does not interfere with measurements of FRα. As can be seen for the control of pure antigen (Std Ag), this methodology of treatment with guanidine and subsequent dilution has no effect on the measurement of FRα. Additionally, it will be noted that in all three (3) urine samples evaluated, the levels of FRα were higher in samples treated (solubilized) with guanidine compared to samples not treated with guanidine.
[00299] The reliability of guanidine pretreatment of urine samples was further assessed by exposing three samples to guanidine and measuring the FRα concentration of each guanidine sample treated 3 times using the ECLIA assay. The results are reflected in table 9 below: Table 9: Intra-assay reproducibility of urine treated with guanidine


[00300] As previously presented, the results indicate that urine treatment with guanidine before the FRα assay provided consistent measurements of FRα concentration with very low CV’s. EXAMPLE 7. DETERMINATION OF FRα LEVELS IN URINE SAMPLES TREATED WITH GUANIDINE OF HUMAN SUBJECTS WITH AND WITHOUT OVAC CANCER MEASURED BY ELECTROCHEMIOLUMINESCENT IMMUNOENSATION (ECLIA).
[00301] Based on the results of example 6 in which guanidine treatment was shown not to interfere with FRα assays, a modified assay protocol was employed to measure FRα in the urine samples of subjects with and without ovarian cancer in example 1. The following test protocol was used: Materials and Methods
[00302] The methodology used was as described in example 1, except that the urine samples were mixed in a 1: 1 ratio with a 6 M guanidine buffer and subsequently diluted 1:26 in reaction buffer. Results of
[00303] Table 10 represents the levels of FRα in urine samples treated with guanidine from subjects suffering from ovarian cancer and healthy female control subjects. Table 10: Urine level of FRα in a control group with normal and ovarian cancer



[00304] Figure 4 shows the distribution of FRα levels in the urine of subjects suffering from female control subjects with normal and ovarian cancer using the modified guanidine treatment protocol. A statistically significant difference between groups was observed. Table 11 summarizes these results. Table 11: Summary of measurement of FRα in urine

[00305] Using the data from this experiment, a characteristic operational analysis of the receiver (ROC) was performed. Figure 5 shows a ROC curve of the sensitivity and specificity of ECLIA measurement of FRα levels in urine after urine has been treated with guanidine. AUC is the area under the curve, which measures the accuracy of the test in separating ovarian cancer from control subjects.
[00306] Using an arbitrary cutoff value of 9,100 pg FRα / mL, the AUC was 0.70 with a positive predictive value of 70% and a negative predictive value of 80%, as shown in table 12. Using this cutoff value , 15/19 patients with ovarian cancer had a concentration of FRα above 9,100 pg / mL and 8/10 normal subjects had a concentration of FRα less than 9,100 pg / mL. Table 12: Treatment with guanidine for urine measurement

EXAMPLE 8: CREATININE CORRECTION OF FRα CONCENTRATIONS DETERMINED IN URINE SAMPLES TREATED WITH GUANIDINE BY ELECTROCHEMIOLUMINESCENT IMMUNOENSATION (ECLIA).
[00307] FRα concentrations were previously determined using ECLIA from urine samples treated with guanidine from controls of patients with ovarian cancer and normal female (See example 7, table 10). Here, these concentrations of FRα have been corrected for urine creatinine levels in order to normalize to the glomerular filtration rate. The resulting values were subjected to a ROC analysis. Methods
[00308] The level of creatinine in the urine was determined by the Ministry of Health, Labor and Welfare approved test kit, L CRE determiner (Kyowa Medex, Japan). The corrected value for urine FRα concentration was calculated as follows: = Correction of Creatinine FRα in Urine (ng / g) = (FRα in Urine (ng / L) x 1,000) / (Creatinine in Urine (mg / dL) x10) = (Urine FRα (ng / L) x 1,000) / Urine Creatinine (mg / L) = Urine FRα (ng / L) / Urine Creatinine (g / L) = Urine FRα (ng) / Creatine in Urine (g) ou = 1 / 1,000 x FRα of Urine (μg) / Creatinine in Urine (g) Results
[00309] Table 13 shows the resulting levels of FRα corrected by cretinin. Table 13: Creatinine-corrected FRα levels determined using ECLIA from urine samples treated with guanidine


[00310] Figure 6 shows the distribution of FRα levels in ovarian cancer (OC) and normal female control subjects after correction for urine creaminin levels. There is a statistically significant difference between patients with ovarian cancer and controls at creatinine-corrected FRα levels (p = 0.007).
[00311] Summary data for ovarian cancer and normal control subjects are provided in table 14. Table 14: Summary statistics for creatinine-corrected FRα levels

[00312] Creatinine-corrected FRα levels were additionally subjected to a ROC analysis. The ROC curve is shown in figure 7. Table 15 presents the sensitivity, specificity, and area under the curve (AUC) for various values of the creatinine-corrected test. Table 15: Sensitivity, specificity, and AUC for various cut values of the FRα test corrected for creatinine


[00313] As previously noted, there is a clear discrimination between urine from patients with ovarian cancer and those from healthy female control subjects. EXAMPLE 9: ENZYME IMMUNOENSATION (EIA) AND OPTIMIZATION OF THIS 1. Enzyme Immunoassay (EIA) Antibody coating on microtiter plates
[00314] The monoclonal anti-folate alpha receptor antibody was coated on the surface of microtiter plates (Nunc-immunoplate, Thermo Scientific) as follows. A hundred microliters of antibody (absorbance 0.02 to 280 nm) in 50 mmol / L of carbonate buffer pH 9.4 were dispensed into the wells, followed by coating for 16 hours at 4 ° C. The microplates were then washed 2 times with PBS containing 0.05% Tween 20 (PBS-T). Then 0.15 mL of PBS containing 20% normal rabbit serum pH 7.8 was dispensed into the wells to block the unbound surface, followed by blocking for 1 hour at room temperature. Finally, the microplates were washed 2 times with PBS-T. The antibody-coated plates were dried and kept at 4 ° C in aluminum bags until use. Biotin labeling
[00315] Biotin labeling was conducted according to the manufacturer's recommendations for EZ-Link Sulfo-NHS-LC-LC-Biotin (Product No. 21338, Thermo Scientific). Briefly, 1 mg of antibody in 0.4 ml of PBS was mixed with 0.013 ml of 10 mM Sulfo-NHS-LC-LC-Biotin, with an initial antibody to biotin molar ratio of 1:20, followed by incubation for 30 minutes at room temperature. The biotin-coupled antibody was purified by gel filtration using a PD-10 column (GE Healthcare) eluted with PBS to remove unreacted biotin. In order to determine the level of biotin incorporation, the EZ Biotin quantitation kit (Product No. 28005, Thermo Scientific) was used. The biotin-labeled antibody was stored at -80 ° C until use. Two-step immunoassay
[00316] For the first reaction, 40 μL of plasma or standard antigen and 60 μL of 50 mM HEPES buffer containing 10% NRS, 150 mmol / L NaCl, 10 mmol / L EDTA-2Na, 0.01% Tween 20 pH 7.5 (reaction buffer) was dispensed in the wells coated with antibody. The plate was incubated for 18 hours at 4 ° C, and subsequently washed 5 times with PBS-T. For the second reaction, 100 μL of 10 μg / mL of biotin-labeled antibody in reaction buffer was dispensed. The plate was incubated for 1 hour at room temperature, and subsequently washed 5 times with PBS-T. 100 μL of horseradish peroxidase (Pierce) was dispensed. After 30 minutes of incubation at room temperature, the plates were washed 5 times with PBS-T. Finally, for color development, 100 μL of TMB solution (KPL) was dispensed and left for 15 minutes in the dark. After stopping color development by adding 100 μL of 1N HCl, the absorption at 450 nm was read using a plate reader. All washing steps were automatically performed by a plate washing machine (AMW-8, BioTec, Japan), and all EIA measurements were performed in duplicate.
[00317] Figure 8 represents the EIA assay using MOV18 as the capture antibody and biotinylated MORAb-003 as the detector antibody. 2. Optimization of EIA procedures
[00318] The previous EIA procedures arrived in part based on the following experiments designed to optimize the procedure.
[00319] First, avidin-HRP, biotin-labeled antibody and HRP-labeled antibody were compared. Compared with HRP-labeled antibody, biotin-labeled antibody and avidin-HRP provided a greater signal; therefore, biotin-labeled antibody and avidin-HRP were employed.
[00320] Second, one- and two-step incubation procedures were compared. As shown in figure 9, a two-step incubation procedure produced a larger signal and was thus employed.
[00321] Third, to optimize the second incubation time, incubation times of one to four hours were compared. The results indicated that an hourly incubation times provided the highest signal for noise ratio and therefore an hourly incubation time was subsequently employed.
[00322] Fourth, in order to optimize the functional concentration of antibody labeled with biotin, antibody labeled with HRP and sample volume, several concentrations were used presented in the previous description of the EIA assay. The concentrations of ideal values are described previously. EXAMPLE 10: COMPARISON OF FRα IN HUMAN PLASMA USING ELECTROCHEMIOLUMINESCENT IMMUNOENSATION (ECLIA) AND ENZYME IMMUNOENSATION (EIA)
[00323] FRα levels were measured in human plasma samples taken from patients with ovarian cancer and healthy female controls using the electrochemiluminescence assay (ECLIA) described in example 1 and figure 1 (using MORAb-003 as the antibody of capture and ruthenium-labeled MOV-18 (Ru) as the labeled detector antibody) and the enzyme immunoassay (EIA) described in Example 9 and Figure 8. In both assays, 40 μL of plasma were assayed.
[00324] Table 16 shows plasma levels of FRα in subjects with ovarian cancer and normal controls, determined using EIA and ECLIA. Table 16: FRα plasma concentrations determined using EIA and ECLIA methods


[00325] With only one exception, the results for all subjects indicated that the concentrations of FRα detected in serum using EIA are lower than the levels detected using ECLIA, demonstrating that the EIA assay, as formatted, is not as sensitive as the ECLIA assay when this combination of particular capture (MOV-18) and antibody detectors (MORAb-003) is used. Therefore, additional experiments with other types of antibodies were conducted to develop a more sensitive EIA procedure. EXAMPLE 11: FEASIBILITY OF DIFFERENT TYPES OF ANTIBODIES FOR EIA DE FRα MEASUREMENTS IN HUMAN PLASMA 1. Preliminary Experiments of Antibody Combinations
[00326] Various combinations of antibody capture / detectors have been considered. Preliminary experiments produced the results shown in table 17. Table 17
2. Comparison of EIA assays using various antibody combinations and comparison with the ECLIA assay
[00327] FRα levels in plasma from ovarian cancer patients and normal healthy female controls were measured using an enzyme-linked immunosorbent assay (EIA) with different combinations of biotin-labeled antibody and capture and compared with levels of FRα measured using the ECLIA assay. Materials and methods
[00328] The ECLIA method was as described in example 1 and represented in figure 1 (using the MORAb-003 antibody as the capture antibody and the Mov-18 antibody as the labeled detector antibody). The EIA method was as described in example 9, except that three different combinations of antibody capture / detectors were employed, as shown in figure 10: MOV18 / MORAb-003, 548908 / MORAb-003 and 6D398 / MORAb-003. Antibodies 548908 and 6D398 are commercially available. Antibody 548908 was obtained by R&D Systems (North Las Vegas, NV) and antibody 6D398 was obtained by US Biological (Swampscott, MA 01907). Results of
[00329] FRα concentrations (pg / mL) determined using the EIA and ECLIA methods are shown in table 18. Furthermore, FRα concentrations (pg / mL) determined by EIA using various antibody capture / detector combinations are plotted in figure 11. Table 18: FRα plasma concentrations (pg / mL) determined using the EIA and ECLIA methods with various combinations of capture antibodies and detectors.

[00330] The data in Table 18 indicates that measurements of FRα levels with EIA using the combination 54908-MORAb-003 produced results that are more similar to the results obtained using the ECLIA assay. Quantitative analyzes were performed, confirming this observation. Additionally, these data demonstrate that the detection of FRα is highly dependent on the antibodies and antibody combination employed. In this way, different combinations of antibody can be used for the determination of FRα in biological fluids. Furthermore, since the data obtained from the EIA and ECLIA test formats are similar, several test formats can be used for the determination of FRα.
[00331] For each of the three combinations of capture antibodies and detectors used for the EIA method, a regression analysis was performed, and the concentrations of FRα (pg / mL) determined with EIA were correlated with the concentrations determined with the assay. ECLIA. The results of this analysis are shown in table 19 and figure 12. Table 19: Correlations of FRα concentrations in plasma measured by ECLIA with concentrations measured by EIA using three combinations of capture antibodies and detectors

[00332] The results for EIA using the capture-detector combination of 548098-MORAb-003 correlated highly (r = 0.96) with the results for ECLIA. EXAMPLE 12: FRα PLASMA LEVELS DETERMINED BY EIA AND ECLIA IN SAMPLES OF PATIENTS WITH OVARY CANCER
[00333] Measurements of serum levels of FRα were determined in a group of patients with ovarian cancer (n = 17) and normal controls (n = 35) using ECLIA and EIA. For EIA measurements, the 548908 capture / MORAb-003 detector antibody combination was employed. The EIA procedure was otherwise as described in example 9. The ECLIA procedure was as described in example 1. The results are shown in table 20. Table 20: FRα plasma concentrations in patients with ovarian cancer and normal controls, determined using EIA and ECLIA



[00334] Figure 13 shows the distribution of FRα concentrations in plasma in female control subjects with ovarian and normal cancer determined using EIA.
[00335] Table 21 shows summary descriptive statistics for plasma plasma concentrations of FRα in female control subjects with ovarian and normal cancer determined using EIA. Table 21: Summary of FRα plasma concentrations in female control subjects with ovarian and normal cancer determined using EIA.

[00336] Figure 14 additionally represents the correlation between FRα plasma concentrations determined using EIA and ECLIA. The correlation is high (r = 0.95). EXAMPLE 13. DETERMINATION OF FRα LEVELS IN MARRIED SERUM AND URINE SAMPLES AND PATIENTS WITH LUNG CANCER AND PATIENTS WITH OVARY CANCER MEASURED BY ELECTROCHEMIOLUMINESCENT IMMUNOSENSE (ECLIA).
[00337] FRα levels were determined in married urine and serum samples in patients with lung cancer and ovarian cancer using ECLIA where the samples were taken from the same patient. The correlation between serum and urine levels of FRα was also determined. Materials and methods
[00338] The ECLIA methodology used is as described in example 1. Guanidine was used to solubilize urine sediments as described in example 6. Results
[00339] The results of the ECLIAs trial of serum and urine from patients with lung cancer and ovarian cancer are shown in table 22. Table 22: FRα concentrations in married urine and serum samples from patients with lung cancer and patients with ovarian cancer, determined by ECLIA


[00340] Summary data for FRα serum and urine levels of lung cancer patients are shown in table 23. Table 23: Summary statistics for FRα concentrations in married urine and serum samples from lung cancer patients, determined by ECLIA

[00341] Summary data for serum and FRα urine levels of patients with ovarian cancer are shown in Table 24. Table 24: Summary statistics for FRα concentrations in married urine and serum samples from patients with ovarian cancer, determined by ECLIA

[00342] Figure 15 shows correlations between ECLIA measurements of FRα levels in married urine and serum samples taken from the same patient. The correlation for patients with lung cancer was r = 0.24 (upper panel) and the correlation for patients with ovarian cancer was r = -0.76 (lower panel).
[00343] These data demonstrate the relative lack of correlation between FRα concentrations measured in urine versus serum, especially as shown for lung cancer patients. Additionally, these data demonstrate that FRα is basically undetectable above background levels in the serum of patients with lung cancer versus normal controls while FRα is detectable in the urine of these patients. EXAMPLE 14. EVALUATION OF FRα LEVELS IN SERUM SAMPLES OF OVARY CANCER PATIENTS, LUNG CANCER PATIENTS, AND NORMAL CONTROLS
[00344] Serum FRα levels in ovarian cancer patients, lung cancer patients, and normal controls were assessed. Serum FRα levels were assessed using ECLIA with two different pairs of capture detector antibody: Par 1, in which 9F3 was the capture antibody and 24F12 was the detector antibody, and Par 2, in which 26B3 was the capture antibody and 19D4 was the detector antibody.
[00345] The FRα pairs were tested with total calibration curves and 196 individual sera diluted 1: 4. In one experiment, 26B3 was used as the capture antibody after CR processing in a plate batch (75 μg / mL, + B, + T) and 19D4 was used as the detector antibody at 1.0 μg / mL. In another experiment, 9F3 was used as the capture antibody and 24F12 was used as the detector antibody at 1.0 μg / mL. Each was processed with CR (batch 10,070) with a marker for a protein ratio (L / P) of 13.3. Diluent 100 (Meso Scale Discovery, Gaithersburg, Maryland) + anti-human mouse antibody (HAMA) + mIgG was used for samples and calibrator. Diluent 3 (Meso Scale Discovery, Gaithersburg, Maryland) was used for detections.
[00346] The following protocol was used for ECLIA. Samples were added at 50 μL / well. The samples were stirred for 2 hours and subsequently washed with Phosphate Buffered Saline (PBS) with Tween 20 detergent (PBST). The detector antibody was added at 25 μL / well. The samples were shaken for 2 hours and then washed with PBST. Finally, the electrochemiluminescence (ECL) emission of the samples was read with 2X MSD® Buffer T.
[00347] The results are shown in table 25 below. table 25: FRα levels in serum of patients with ovarian cancer, patients with lung cancer, and normal controls
















1 LLOQ is the lower limit of quantitation 2 The retro-adjustment concentration is made to take into account the sample dilution.
[00348] Based on the previous data, it became apparent that antibodies 9F3, 2412, 26B3 and 19D4 were used to detect levels of FRα in biological samples, for example, serum, derived from a subject. In addition, the particular combinations of (i) 9F3 as a capture antibody and 24F12 as a detector antibody and (ii) 26B3 as a capture antibody and 19D4 as a detector antibody were capable and particularly effective in assessing FRα levels in biological samples. Example 15. Evaluation of FRα Levels in Urine USING THREE DIFFERENT PAIRS OF DETECTOR AND CAPTURE ANTIBODY
[00349] The ability of three pairs of anti-FRα antibodies to detect FRα levels in urine samples was assessed. The antibody pairs used were as follows: (1) 26B3 as a detector antibody and 9F3 as a capture antibody, and (2) 24F12 as a detector antibody and 9F3 as a capture antibody. Method
[00350] Two pairs of antibody were tested with total calibration curves and pre-treated urine with a 1: 1 dilution for 2 minutes in both 6 M guanidine, 3 M guanidine and PBS control. The following urine samples were tested: three clusters of human urine diluted 1:80, and five individual human urines diluted 1:80 (one male, four female).
[00351] Plates were Biodotted at 150 μg / mL, + B, + T, in 4spot STD ((Meso Scale Discovery, Gaithersburg, Maryland)), one capture per well. Detections were run at 1 μg / mL. Diluent 100 + HAMA + mIgG was used for samples and calibrator. Diluent 3 was used for detections. Diluents were commercially available diluents obtained by Meso Scale Discovery.
[00352] The following protocol was used for ECLIA. Samples were added at 50 μL / well. The samples were stirred for 2 hours. The samples were washed with Phosphate-Buffered Saline (PBS) with Tween 20 detergent (PBST). The detector antibody was added at 25 μL / well. The samples were shaken for 2 hours and subsequently washed with PBST. The electrochemiluminescence (ECL) emission of the samples was read with 2X MSD Buffer T.
[00353] The results of these experiments are shown in tables 2627. Table 26: Detection of FRα levels in urine using 26B3 as a detector antibody and 9F3 as a capture antibody

table 27: Detection of FRα levels in urine using 24F12 as the detector antibody and 9F3 as the capture antibody


[00354] Based on the previous data, it became apparent that antibodies 9F3, 24F12, 26B3 and 19D4s were used to detect FRα levels in biological samples derived from a subject. In addition, combinations of (1) 26B3 as a detector antibody and 9F3 as a capture antibody and (2) 24F12 as a detector antibody and 9F3 as a capture antibody were able and particularly effective in assessing the levels of FRα in biological samples.
[00355] A second set of experiments, following the protocol described above and using the same two pairs of antibodies, were conducted using four of the individual female human urines diluted 1:80. The urine was pretreated with a 1: 1 dilution for 2 minutes in both 3 M guanidine and PBSe control. The results are shown in tables 28-29. Table 28: Detection of FRα levels in urine using 26B3 as a detector antibody and 9 F3 as a capture antibody
Table 29: Detection of FRα levels in urine using 24F12 as the detector antibody and 9 F3 as the capture antibody


[00356] The results of this second set of experiments further confirm the results of the first set of experiments and demonstrate that the level of FRα that is not bound to a cell can be reliably assessed, for example, in urine, using assays such as the assay ECLIA and using the 26B3, 9F3, 24F12s antibody. In addition, the results demonstrate that such assays can effectively detect FRα using pairs of detector and capture antibodies that bind FRα (such as, for example, 26B3 as a detector antibody and 9F3 as a capture antibody, and 24F12 as a detector antibody and 9F3 as an antibody capture). EXAMPLE 16. EVALUATION OF FRα LEVELS IN SERUM AND PLASMA
[00357] FRα levels were assessed in serum and plasma samples on the two separate days. The subjects from which the samples were derived were both normal subjects and patients with ovarian or lung cancer.
[00358] The protocol for assessing FRα levels was the same as that presented in example 14 previously. The pairs of antibodies used to assess FRα levels were also the same as in Example 14, that is, Par 1, in which 9F3 was the capture antibody and 24F12 was the detector antibody, and Par 2, in which 26B3 was the anti- capture body and 19D4 was the detector antibody.
[00359] The results are provided in table 30. Table 30: FRα levels assessed in serum and plasma samples on different days






[00360] Based on the previous data, it became apparent that antibodies 9F3, 2412, 26B3 and 19D4 were used to detect FRα levels in biological samples, for example, serum or plasma, derived from a subject. In addition, particular combinations of (i) 9F3 as a capture antibody and 24F12 as a detector antibody and (ii) 26B3 as a capture antibody and 19D4 as a detector antibody were capable and particularly effective in assessing FRα levels in biological samples.
[00361] For assays conducted using both Par 1 and Par 2, there was a high correlation between serum and plasma levels of FRα. Figure 16 shows the correlation in FRα levels in serum versus plasma for assays conducted using Par 1 (see example 16). The R2 value was 0.8604. Figure 17 shows the correlation in FRα levels in serum versus plasma for assays conducted using Par 2 (see example 16). The R2 value was 0.9766.
[00362] For both serum and plasma samples, there was a high correlation between FRα levels measured using Par 1 and Par 2. Figure 18 shows the correlation in FRα levels in serum for assays conducted using Par 1 versus Par 2 (see example 16). The R2 value was 0.9028. Figure 19 shows the correlation in plasma levels of FRα for assays conducted using pair 1 versus pair 2 (see example 16). The R2 value was 0.8773.
[00363] The results also showed that there was a high correlation between FRα levels measured on the different days. Figure 20 shows the intermediate correlation in serum levels of FRα for assays conducted using pair 2. The R2 value was 0.9839. EQUIVALENTS
[00364] Those skilled in the art will realize, or be able to certify, using no more than a routine experiment, many equivalents of the specific modalities of the invention described here. Such equivalents are considered to be encompassed by the following claims. Any combination of the modalities disclosed in the dependent claims is contemplated in the scope of the invention. Table 33: SEQUENCES

权利要求:
Claims (39)
[0001]
1. Method of assessing whether a subject is suffering from ovarian or lung cancer, the method CHARACTERIZED by the fact that it comprises: placing a urine sample derived from said subject in contact with an antibody that binds to FRα, thus determining the level of alpha folate receptor (FRα) that is not bound to a cell in said urine sample; comparing the level of alpha folate receptor (FRα) that is not bound to a cell in said urine sample derived from said subject with the level of FRα in a control sample; identify an increase in the level of FRα in the urine sample derived from said subject compared to the level of FRα in the control sample; and determining that the subject is suffering from ovarian or lung cancer based on said increase.
[0002]
2. Method, according to claim 1, CHARACTERIZED by the fact that the subject is identified as being suffering from ovarian cancer.
[0003]
3. Method, according to claim 2, CHARACTERIZED by the fact that the subject is identified as suffering from lung cancer.
[0004]
4. Method according to claim 3, CHARACTERIZED by the fact that lung cancer is non-small cell lung cancer.
[0005]
5. Method according to claim 4, CHARACTERIZED by the fact that non-small cell lung cancer is adenocarcinoma.
[0006]
6. Method, according to claim 1, CHARACTERIZED by the fact that the presence of FRα in said sample at a concentration greater than about 9,500 pg / mL, about 10,000 pg / mL, about 11,000 pg / mL, about of 12,000 pg / mL, about 13,000 pg / mL, about 14,000 pg / mL, about 15,000 pg / mL, about 16,000 pg / mL, about 17,000 pg / mL, about 18,000 pg / mL, about 19,000 pg / mL, or about 20,000 pg / mL, is an indication that the subject is suffering from ovarian cancer.
[0007]
7. Method, according to claim 1, CHARACTERIZED by the fact that the antibody is: (a) an antibody that binds in the same epitope as the MORAb-003 antibody; (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRLH1 SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3; (c) the MOV18 antibody; (d) an antibody that binds to the same epitope as the MOV18 antibody; (e) antibody 548908; (f) an antibody that binds to the same epitope as the 548908 antibody; (g) the 6D398 antibody; (h) an antibody that binds to the same epitope as the 6D398 antibody; (i) an antibody that binds to the same epitope as the 26B3 antibody; (j) an antibody comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as CDRL2 and SEQ ID NO: 53 (QHHYAFPWT) as CDRL3; (k) the 26B3 antibody; (l) an antibody that binds to the same epitope as the 19D4 antibody; (m) an antibody comprising SEQ ID NO: 39 (HPYMH) as CDRH1, SEQ ID NO: 40 (RIDPANGNTKYDPKFQG) as CDRH2, SEQ ID NO: 41 (EEVADYTMDY) as CDRH3, SEQ ID NO: 35 (RASESVDTYGNNFIH) as CDRL1 SEQ ID NO: 36 (LASNLES) as CDRL2 and SEQ ID NO: 37 (QQNNGDPWT) as CDRL3; (n) the 19D4 antibody; (o) an antibody that binds to the same epitope as the 9F3 antibody; (p) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3; (q) the 9F3 antibody; (r) an antibody that binds to the same epitope as the 24F12 antibody; (s) an antibody comprising SEQ ID NO: 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 (ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3; (t) the 24F12 antibody; (u) an antibody comprising a light chain variable region selected from the group consisting of LK26HuVK (SEQ ID NO: 13); LK26HuVKY (SEQ ID NO: 14); LK26HuVKPW (SEQ ID NO: 15); and LK26HuVKPW, Y (SEQ ID NO: 16); (v) an antibody comprising a heavy chain variable region selected from the group consisting of LK26HuVH (SEQ ID NO: 17); LK26HuVH FAIS, N (SEQ ID NO: 18); LK26HuVH SLF (SEQ ID NO: 19); LK26HuVH I, I (SEQ ID NO: 20); and LK26KOLHuVH (SEQ ID NO: 21); (w) an antibody comprising the heavy chain variable region LK26KOLHuVH (SEQ ID NO: 21) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); (x) an antibody comprising the heavy chain variable region LK26HuVH SLF (SEQ ID NO: 19) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16); or (y) an antibody comprising the heavy chain variable region LK26HuVH FAIS, N (SEQ ID NO: 18) and the light chain variable region LK26HuVKPW, Y (SEQ ID NO: 16).
[0008]
8. Method according to claim 1, CHARACTERIZED by the fact that the antibody is a murine antibody, a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, Fab'2, ScFv, na- the body or an antibody in the domain.
[0009]
9. Method, according to claim 1, CHARACTERIZED by the fact that the antibody is labeled.
[0010]
10. Method according to claim 9, CHARACTERIZED by the fact that the antibody is labeled with a radio marker, a biotin marker, a chromophore marker, a fluorophore marker, an ECL marker or an enzyme marker.
[0011]
11. Method, according to claim 1, CHARACTERIZED by the fact that the level of FRα is determined using a technique that is western blot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, solution phase assay, immunoassay electrochemiluminescent (ECLIA) or ELISA assay.
[0012]
12. Method, according to claim 1, CHARACTERIZED by the fact that the control sample comprises a standardized level of control of FRα in a healthy subject.
[0013]
13. Method, according to claim 1, CHARACTERIZED by the fact that the urine sample derived from the subject is treated with guanidine before determining the level of FRα.
[0014]
14. Method, according to claim 1, CHARACTERIZED by the fact that the urine sample is diluted before determining the level of FRα.
[0015]
15. Method, according to claim 1, CHARACTERIZED by the fact that the urine sample is centrifuged, swirled, or both, before determining the level of FRα.
[0016]
16. Method, according to claim 1, CHARACTERIZED by the fact that the level of FRα in the urine sample derived from said subject is evaluated by placing the urine sample derived from said subject in contact with a pair of antibodies which are: ( a) MOV18 antibody immobilized on a solid support and labeled MORAB-003 antibody, (b) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 ( EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3 immobilized on a solid support and a labeled antibody comprising SEQ ID NO : 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 (ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3, or (c) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3 immobilized on a solid support and an antibody marked comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as CDRL2 and SEQ ID NO: 53 (QHHYAFPWT) as CDRL3.
[0017]
17. Method of evaluating the progression of ovarian cancer in a subject suffering from ovarian cancer, the method CHARACTERIZED by the fact that it comprises: putting a urine sample derived from said subject in contact with an antibody that binds to FRα , thereby determining the level of alpha folate receptor (FRα) that is not bound to a cell in said urine sample; comparing the level of alpha folate receptor (FRα) that is not bound to a cell in said urine sample derived from said subject with the level of FRα in a control sample; and (i) identify an increase in the level of FRα in the urine sample derived from said subject compared to the level of FRα in the control sample and determine that ovarian cancer will progress rapidly based on said increase; or (ii) identify a decrease in the level of FRα in the urine sample derived from said subject compared to the level of FRα in the control sample and determine that ovarian cancer will progress slowly based on said decrease.
[0018]
18. Method of stratifying a subject suffering from ovarian cancer into one of at least four cancer therapy groups, CHARACTERIZED by the fact that it comprises: placing a urine sample derived from said subject in contact with an antibody that binds to FRα , thereby determining the level of alpha folate receptor (FRα) that is not bound to a cell in said urine sample; comparing the level of alpha folate receptor (FRα) that is not bound to a cell in said urine sample derived from said subject with the level of FRα in a control sample; and stratifying the subject into one of at least four cancer therapy groups based on the level of alpha folate receptor (FRα) that is not bound to a cell.
[0019]
19. Method, according to claim 18, CHARACTERIZED by the fact that the subject is stratified in Stage I, Stage II, Stage III or Stage IV ovarian cancer.
[0020]
20. Method of monitoring the effectiveness of treatment with MORAb-003 for ovarian cancer in a subject suffering from ovarian cancer, the method CHARACTERIZED by the fact that it comprises: putting a urine sample derived from said subject in contact with an antibody that binds to FRα, thus determining the level of alpha folate receptor (FRα) that is not bound to a cell in said urine sample, in which said subject was previously administered with MORAb-003; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in said urine sample derived from said subject with the level of FRα in a control sample; and (i) identify an increase in the level of FRα in the urine sample derived from said subject compared with the level of FRα in the control sample and determine that treatment with MORAb-003 is not efficient; or (ii) identify a decrease in the level of FRα in the urine sample derived from said subject compared to the level of FRα in the control sample and determine that treatment with MORAb-003 is efficient.
[0021]
21. Method for predicting whether a subject suffering from ovarian cancer will respond to treatment with MORAb-003, the method CHARACTERIZED by the fact that it comprises: putting a urine sample derived from said subject in contact with an antibody that binds to FRα, thereby determining the level of alpha folate receptor (FRα) that is not bound to a cell in said urine sample; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in said urine sample derived from said subject with the level of FRα in a control sample; and identifying an increase in the level of FRα in the urine sample derived from said subject compared to the level of FRα in the control sample and determining that the subject will respond to treatment with MORAb-003.
[0022]
22. Method of assessing whether a subject is suffering from ovarian or lung cancer that expresses alpha folate receptor (FRα), the method CHARACTERIZED by the fact that it comprises: determining the level of FRα that is not bound to a cell in a serum or plasma sample derived from said subject; and compare the level of FRα that is not bound to a cell with the level of FRα in a control sample, identify an increase in the level of FRα in the serum or plasma sample derived from said subject compared to the level of FRα in the sample of control; and determine that the subject is suffering from ovarian cancer or lung cancer based on said increase, in which the level of FRα that is not bound to a cell in the serum or plasma sample derived from said subject is evaluated by placing the sample of serum or plasma in contact with a pair of antibodies that bind to FRα in a two-antibody sandwich assay, in which the two-antibody sandwich assay is an electrochemical chemiluminescent assay (ECLIA) or enzyme immunoassay (EIA).
[0023]
23. Method, according to claim 22, CHARACTERIZED by the fact that the subject is identified as suffering from ovarian cancer.
[0024]
24. Method, according to claim 22, CHARACTERIZED by the fact that the subject is identified as suffering from lung cancer.
[0025]
25. Method according to claim 24, CHARACTERIZED by the fact that lung cancer is non-small cell lung cancer.
[0026]
26. Method, according to claim 25, CHARACTERIZED by the fact that non-small cell lung cancer is adenocarcinoma.
[0027]
27. Method, according to claim 22, CHARACTERIZED by the fact that antibodies that bind to FRα are selected from the group consisting of: (a) an antibody that specifically binds to the same epitope as the MORAb-003 antibody; (b) an antibody comprising SEQ ID NO: 1 (GFTFSGYGLS) as CDRH1, SEQ ID NO: 2 (MISSGGSYTYYADSVKG) as CDRH2, SEQ ID NO: 3 (HGDDPAWFAY) as CDRH3, SEQ ID NO: 4 (SVSSSISSNNLH) as CDRLH1 SEQ ID NO: 5 (GTSNLAS) as CDRL2 and SEQ ID NO: 6 (QQWSSYPYMYT) as CDRL3; (c) the MOV18 antibody; (d) an antibody that specifically binds to the same epitope as the MOV18 antibody; (e) antibody 548908; (f) an antibody that specifically binds to the same epitope as the 548908 antibody; (g) the 6D398 antibody; (h) an antibody that specifically binds to the same epitope as the 6D398 antibody; (i) an antibody that specifically binds to the same epitope as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 58 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 54 ; (j) an antibody comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as CDRL2 and SEQ ID NO: 53 (QHHYAFPWT) as CDRL3; (k) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 58 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 54; (l) an antibody that specifically binds to the same epitope as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 42 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 ; (m) an antibody comprising SEQ ID NO: 39 (HPYMH) as CDRH1, SEQ ID NO: 40 (RIDPANGNTKYDPKFQG) as CDRH2, SEQ ID NO: 41 (EEVADYTMDY) as CDRH3, SEQ ID NO: 35 (RASESVDTYGNNFIH) as CDRL1 SEQ ID NO: 36 (LASNLES) as CDRL2 and SEQ ID NO: 37 (QQNNGDPWT) as CDRL3; (n) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 42 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 38; (o) an antibody that specifically binds to the same epitope as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 30 ; (p) an antibody comprising SEQ ID NO: 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) as CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3; (q) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 30; (r) an antibody that specifically binds to the same epitope as an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 50 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 46 ; (s) an antibody comprising SEQ ID NO: 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 (ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3; (t) an antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 50 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 46; (u) an antibody comprising a light chain variable region of SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; or SEQ ID NO: 16; (v) an antibody comprising a heavy chain variable region of SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; or SEQ ID NO: 21; (w) an antibody comprising the heavy chain variable region SEQ ID NO: 21 and the light chain variable region SEQ ID NO: 16; (x) an antibody comprising the heavy chain variable region SEQ ID NO: 19 and the light chain variable region SEQ ID NO: 16; and (y) an antibody comprising the heavy chain variable region SEQ ID NO: 18 and the light chain variable region SEQ ID NO: 16.
[0028]
28. Method according to claim 22, CHARACTERIZED by the fact that antibodies that bind to FRα are selected from the group consisting of a murine antibody, a human antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a Fab, Fab'2, ScFv, nanobody or an antibody in the domain.
[0029]
29. Method, according to claim 22, CHARACTERIZED by the fact that the level of FRα is determined by an electrochemical chemiluminescent immunoassay (ECLIA).
[0030]
30. Method, according to claim 22, CHARACTERIZED by the fact that the control sample comprises a standardized level of control of FRα in a healthy subject.
[0031]
31. Method according to claim 22, CHARACTERIZED by the fact that (i) the sample of serum or plasma derived from the subject is treated with guanidine before determining the level of FRα, (ii) the sample of serum or plasma derived of the subject is diluted before the determination of the level of FRα, and / or (iii) the serum or plasma sample derived from the subject is centrifuged, swirled, or both, before the determination of the level of FRα.
[0032]
32. Method according to claim 22, CHARACTERIZED by the fact that the antibody pair is selected from: (a) MOV18 antibody immobilized on a solid support and labeled MORAB-003 antibody, (b) an antibody comprising SEQ ID NO : 31 (SGYYWN) as CDRH1, SEQ ID NO: 32 (YIKSDGSNNYNPSLKN) as CDRH2, SEQ ID NO: 33 (EWKAMDY) as CDRH3, SEQ ID NO: 27 (RASSTVSYSYLH) as CDRL1, SEQ ID NO: 28 (GTSNLAS) CDRL2 and SEQ ID NO: 29 (QQYSGYPLT) as CDRL3 immobilized on a solid support and a labeled antibody comprising SEQ ID NO: 47 (SYAMS) as CDRH1, SEQ ID NO: 48 (EIGSGGSYTYYPDTVTG) as CDRH2, SEQ ID NO: 49 ( ETTAGYFDY) as CDRH3, SEQ ID NO: 43 (SASQGINNFLN) as CDRL1, SEQ ID NO: 44 (YTSSLHS) as CDRL2 and SEQ ID NO: 45 (QHFSKLPWT) as CDRL3, or (c) an antibody comprising SEQ ID NO: 55 (GYFMN) as CDRH1, SEQ ID NO: 56 (RIFPYNGDTFYNQKFKG) as CDRH2, SEQ ID NO: 57 (GTHYFDY) as CDRH3, SEQ ID NO: 51 (RTSENIFSYLA) as CDRL1, SEQ ID NO: 52 (NAKTLAE) as SEQ ID NO: 53 (QHHYAFPWT) as CDRL3 immobilized on a solid support and a labeled antibody comprising SEQ ID NO: 39 (HPYMH) as CDRH1, SEQ ID NO: 40 (RIDPANGNTKYDPKFQG) as CDRH2, SEQ ID NO: 41 (EEVADYTMDY) as CDRH3, SEQ ID NO: 35 (RASESVDTYGNNFIH) as CDRL1, SEQ ID NO: 36 (LASNLES) as CDRL2 and SEQ ID NO: 37 (QQNNGDPWT) as CDRL3.
[0033]
33. Method of evaluating the progression of ovarian cancer in a subject suffering from ovarian cancer, the method CHARACTERIZED by the fact that it comprises: determining the level of alpha folate receptor (FRα) that is not bound to a cell in a sample serum or plasma derived from said subject by means of a two antibody sandwich assay, wherein the two antibody sandwich assay is an electrochemiluminescent assay (ECLIA) or enzyme immunoassay (EIA); compare the level of alpha folate receptor (FRα) that is not bound to a cell with the level of FRα in a control sample, (i) identify an increase in the level of FRα in the serum or plasma sample derived from said subject compared to the level of FRα in the control sample and determining that ovarian cancer will progress rapidly based on said increase; or (ii) identify a decrease in the level of FRα in the serum or plasma sample derived from said subject compared to the level of FRα in the control sample and determine that ovarian cancer will progress slowly based on said decrease.
[0034]
34. Method of stratifying a subject suffering from ovarian cancer into one of at least four cancer therapy groups, CHARACTERIZED by the fact that it comprises: determining the level of alpha folate receptor (FRα) that is not bound to a cell in a serum or plasma sample derived from said subject by means of a two antibody sandwich assay, wherein the two antibody sandwich assay is an electrochemiluminescent assay (ECLIA) or enzyme immunoassay (EIA); and stratifying the subject into one of at least four cancer therapy groups based on the level of alpha folate receptor (FRα) that is not bound to a cell.
[0035]
35. Method, according to claim 34, CHARACTERIZED by the fact that the subject is stratified in Stage I, Stage II, Stage III or Stage IV ovarian cancer.
[0036]
36. Method of monitoring the effectiveness of treatment with MORAb-003 for ovarian cancer in a subject suffering from ovarian cancer, the method CHARACTERIZED by the fact that it comprises: determining the level of alpha folate receptor (FRα) that is not bound to a cell in a serum or plasma sample derived from said subject by means of a two-antibody sandwich assay, in which the two-antibody sandwich assay is an electrochemiluminescent assay (ECLIA) or enzyme immunoassay (EIA), in which the said subject was previously administered with MORAb-003; and comparing the level of alpha folate receptor (FRα) that is not bound to a cell in said serum or plasma sample derived from said subject with the level of FRα in a control sample; and (i) identify an increase in the level of FRα in the serum or plasma sample derived from said subject compared to the level of FRα in the control sample and determine that treatment with MORAb-003 is not efficient; or (ii) identify a decrease in the level of FRα in the serum or plasma sample derived from said subject compared to the level of FRα in the control sample and determine that treatment with MORAb-003 is efficient.
[0037]
37. Method to predict whether a subject suffering from ovarian cancer will respond to treatment with MORAb-003, the method CHARACTERIZED by the fact that it comprises: determining the level of alpha folate receptor (FRα) that is not bound to a cell in a serum or plasma sample derived from said subject; and compare the level of alpha folate receptor (FRα) that is not bound to a cell in the serum or plasma sample derived from said subject with the level of FRα in a control sample, identify an increase in the level of FRα in the serum sample or plasma derived from said subject compared to the level of FRα in the control sample and determining that the subject will respond to treatment with MORAb-003, wherein the level of FRα that is not bound to a cell in the serum or plasma sample derived the said subject is evaluated by placing the serum or plasma sample in contact with a pair of antibodies that bind to FRα in a two-antibody sandwich assay, in which the two-antibody sandwich assay is an electrochemical chemiluminescent assay (ECLIA) or enzyme immunoassay (EIA).
[0038]
38. Method according to claim 22, CHARACTERIZED by the fact that at least one of the antibodies that bind to FRα is labeled.
[0039]
39. Method according to claim 38, CHARACTERIZED by the fact that the marker is a radio marker, a biotin marker, a chromophore marker, a fluorophore marker, an ECL marker or an enzyme marker.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-06-19| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|

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2019-02-05| B25A| Requested transfer of rights approved|Owner name: EISAI INC. (US) |

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2019-07-30| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-12-08| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-02-02| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 04/11/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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