Methods for detecting human folate receptor 1 in patient samples - Patents.com

Abstract

The present invention relates generally to methods and kits for detecting in a sample human folate receptor 1. Peptides of human folate receptor 1 are further provided. [Selection diagram] None

Description

[Technical field]

[0001] The field of the invention relates generally to methods and kits for detecting human folate receptor 1 (FOLR1) in a sample. [Background technology]

[0002] Cancer is one of the leading causes of death in developed countries, with over one million people diagnosed with cancer annually in the United States alone, and half a million deaths. Overall, it is estimated that more than one in three people will develop some form of cancer in their lifetime. There are over 200 different types of cancer, with four accounting for more than half of all new cases: breast, lung, colorectal, and prostate (Jemal et al., 2003, Cancer J. Clin. 53:5-26).

[0003] Folate receptor 1 (FOLR1), also known as folate receptor alpha or folate binding protein, is an N-glycosylated protein expressed on the plasma membrane of cells. FOLR1 has high affinity for folate and several reduced folate derivatives. FOLR1 mediates the delivery of 5-methyltetrahydrofolate, the physiological folate, into cells.

[0004] FOLR1 is overexpressed in the majority of ovarian cancers, as well as many uterine, endometrial, pancreatic, renal, lung, and breast cancers, whereas expression of FOLR1 in normal tissues is restricted to the apical membrane of epithelial cells in renal proximal tubules, alveolar pneumocytes in the lung, bladder, testis, choroid plexus, and thyroid (Weitman SD, et al., Cancer Res 52:3396-3401(1992); Antony AC, Annu Rev Nutr 16:501-521(1996); Kalli KR, et al. Gynecol Oncol 108:619-626(2008)). This expression pattern of FOLR1 makes it a desirable target for cancer therapy directed against FOLR1.

[0005] Ovarian cancer is usually asymptomatic until advanced stages, and therefore is often diagnosed at a late stage and has a poor prognosis when treated with chemotherapy agents after conventional surgical debulking, which is the currently available approach (von Gruenigen V et al., Cancer 112:2221-2227(2008); Ayhan A et al., Am J Obstet Gynecol 196:81 e81-86(2007); Harry VN et al., Obstet Gynecol Surv 64:548-560(2009)). Thus, there is a clear unmet medical need for more effective treatments for ovarian cancer.

[0006] Some of the previous assays used to detect shed FOLR1 are not specific enough for FOLR1. For example, some assays do not distinguish between FOLR1 and other folate receptor family members (FOLR2, 3, and 4) or do not report values ​​for total FBP (folate binding protein). In addition, some assays require human samples (e.g., plasma) to be pretreated with a mild acid wash step to dissociate folate from the receptor. Some assay results may further have inaccuracies due to competitive effects between antibody therapeutics and diagnostic antibodies. Furthermore, many commercial kits are unreliable both in terms of their conventional reagents and their lot-to-lot stability. Evaluation of these kits has given very mixed results and is intended for research use only. In many cases, human samples need to be prediluted before analysis to reduce the possibility of false positives due to "matrix effects". Furthermore, many current assays, e.g., ELISA-based assays, do not provide sufficient sensitivity to distinguish between differences in shed FOLR1 at physiological levels and are limited by false positive results. Thus, there is a clear need for sensitive and accurate methods to detect FOLR1 in patient samples. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Jemal et al.,2003,CancerJ.Clin.53:5-26 [Non-Patent Document 2] Weitman SD, et al., Cancer Res 52:3396-3401(1992) [Non-Patent Document 3] Antony AC,Annu Rev Nutr 16:501-521(1996) [Non-Patent Document 4] Kalli KR, et al. Gynecol Oncol 108:619-626(2008) [Non-Patent Document 5] von Gruenigen V et al.,Cancer 112:2221-2227(2008) [Non-Patent Document 6] Ayhan A et al., Am J Obstet Gynecol 196:81 e81-86(2007) [Non-Patent Document 7] Harry VN et al., Obstet Gynecol Surv 64:548-560(2009)) Summary of the Invention [Means for solving the problem]

[0008] The present invention provides methods for detecting FOLR1 in a sample, which can be used, for example, to quantify the level of human FOLR1 in a sample.

[0009] In one embodiment, a method for detecting human folate receptor 1 (FOLR1) in a sample includes (a) capturing the folate receptor 1 (FOLR1) using an immunocapture reagent bound to a solid support, (b) eluting the FOLR1 from the solid support, (c) digesting the eluted FOLR1, and (d) performing liquid chromatography mass spectrometry (LC / MS) analysis on the digested FOLR1, wherein the FOLR1 is detected by monitoring chromatographic separation and mass spectrometry response of at least one signature FOLR1 peptide.

[0010] In one embodiment, the level of FOLR1 in the sample is quantified by LC / MS analysis. In one embodiment, the level of FOLR1 in the sample is quantified by comparing the level of FOLR1 in the sample with a reference level of FOLR1.

[0011] In one embodiment, the immunocapture reagent comprises an antibody or antigen-binding fragment that binds to FOLR1. In one embodiment, the binding of the antibody or antigen-binding fragment to FOLR1 is not competitively inhibited by the binding of IMGN853 to FOLR1. In one embodiment, the binding of the antibody or antigen-binding fragment to FOLR1 is not competitively inhibited by the binding of huMov19 to FOLR1. In one embodiment, the binding of the antibody or antigen-binding fragment to FOLR1 is not competitively inhibited by the binding of a second antibody or antigen-binding fragment to FOLR1, the second antibody or antigen-binding fragment comprising a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO:59, a VL CDR-2 of SEQ ID NO:60, a VL CDR-3 of SEQ ID NO:61, a variable heavy chain (VH) CDR-1 of SEQ ID NO:62, a VH CDR-2 of SEQ ID NO:64, and a VH CDR-3 of SEQ ID NO:65. In one embodiment, the binding of the antibody or antigen-binding fragment to FOLR1 is not competitively inhibited by the binding of a second antibody or antigen-binding fragment to FOLR1, the second antibody or antigen-binding fragment comprising a variable heavy chain (VH) having the sequence of SEQ ID NO: 56, and a variable light chain (VL) having the sequence of SEQ ID NO: 57 or SEQ ID NO: 58. In one embodiment, the binding of the antibody or antigen-binding fragment to FOLR1 is not competitively inhibited by the binding of a second antibody or antigen-binding fragment to FOLR1, the second antibody or antigen-binding fragment comprising (i) a heavy chain comprising the same amino acid sequence as the amino acid sequence of the heavy chain encoded by the plasmid deposited with the American Type Culture Collection (ATCC) as PTA-10772, and (ii) a light chain comprising the same amino acid sequence as the amino acid sequence of the light chain encoded by the plasmid deposited with the ATCC as PTA-10774. In one embodiment, the binding of the antibody or antigen-binding fragment to FOLR1 is not inhibited by the binding of folic acid to FOLR1.

[0012] In one embodiment, the antibody is muFR1-9. In one embodiment, the antibody is muFR1-13. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 1, a VH CDR-2 of SEQ ID NO: 2, a VH CDR-3 of SEQ ID NO: 3, a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 13, a VL CDR-2 of SEQ ID NO: 14, and a VL CDR-3 of SEQ ID NO: 15. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 4, a VH CDR-2 of SEQ ID NO: 5, a VH CDR-3 of SEQ ID NO: 6, a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 16, a VL CDR-2 of SEQ ID NO: 17, and a VL CDR-3 of SEQ ID NO: 18.

[0013] In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 90% identity with the sequence of SEQ ID NO:25, and a variable light chain (VL) having at least 90% identity with the sequence of SEQ ID NO:29. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 95% identity with the sequence of SEQ ID NO:25, and a variable light chain (VL) having at least 95% identity with the sequence of SEQ ID NO:29. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 90% identity with the sequence of SEQ ID NO:26, and a variable light chain (VL) having at least 90% identity with the sequence of SEQ ID NO:30. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 95% identity with the sequence of SEQ ID NO:26, and a variable light chain (VL) having at least 95% identity with the sequence of SEQ ID NO:30.

[0014] In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO: 25, and a variable light chain (VL) having the sequence of SEQ ID NO: 29. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO: 26, and a variable light chain (VL) having the sequence of SEQ ID NO: 30. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain having the sequence of SEQ ID NO: 33, and a light chain having the sequence of SEQ ID NO: 37. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain having the sequence of SEQ ID NO: 34, and a light chain having the sequence of SEQ ID NO: 38.

[0015] In one embodiment, the antibody or antigen-binding fragment binds to human FOLR1 with a Kd of about 1.0 nM to about 10 nM. In one embodiment, the antibody or antigen-binding fragment binds to human FOLR1 with a Kd of about 0.5 nM to about 5 nM.

[0016] In one embodiment, the solid support comprises a mass spectrometry immunoassay (MSIA) microcolumn. In one embodiment, the solid support comprises magnetic beads.

[0017] In one embodiment, at least one wash step is performed before eluting FOLR1 from the solid support. In one embodiment, two or more wash steps are performed before eluting FOLR1 from the solid support. In one embodiment, the wash step comprises contacting the solid support-bound FOLR1 with a wash buffer, a salt solution, and a detergent. In one embodiment, FOLR1 is eluted from the solid support using an acidic solution.

[0018] In one embodiment, FOLR1 is reduced and alkylated prior to digesting FOLR1. In one embodiment, FOLR1 is digested with trypsin / Lys-C. In one embodiment, digesting FOLR1 produces a peptide comprising the sequence of SEQ ID NO:42. In one embodiment, digesting FOLR1 produces a peptide comprising the sequence of SEQ ID NO:43. In one embodiment, digesting FOLR1 produces a peptide comprising the sequence of SEQ ID NO:44. In one embodiment, digesting FOLR1 produces a peptide comprising the sequence of SEQ ID NO:45.

[0019] In one embodiment, at least two signature peptides of FOLR1 are selected and monitored in the LC / MS analytical step. In one embodiment, at least three signature peptides of FOLR1 are selected and monitored in the LC / MS analytical step. In one embodiment, at least four signature peptides of FOLR1 are selected and monitored in the LC / MS analytical step.

[0020] In one embodiment, the signature peptides include (a) a peptide comprising the sequence of SEQ ID NO: 42, (b) a peptide comprising the sequence of SEQ ID NO: 43, (c) a peptide comprising the sequence of SEQ ID NO: 44, and (d) a peptide comprising the sequence of SEQ ID NO: 45.

[0021] In one embodiment, the sample comprises a bodily fluid. In one embodiment, the bodily fluid is plasma. In one embodiment, the bodily fluid is serum. In one embodiment, the bodily fluid is ascites.

[0022] In one embodiment, the sample comprises a peripheral blood sample.

[0023] In one embodiment, the sample is obtained from a patient with cancer. In one embodiment, the cancer is selected from the group consisting of ovarian, brain, breast, uterine, endometrial, pancreatic, renal, lung, and peritoneal cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, detecting FOLR1 is not inhibited by IMGN853 present in the sample. In one embodiment, detecting FOLR1 is not inhibited by huMov19 present in the sample.

[0024] In one embodiment, detecting FOLR1 is not inhibited by an antibody or antigen-binding fragment present in the sample, the antibody or antigen-binding fragment comprising a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 59, a VL CDR-2 of SEQ ID NO: 60, a VL CDR-3 of SEQ ID NO: 61, a variable heavy chain (VH) CDR-1 of SEQ ID NO: 62, a VH CDR-2 of SEQ ID NO: 64, and a VH CDR-3 of SEQ ID NO: 65. In one embodiment, detecting FOLR1 is not inhibited by an antibody or antigen-binding fragment present in the sample, the antibody or antigen-binding fragment comprising a variable heavy chain (VH) having the sequence of SEQ ID NO: 56, and a variable light chain (VL) having the sequence of SEQ ID NO: 57 or SEQ ID NO: 58. In one embodiment, detecting FOLR1 is not inhibited by an antibody or antigen-binding fragment present in the sample, which comprises (i) a heavy chain that comprises the same amino acid sequence as the amino acid sequence of a heavy chain encoded by the plasmid deposited with the American Type Culture Collection (ATCC) as PTA-10772, and (ii) a light chain that comprises the same amino acid sequence as the amino acid sequence of a light chain encoded by the plasmid deposited with the ATCC as PTA-10774.

[0025] In one embodiment, detecting FOLR1 is not inhibited by folic acid present in the sample.

[0026] In one embodiment, the method is capable of detecting at least 0.5 ng / mL of FOLR1 in a sample. In one embodiment, the method is capable of detecting at least 0.3 ng / mL of FOLR1 in a sample. In one embodiment, the method is capable of detecting at least 0.25 ng / mL of FOLR1 in a sample.

[0027] In one embodiment, the signal to noise ratio is at least 5. In one embodiment, the signal to noise ratio is at least 10.

[0028] In one embodiment, the FOLR1 is shed FOLR1.

[0029] The present invention further provides novel peptides of FOLR1. In one embodiment, the peptide consists of the sequence of SEQ ID NO: 42. In one embodiment, the peptide consists of the sequence of SEQ ID NO: 43. In one embodiment, the peptide consists of the sequence of SEQ ID NO: 44. In one embodiment, the peptide consists of the sequence of SEQ ID NO: 45.

[0030] The present invention further provides a kit for detecting FOLR1 in a sample. In one embodiment, the kit comprises an immunocapture reagent that binds to FOLR1, a digestion reagent, and at least one peptide selected from the group consisting of a) a peptide comprising the sequence of SEQ ID NO: 42, b) a peptide comprising the sequence of SEQ ID NO: 43, c) a peptide comprising the sequence of SEQ ID NO: 44, and d) a peptide comprising the sequence of SEQ ID NO: 45.

[0031] In one embodiment, the immunocapture reagent comprises an antibody or antigen-binding fragment that binds to FOLR1. In one embodiment, the antibody is muFR1-9. In one embodiment, the antibody is muFR1-13.

[0032] In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 1, a VH CDR-2 of SEQ ID NO: 2, a VH CDR-3 of SEQ ID NO: 3, a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 13, a VL CDR-2 of SEQ ID NO: 14, and a VL CDR-3 of SEQ ID NO: 15. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 4, a VH CDR-2 of SEQ ID NO: 5, a VH CDR-3 of SEQ ID NO: 6, a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 16, a VL CDR-2 of SEQ ID NO: 17, and a VL CDR-3 of SEQ ID NO: 18.

[0033] In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 90% identity with the sequence of SEQ ID NO:25, and a variable light chain (VL) having at least 90% identity with the sequence of SEQ ID NO:29. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 95% identity with the sequence of SEQ ID NO:25, and a variable light chain (VL) having at least 95% identity with the sequence of SEQ ID NO:29. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 90% identity with the sequence of SEQ ID NO:26, and a variable light chain (VL) having at least 90% identity with the sequence of SEQ ID NO:30. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 95% identity with the sequence of SEQ ID NO:26, and a variable light chain (VL) having at least 95% identity with the sequence of SEQ ID NO:30.

[0034] In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO: 25, and a variable light chain (VL) having the sequence of SEQ ID NO: 29. In one embodiment, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO: 26, and a variable light chain (VL) having the sequence of SEQ ID NO: 30.

[0035] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain having the sequence of SEQ ID NO: 33, and a light chain having the sequence of SEQ ID NO: 37. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain having the sequence of SEQ ID NO: 34, and a light chain having the sequence of SEQ ID NO: 38.

[0036] In one embodiment, the antibody or antigen-binding fragment binds to human FOLR1 with a Kd of about 1.0 nM to about 10 nM. In one embodiment, the antibody or antigen-binding fragment binds to human FOLR1 with a Kd of about 0.5 nM to about 5 nM. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for detecting human folate receptor 1 (FOLR1) in a sample, comprising: (a) capturing said folate receptor 1 (FOLR1) with an immunocapture reagent bound to a solid support; (b) eluting FOLR1 from the solid support; and (c) digesting the eluted FOLR1; and (d) performing liquid chromatography mass spectrometry (LC / MS) analysis on the digested FOLR1, wherein the FOLR1 is detected by monitoring chromatographic separation and mass spectrometry response of at least one signature FOLR1 peptide. (Item 2) 2. The method of claim 1, wherein the level of FOLR1 in the sample is quantified by LC / MS analysis. (Item 3) 3. The method of claim 2, wherein the level of FOLR1 in the sample is quantified by comparing the level of FOLR1 in the sample to a reference level of FOLR1. (Item 4) 4. The method according to any one of items 1 to 3, wherein the immunocapture reagent comprises an antibody or antigen-binding fragment that binds to FOLR1. (Item 5) The method of claim 4, wherein the binding of the antibody or antigen-binding fragment to FOLR1 is not competitively inhibited by the binding of IMGN853 to FOLR1. (Item 6) The method of claim 4, wherein the binding of the antibody or antigen-binding fragment to FOLR1 is not competitively inhibited by the binding of huMov19 to FOLR1. (Item 7) 5. The method of claim 4, wherein the binding of the antibody or antigen-binding fragment to FOLR1 is not competitively inhibited by the binding of a second antibody or antigen-binding fragment to FOLR1, and the second antibody or antigen-binding fragment comprises a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 59, a VL CDR-2 of SEQ ID NO: 60, a VL CDR-3 of SEQ ID NO: 61, a variable heavy chain (VH) CDR-1 of SEQ ID NO: 62, a VH CDR-2 of SEQ ID NO: 64, and a VH CDR-3 of SEQ ID NO: 65. (Item 8) The method of claim 4, wherein the binding of the antibody or antigen-binding fragment to FOLR1 is not competitively inhibited by the binding of a second antibody or antigen-binding fragment to FOLR1, and the second antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO: 56 and a variable light chain (VL) having the sequence of SEQ ID NO: 57 or SEQ ID NO: 58. (Item 9) The method of claim 4, wherein the binding of the antibody or antigen-binding fragment to FOLR1 is not competitively inhibited by the binding of a second antibody or antigen-binding fragment to FOLR1, and the second antibody or antigen-binding fragment comprises (i) a heavy chain that comprises the same amino acid sequence as the heavy chain encoded by the plasmid deposited with the American Type Culture Collection (ATCC) as PTA-10772, and (ii) a light chain that comprises the same amino acid sequence as the light chain encoded by the plasmid deposited with the ATCC as PTA-10774. (Item 10) 5. The method of claim 4, wherein the binding of the antibody or antigen-binding fragment to FOLR1 is not inhibited by the binding of folic acid to FOLR1. (Item 11) The method of claim 4, wherein the antibody is muFR1-9. (Item 12) The method of claim 4, wherein the antibody is muFR1-13. (Item 13) 5. The method of claim 4, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 1, a VH CDR-2 of SEQ ID NO: 2, a VH CDR-3 of SEQ ID NO: 3, a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 13, a VL CDR-2 of SEQ ID NO: 14, and a VL CDR-3 of SEQ ID NO: 15. (Item 14) 5. The method of claim 4, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 4, a VH CDR-2 of SEQ ID NO: 5, a VH CDR-3 of SEQ ID NO: 6, a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 16, a VL CDR-2 of SEQ ID NO: 17, and a VL CDR-3 of SEQ ID NO: 18. (Item 15) 5. The method of claim 4, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 90% identity to the sequence of SEQ ID NO: 25 and a variable light chain (VL) having at least 90% identity to the sequence of SEQ ID NO: 29. (Item 16) 5. The method of claim 4, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 95% identity to the sequence of SEQ ID NO: 25 and a variable light chain (VL) having at least 95% identity to the sequence of SEQ ID NO: 29. (Item 17) 5. The method of claim 4, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 90% identity to the sequence of SEQ ID NO: 26 and a variable light chain (VL) having at least 90% identity to the sequence of SEQ ID NO: 30. (Item 18) 5. The method of claim 4, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 95% identity to the sequence of SEQ ID NO: 26 and a variable light chain (VL) having at least 95% identity to the sequence of SEQ ID NO: 30. (Item 19) 5. The method of claim 4, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO: 25 and a variable light chain (VL) having the sequence of SEQ ID NO: 29. (Item 20) 5. The method of claim 4, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO: 26 and a variable light chain (VL) having the sequence of SEQ ID NO: 30. (Item 21) 5. The method of claim 4, wherein the antibody or antigen-binding fragment comprises a heavy chain having the sequence of SEQ ID NO: 33 and a light chain having the sequence of SEQ ID NO: 37. (Item 22) 5. The method of claim 4, wherein the antibody or antigen-binding fragment comprises a heavy chain having the sequence of SEQ ID NO: 34 and a light chain having the sequence of SEQ ID NO: 38. (Item 23) 5. The method of claim 4, wherein the antibody or antigen-binding fragment binds to human FOLR1 with a Kd of about 1.0 nM to about 10 nM. (Item 24) 5. The method of claim 4, wherein the antibody or antigen-binding fragment binds to human FOLR1 with a Kd of about 0.5 nM to about 5 nM. (Item 25) 25. The method of any one of items 1 to 24, wherein the solid support comprises a mass spectrometry immunoassay (MSIA) microcolumn. (Item 26) 25. The method according to any one of items 1 to 24, wherein the solid support comprises magnetic beads. (Item 27) 27. The method according to any one of items 1 to 26, wherein at least one washing step is performed before eluting FOLR1 from the solid support. (Item 28) 28. The method of claim 27, wherein two or more washing steps are performed before eluting FOLR1 from the solid support. (Item 29) 29. The method of claim 27 or 28, wherein the washing step comprises contacting the FOLR1 bound to the solid support with a wash buffer, a salt solution, and a detergent. (Item 30) 30. The method according to any one of items 1 to 29, wherein FOLR1 is eluted from the solid support using an acidic solution. (Item 31) 31. The method of claim 30, wherein the FOLR1 is reduced and alkylated prior to digesting the FOLR1. (Item 32) 32. The method according to any one of items 1 to 31, wherein FOLR1 is digested with trypsin / Lys-C. (Item 33) 33. The method according to any one of items 1 to 32, wherein a peptide comprising the sequence of SEQ ID NO: 42 is generated by digesting the FOLR1. (Item 34) 34. The method according to any one of items 1 to 33, wherein a peptide comprising the sequence of SEQ ID NO: 43 is generated by digesting the FOLR1. (Item 35) 35. The method according to any one of items 1 to 34, wherein a peptide comprising the sequence of SEQ ID NO: 44 is generated by digesting the FOLR1. (Item 36) 36. The method according to any one of items 1 to 35, wherein a peptide comprising the sequence of SEQ ID NO: 45 is generated by digesting the FOLR1. (Item 37) 37. The method according to any one of items 1 to 36, wherein at least two signature peptides of FOLR1 are selected and monitored in the LC / MS analysis step. (Item 38) 38. The method according to any one of items 1 to 37, wherein at least three signature peptides of FOLR1 are selected and monitored in the LC / MS analysis step. (Item 39) 39. The method according to any one of items 1 to 38, wherein at least four signature peptides of FOLR1 are selected and monitored in the LC / MS analysis step. (Item 40) The signature peptide is (a) a peptide comprising the sequence of SEQ ID NO: 42; (b) a peptide comprising the sequence of SEQ ID NO: 43; (c) a peptide comprising the sequence of SEQ ID NO: 44, and (d) The method according to item 39, comprising a peptide comprising the sequence of SEQ ID NO: 45. (Item 41) 41. The method according to any one of items 1 to 40, wherein the sample comprises a body fluid. (Item 42) 42. The method of claim 41, wherein the body fluid is plasma. (Item 43) 42. The method of claim 41, wherein the body fluid is serum. (Item 44) 42. The method of claim 41, wherein the body fluid is ascites. (Item 45) 41. The method according to any one of items 1 to 40, wherein the sample comprises a peripheral blood sample. (Item 46) 46. ​​The method according to any one of items 1 to 45, wherein the sample is obtained from a patient having cancer. (Item 47) 47. The method of claim 46, wherein the cancer is selected from the group consisting of ovarian, brain, breast, uterine, endometrial, pancreatic, renal, lung, and peritoneal cancer. (Item 48) 48. The method of claim 47, wherein the cancer is ovarian cancer. (Item 49) 49. The method according to any one of items 1 to 48, wherein detecting FOLR1 is not inhibited by IMGN853 present in the sample. (Item 50) 49. The method according to any one of items 1 to 48, wherein detecting FOLR1 is not inhibited by huMov19 present in the sample. (Item 51) 49. The method according to any one of items 1 to 48, wherein detecting FOLR1 is not inhibited by an antibody or antigen-binding fragment present in the sample, and wherein the antibody or antigen-binding fragment comprises a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 59, a VL CDR-2 of SEQ ID NO: 60, a VL CDR-3 of SEQ ID NO: 61, a variable heavy chain (VH) CDR-1 of SEQ ID NO: 62, a VH CDR-2 of SEQ ID NO: 64, and a VH CDR-3 of SEQ ID NO: 65. (Item 52) 49. The method according to any one of items 1 to 48, wherein detecting FOLR1 is not inhibited by an antibody or antigen-binding fragment present in the sample, and wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO: 56, and a variable light chain (VL) having the sequence of SEQ ID NO: 57 or SEQ ID NO: 58. (Item 53) 49. The method of any one of items 1 to 48, wherein detecting FOLR1 is not inhibited by an antibody or antigen-binding fragment present in the sample, and the antibody or antigen-binding fragment comprises (i) a heavy chain that comprises an amino acid sequence identical to the amino acid sequence of a heavy chain encoded by a plasmid deposited with the American Type Culture Collection (ATCC) as PTA-10772, and (ii) a light chain that comprises an amino acid sequence identical to the amino acid sequence of a light chain encoded by a plasmid deposited with the ATCC as PTA-10774. (Item 54) 54. The method according to any one of items 1 to 53, wherein detecting FOLR1 is not inhibited by folic acid present in the sample. (Item 55) 55. The method according to any one of items 1 to 54, wherein at least 0.5 ng / mL of FOLR1 in a sample can be detected. (Item 56) 55. The method according to any one of items 1 to 54, wherein at least 0.3 ng / mL of FOLR1 in a sample can be detected. (Item 57) 55. The method according to any one of items 1 to 54, wherein at least 0.25 ng / mL of FOLR1 in a sample can be detected. (Item 58) 58. The method according to any one of items 1 to 57, wherein the signal to noise ratio is at least 5. (Item 59) 58. The method according to any one of items 1 to 57, wherein the signal to noise ratio is at least 10. (Item 60) 60. The method of any one of items 1 to 59, wherein the FOLR1 is shed FOLR1. (Item 61) A peptide consisting of the sequence of SEQ ID NO:42. (Item 62) A peptide consisting of the sequence of SEQ ID NO:43. (Item 63) A peptide consisting of the sequence of SEQ ID NO:44. (Item 64) A peptide consisting of the sequence of SEQ ID NO:45. (Item 65) Immunocapture reagents that bind to FOLR1, digestion reagents, and (a) a peptide comprising the sequence of SEQ ID NO: 42; (b) a peptide comprising the sequence of SEQ ID NO: 43; (c) a peptide comprising the sequence of SEQ ID NO: 44, and (d) at least one peptide selected from the group consisting of peptides comprising the sequence of SEQ ID NO: 45. (Item 66) 66. The kit of claim 65, wherein the immunocapture reagent comprises an antibody or antigen-binding fragment that binds to FOLR1. (Item 67) 67. The kit of claim 66, wherein the antibody is muFR1-9. (Item 68) 67. The kit of claim 66, wherein the antibody is muFR1-13. (Item 69) 67. The kit of item 66, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 1, a VH CDR-2 of SEQ ID NO: 2, a VH CDR-3 of SEQ ID NO: 3, a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 13, a VL CDR-2 of SEQ ID NO: 14, and a VL CDR-3 of SEQ ID NO: 15. (Item 70) 67. The kit of claim 66, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) complementarity determining region (CDR)-1 of SEQ ID NO:4, a VH CDR-2 of SEQ ID NO:5, a VH CDR-3 of SEQ ID NO:6, a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO:16, a VL CDR-2 of SEQ ID NO:17, and a VL CDR-3 of SEQ ID NO:18. (Item 71) 67. The kit of item 66, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 90% identity to the sequence of SEQ ID NO: 25 and a variable light chain (VL) having at least 90% identity to the sequence of SEQ ID NO: 29. (Item 72) 67. The kit of item 66, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 95% identity to the sequence of SEQ ID NO: 25 and a variable light chain (VL) having at least 95% identity to the sequence of SEQ ID NO: 29. (Item 73) 67. The kit of item 66, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 90% identity to the sequence of SEQ ID NO: 26 and a variable light chain (VL) having at least 90% identity to the sequence of SEQ ID NO: 30. (Item 74) 67. The kit of item 66, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having at least 95% identity to the sequence of SEQ ID NO: 26 and a variable light chain (VL) having at least 95% identity to the sequence of SEQ ID NO: 30. (Item 75) 67. The kit of item 66, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO: 25 and a variable light chain (VL) having the sequence of SEQ ID NO: 29. (Item 76) 67. The kit of item 66, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO: 26 and a variable light chain (VL) having the sequence of SEQ ID NO: 30. (Item 77) 67. The kit of item 66, wherein the antibody or antigen-binding fragment comprises a heavy chain having the sequence of SEQ ID NO: 33 and a light chain having the sequence of SEQ ID NO: 37. (Item 78) 67. The kit of item 66, wherein the antibody or antigen-binding fragment comprises a heavy chain having the sequence of SEQ ID NO: 34 and a light chain having the sequence of SEQ ID NO: 38. (Item 79) 67. The kit of item 66, wherein the antibody or antigen-binding fragment binds to human FOLR1 with a Kd of about 1.0 nM to about 10 nM. (Item 80) 67. The kit of item 66, wherein the antibody or antigen-binding fragment binds to human FOLR1 with a Kd of about 0.5 nM to about 5 nM. [Brief description of the drawings]

[0037] [Figure 1] Schematic diagram of FOLR1 immunocapture-LC / MS assay. [Diagram 2] 1 shows an ion chromatogram extracted from a sample containing low levels of soluble FOLR1 in an alternative matrix at 0.3 ng / mL. [Diagram 3]1 shows an extracted ion chromatogram from a normal human plasma sample. [Figure 4] 1 shows an ion chromatogram extracted from a pre-dose sample of a patient with increased soluble FOLR1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The present invention provides a novel method for detecting human folate receptor 1 (FOLR1) in patient samples. An initial immunocapture step of FOLR1 is performed, followed by digestion of the captured FOLR1 into peptides and quantitative analysis of the peptides via liquid chromatography mass spectrometry (LC / MS). Antibodies that do not competitively inhibit the binding of anti-FOLR1 active agents (e.g., active agents including antibody huMov19, such as IMGN853) to FOLR1 are useful in the immunocapture step of the present invention. Antibodies that do not competitively inhibit the binding of anti-FOLR1 active agents are particularly useful for capturing FOLR1 (e.g., shed FOLR1) in samples from patients treated with anti-FOLR1 active agents. Further disclosed are peptides of FOLR1 produced by the methods of the present invention. Such peptides are useful in detecting FOLR1 levels in patient samples by LC / MS. Further provided are kits that include an agent that binds to FOLR1 and a FOLR1 peptide.

[0039] I. Definition To facilitate the understanding of this invention, several terms and phrases are defined below.

[0040] The terms "human folate receptor 1", "FOLR1", or "folate receptor alpha (FR-α)" as used herein refer to any naturally occurring human FOLR1 unless otherwise specified. Thus, all of these terms can refer to any of the proteins or nucleic acid sequences set forth herein. The term "FOLR1" encompasses "full-length" unprocessed FOLR1 as well as any form of FOLR1 that is processed within a cell. The term further encompasses naturally occurring variants of FOLR1, such as splice variants (excluding those variants that include FOLR2, FOLR3, or FOLR4), allelic variants, and isoforms. The FOLR1 polypeptides described herein can be isolated from various sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Examples of FOLR1 sequences include, but are not limited to, NCBI reference numbers P15328, NP_001092242.1, AAX29268.1, AAX37119.1, NP_057937.1, and NP_057936.1. The human FOLR1 sequence is as follows: MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS (sequence number 41).

[0041] The terms "shed antigen" and "shed FOLR1" ("sFOLR1" or "sFRα") are used interchangeably herein. These terms refer to FOLR1 protein that is soluble and not associated with cells. In some embodiments, it includes an extracellular domain (ECD) and a glycosylphosphatidylinositol (GPI) linker. In one embodiment, shed FOLR1 includes only the ECD. FOLR1 includes a signal peptide (amino acids 1-24), the FOLR1 protein chain (amino acids 25-233 or 234), and a cleavable propeptide (amino acids 235-257). Shed FOLR can include amino acids 1-257, 1-233, 1-234, 25-233, 25-234, or any other fragment thereof. In some embodiments, the signal sequence is cleaved. In other embodiments, the ECD and GPI portions can be embedded in a membrane (e.g., a soluble lipid raft). In one embodiment, the shed FOLR1 can include amino acids 1-233 or a fragment thereof.

[0042] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the above, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antibody, and any other modified immunoglobulin molecule, so long as the antibody exhibits the desired biological activity. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or can be conjugated to other molecules, such as toxins, radioisotopes, etc.

[0043] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can include the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies.

[0044] The term "anti-FOLR1 antibody" or "antibody that binds FOLR1" refers to an antibody capable of binding to FOLR1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting FOLR1 (e.g., the huMov19(M9346A) antibody). The extent of binding of an anti-FOLR1 antibody to an unrelated, non-FOLR1 protein may be less than about 10% of the binding of the antibody to FOLR1 as measured, for example, by radioimmunoassay (RIA).

[0045] The term "IMGN853" (also known as mirvetuximab soravtansine) refers to an immunoconjugate described herein that includes the huMov19(M9346A) antibody, a sulfoSPDB linker, and a DM4 maytansinoid. The polypeptide of SEQ ID NOs:56-58 includes the variable domain of the heavy chain of huMov19(M9346A), the variable domain light chain version 1.00, and the variable domain light chain version 1.60 of huMov19, respectively. In a specific embodiment, the huMov19(M9346A) antibody is an anti-FOLR1 antibody that includes the variable heavy chain sequence SEQ ID NO:56 and the variable light chain sequence SEQ ID NO:58 (huMov19 version 1.60). DM4 refers to N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl) maytansine. "SulfoSPDB" refers to N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate) linker. In certain embodiments, the huMov19(M9346A) antibody is encoded by a plasmid deposited with the American Type Culture Collection (ATCC), under the terms of the Budapest Treaty, on April 7, 2010, located at 10801 University Boulevard, Manassas, VA 20110, and has ATCC deposit numbers PTA-10772 and PTA-10773 or 10774. The following sequence information provides the complementarity determining regions (CDRs), variable heavy and light chains, and complete heavy and light chains of huMov19: huMov19 variable heavy chain (SEQ ID NO:56) QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGTTVTVSS huMov19 variable light chain version 1.00 (SEQ ID NO:57) DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR huMov19 variable light chain version 1.60 (SEQ ID NO:58) DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLEIKR huMov19 variable light chain CDR1 (SEQ ID NO:59) KASQSVSFAGTSLMH huMov19 variable light chain CDR2 (SEQ ID NO:60) RASNLEA huMov19 variable light chain CDR3 (SEQ ID NO:61) QQSREYPYT huMov19 variable heavy chain CDR1 (SEQ ID NO:62) GYFMN huMov19 variable heavy chain CDR2 - Kabat definition (SEQ ID NO:63) RIHPYDGDTFYNQKFQG huMov19 variable heavy chain CDR2-Abm definition (SEQ ID NO:64) RIHPYDGDTF huMov19 variable heavy chain CDR3 (SEQ ID NO:65) YDG SRAMDY huMov19 heavy chain amino acid sequence (SEQ ID NO:66) QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK huMov19 light chain version 1.00 amino acid sequence (SEQ ID NO:67) DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC huMov19 light chain version 1.60 amino acid sequence (SEQ ID NO:68) DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0046] As used herein, the term "immunoconjugate" or "conjugate" refers to a compound or derivative thereof linked to a cell-binding agent (i.e., an anti-FOLR1 antibody or fragment thereof) and defined by the general formula: CLA, where C=cytotoxin, L=linker, and A=antibody or antigen-binding fragment thereof, e.g., an anti-FOLR1 antibody or antibody fragment. An immune conjugate can also be defined by the general formula in the reverse order: ALC.

[0047] A "linker" is any chemical moiety capable of attaching a compound, usually a drug (such as a maytansinoid), to a cell-binding agent (such as an anti-FOLR1 antibody or fragment thereof) in a stable covalent manner. The linker can be, for example, sensitive or substantially resistant to disulfide bond cleavage under conditions under which the compound or antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups and thioether groups.

[0048] A "monoclonal" antibody or antigen-binding fragment thereof refers to a homogeneous antibody or antigen-binding fragment population involved in highly specific recognition and binding at a single antigenic determinant or epitope. This is in contrast to a polyclonal antibody, which usually contains different antibodies directed to different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses both intact, full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) variants, fusion proteins containing antibody portions, and other modified immunoglobulin molecules that contain an antigen recognition site. Furthermore, a "monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof produced in any number of ways, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0049] The term "humanized" antibody or antigen-binding fragment thereof refers to a form of a non-human (e.g., murine) antibody or antigen-binding fragment that is a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof that contains minimal non-human (e.g., murine) sequence. Typically, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin in which residues from a complementarity determining region (CDR) are replaced with residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that has the desired specificity, affinity, and capacity ("CDR grafted") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, Fv framework region (FR) residues of a human immunoglobulin are replaced with corresponding residues of an antibody or fragment from a non-human species having the desired specificity, affinity, and capacity. The humanized antibody or antigen-binding fragment thereof can be further modified by substitution of any additional residues in the Fv framework regions and / or within the replaced non-human residues to refine and optimize the specificity, affinity, and / or capacity of the antibody or antigen-binding fragment. In general, a humanized antibody or antigen-binding fragment thereof will comprise substantially all of at least one, and typically two or three, variable domains, including all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody or antigen-binding fragment thereof can further comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Protein Eng. 9(10):895-904 (1996). In some embodiments, a "humanized antibody" is a resurfaced antibody.

[0050] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two approaches to determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.), "Kabat"), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., J. Molec. Biol. 273:927-948 (1997)). Moreover, a combination of these two approaches is sometimes used in the art to determine CDRs.

[0051] When referring to residues in variable domains (approximately residues 1-107 for the light chain and residues 1-113 for the heavy chain), the Kabat numbering system is commonly used (e.g., Kabat et al., Sequences of Immunological Interest. (5th Ed., 1991, National Institutes of Health, Bethesda, Md.) ("Kabat").

[0052] The numbering of amino acid positions as Kabat refers to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al. (Sequences of Immunological Interest. (5th Ed., 1991, National Institutes of Health, Bethesda, Md.), "Kabat"). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (residues 82a, 82b, 82c, etc. according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the homology regions of the antibody's sequence with the "standard" Kabat numbered sequence. Cothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. [Table A]

[0053] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequences are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to the sequences of an antibody or antigen-binding fragment derived from another (usually human) species to avoid eliciting an immune response in that species.

[0054] The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen that can be recognized and specifically bound by a particular antibody. When an antigen is a polypeptide, epitopes can be formed both from contiguous and non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are usually retained upon denaturation of a protein, whereas epitopes formed by tertiary folding are usually lost upon denaturation of a protein. An epitope typically comprises at least 3, more commonly at least 5, or 8-10 amino acids in a unique spatial conformation.

[0055] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for a partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. In general, low affinity antibodies bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Certain exemplary embodiments are described herein.

[0056] "Or better" as used herein to refer to binding affinity refers to stronger binding between a molecule and its binding partner. "Better" as used herein refers to stronger binding, represented by a smaller numerical Kd value. For example, an antibody may have an affinity for an antigen of "0.6 nM or better," where the affinity of the antibody for the antigen is <0.6 nM, i.e., 0.59 nM, 0.58 nM, 0.57 nM, etc., or any value less than 0.6 nM. In one embodiment, the affinity of the antibody, as determined by Kd, is about 10 -3 ~about 10 -12 M, about 10 -6 ~about 10 -11 M, about 10 -6 ~about 10 -10 M, about 10 -6 ~about 10 -9 M, about 10 -6 ~about 10 -8 M, or about 10 -6 ~about 10 -7 It must be M.

[0057] An antibody is said to "competitively inhibit" the binding of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks the binding of the reference antibody to that epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as, for example, competitive ELISA assay. An antibody can be said to competitively inhibit the binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0058] The phrases "substantially similar" or "substantially the same" as used herein indicate a sufficiently high degree of similarity between two numerical values ​​(typically one associated with an antibody of the invention and the other associated with a reference / comparator antibody) such that one of skill in the art would consider the difference between the two values ​​to be of low or no biological and / or statistical significance in the context of the biological property measured by the values ​​(e.g., Kd values). The difference between the two values ​​is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value for the reference / comparator antibody.

[0059] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0060] As used herein, "substantially pure" refers to a material that is at least 50% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (i.e., free from contaminants).

[0061] The term "increased expression" of FOLR1 refers to a sample that contains an increased FOLR1 expression level compared to a reference sample, a reference FOLR1 level, or a previous FOLR1 level detected from the same control.Thus, for example, an "increased FOLR1 protein level" in a patient sample can have a FOLR1 protein level that is higher than the FOLR1 protein level in a non-cancerous reference sample.An "increased FOLR1 protein level" in a patient sample can also have a FOLR1 protein level that is equal to the FOLR1 protein level in a cancerous sample, for example.

[0062] A "reference sample" can be used to correlate and compare results obtained in the method of the present invention from a test sample. A reference sample can be a cell (e.g., a cell line, a cell pellet), a body fluid, or a tissue. The FOLR1 level in a "reference sample" can be an absolute or relative amount, a range of amounts, a minimum and / or maximum amount, an average amount, and / or a median amount of FOLR1. A "reference sample" also serves as a standard of FOLR1 expression to be compared with a test sample. A "reference sample" can include a previous sample or a reference sample from the same patient, a normal reference, or a reference from a related patient population. In general, FOLR1 levels are expressed as values ​​of a standard curve. A standard curve is a quantitative method of plotting assay data to determine the concentration of FOLR1 in a sample. In one embodiment, the reference sample is an antigen standard purified FOLR1 or FOLR1-Fc. The detection method disclosed herein can involve a comparison of the expression level of FOLR1 in a test sample to a "reference value" or "reference level". In some embodiments, the reference value is the expression level of FOLR1 in a reference sample. The reference value may be a predetermined value and may be determined from a reference sample (e.g., a control biological sample) tested in parallel with the test sample. The reference value may be a single cut-off value, such as a median or mean value, or a range of values, such as a confidence interval. Reference values ​​may be established for various subgroups of individuals, such as individuals with a predisposition to cancer, individuals with early or late stage cancer, male and / or female individuals, or individuals undergoing cancer treatment. Examples of normal reference samples or values ​​and positive reference samples or values ​​are described herein.

[0063] A "sample" or "biological sample" of the present invention is of biological origin, and in certain embodiments, is from, for example, a eukaryotic organism. In a preferred embodiment, the sample is a human sample, but animal samples can also be used in the practice of the present invention. Non-limiting sources of samples for use in the present invention include, but are not limited to, for example, solid tissue, biopsy aspirate, ascites, fluid extract, blood, plasma, serum, cerebrospinal fluid, lymphatic fluid, external section of the skin, respiratory tract, intestinal tract, and genitourinary tract, tears, saliva, milk, tumor, organ, cell culture, and / or cell culture components. The present invention is particularly useful for cancer samples that typically contain bodily fluids such as ascites, where there is a small amount of material available. The method can be used to test aspects of FOLR1 expression or status of a sample, including comparing different types of cells or tissues, comparing different developmental stages, and detecting or determining the presence and / or type of disease or abnormality.

[0064] As used herein, the term "capture reagent" refers to a reagent that, under suitable conditions, is capable of binding to and capturing a target molecule in a sample such that the capture reagent-target molecule complex can be separated from the remainder of the sample. The term "immunocapture reagent" refers to an immunological reagent that, under suitable conditions, is capable of binding to and capturing a target molecule in a sample such that the capture reagent-target molecule complex can be separated from the remainder of the sample. In one embodiment, the immunocapture reagent is an antibody or an antigen-binding fragment. In one embodiment, the capture reagent or immunocapture reagent is immobilized. In one embodiment, the capture reagent or immunocapture reagent is immobilized on a solid support.

[0065] As used herein, the term "detectable antibody" refers to an antibody that can be detected directly, via a label that is amplified by a detection means, or indirectly, for example, via another antibody that is labeled. For direct labeling, the antibody is typically conjugated to a moiety that is detectable by some means. In one embodiment, the detectable antibody is a biotinylated antibody.

[0066] The word "label," as used herein, refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a "labeled" antibody. The label can be detectable in its own right (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze chemical alteration of a substrate compound or composition that is detectable.

[0067] "Correlate" or "correlating" means comparing in any manner the performance and / or results of a first analysis with the performance and / or results of a second analysis, e.g., the results of the first analysis may be used in performing the second analysis, the results of the first analysis may be used to determine whether to perform the second analysis, and / or the results of the first analysis may be compared with the results of the second analysis.

[0068] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals in which a population of cells is characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancer include squamous cell carcinoma of the lung, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. The cancer may be a cancer that expresses FOLR1.

[0069] "Tumor" and "neoplasm" refer to any mass of tissue resulting from the growth or proliferation of excessive cells, either benign (non-cancerous) or malignant (cancerous), including precancerous lesions.

[0070] The terms "cancer cells", "tumor cells" and grammatical equivalents refer to the entire cell population derived from a tumor or precancerous lesion, including both non-tumorigenic cells, which comprise the majority of the tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cells" will be modified by the term "non-tumorigenic" to simply refer to those tumor cells that lack the ability to reproduce and differentiate, and to distinguish them from cancer stem cells.

[0071] The term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to human subjects.

[0072] The term "pharmaceutical formulation" refers to a preparation that is in a form that effectively activates the biological activity of the active ingredient and does not contain additional ingredients that are unacceptably toxic to a subject to which the formulation may be administered. Such formulations may be sterile.

[0073] An "effective amount" of an antibody as disclosed herein is an amount sufficient to carry out a specifically mentioned purpose. An "effective amount" can be determined empirically and in a routine manner in relation to the stated purpose.

[0074] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art, are also included within the definition. Because the polypeptides of the invention are based on antibodies, it is understood that in certain embodiments, the polypeptides can exist as single chains or associated chains. In some embodiments, the polypeptides, peptides, or proteins are not naturally occurring. In some embodiments, the polypeptides, peptides, or proteins are purified from other naturally occurring components. In some embodiments, the polypeptides, peptides, or proteins are recombinantly produced.

[0075] The term "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are the same, or that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (with gaps introduced, if necessary) for maximum correspondence, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certain embodiments, gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software (e.g., using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6).In certain alternative embodiments, the percent identity between two amino acid sequences can be determined using the GAP program in the GCG software package incorporating the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) (e.g., using a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4: 11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using PAM120 with a residue table, a gap length penalty of 12, and a gap penalty of 4. Appropriate parameters for maximum alignment with a particular alignment software can be determined by one of ordinary skill in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percent identity "X" of the first amino acid sequence to the second sequence amino acid is calculated as 100 x (Y / Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (aligned by visual inspection or by a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than that of the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.

[0076] As a non-limiting example, whether any particular polynucleotide has a certain percentage of sequence identity to a reference sequence (e.g., at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical) can be determined in certain embodiments using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482 489 (1981) to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention, parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and allows gaps in homology of up to 5% of the total number of nucleotides in the reference sequence.

[0077] In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection. In certain embodiments, the identity exists over a region of the sequences that is at least about 10, about 20, about 40-60 residues in length, or any integer value therebetween, or over a region longer than 60-80 residues, at least about 90-100 residues, or the sequences are substantially identical over the entire length of the sequences being compared, such as, for example, the coding regions of the nucleotide sequences.

[0078] "Conservative amino acid substitution" refers to a substitution of an amino acid residue with another amino acid residue having a similar side chain. A family of amino acid residues having similar side chains, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) are defined in the art. For example, the substitution of tyrosine for phenylalanine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present invention do not abolish the binding of the polypeptide or antibody containing the amino acid sequence to the antigen(s), i.e., to FOLR1 to which the polypeptide or antibody binds. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1 187 (1993), Kobayashi et al. Protein Eng. 12(10):879-884 (1999), and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0079] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0080] Wherever embodiments are described herein with the language "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0081] When used herein in phrases such as "A and / or B," the term "and / or" is intended to include both "A and B," "A or B," "A," and "B." Similarly, when used in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0082] II. FOLR1-binding agents Agents that specifically bind to human FOLR1 are useful for carrying out the methods of the present invention. Such agents are referred to herein as "FOLR1 binding agents". FOLR1 binding agents that are useful for carrying out the methods of the present invention are described, for example, in WO2014 / 036495 and WO2015 / 031815, which are incorporated herein by reference in their entirety.

[0083] FOLR1 binding agents include those that contain the heavy and light chain CDR sequences of muFR1-9, muFR1-13, muFR1-53, muFR1-62, muFR1-64, and FOLR1-2.1 (also referred to as "IMGN 353.2-1," "353.2-1," "2.1," or "muFRIHC2-1"). The CDR sequences muFR1-9, muFR1-13, muFR1-53, muFR1-62, FOLR1-2.1 are set forth in Tables 1 and 2 below. [Table 1] [Table 2]

[0084] A FOLR1 binding molecule can be an antibody or antigen-binding fragment that specifically binds to FOLR1 containing the CDRs of muFR1-9, muFR1-13, muFR1-53, muFR1-62, muFR1-64, or FOLR1-2.1 (also referred to as "IMGN 353.2-1," "353.2-1," "2.1," or "muFRIHC2-1") with up to four (i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutions per CDR.

[0085] The polypeptides may comprise one of the individual variable light or heavy chains described herein. The antibodies and polypeptides may also comprise both variable light and heavy chains. The variable light and heavy chain sequences of murine muFR1-9, muFR1-13, muFR1-53, muFR1-62, and FOLR1-2.1 (also referred to as "IMGN 353.2-1", "353.2-1", "2.1", or "muFRIHC2-1") antibodies are provided in Tables 3 and 4 below. [Table 3] [Table 4-1] [Table 4-2]

[0086] Further provided are polypeptides comprising (a) a polypeptide having at least about 90% sequence identity to SEQ ID NOs: 25-28, and / or (b) a polypeptide having at least about 90% sequence identity to SEQ ID NOs: 29-32. In certain embodiments, the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 25-32. Thus, in certain embodiments, the polypeptide comprises (a) a polypeptide having at least about 95% sequence identity to SEQ ID NOs: 25-28, and / or (b) a polypeptide having at least about 95% sequence identity to SEQ ID NOs: 29-32. In certain embodiments, the polypeptide comprises (a) a polypeptide having an amino acid sequence of SEQ ID NOs: 25-28, and / or (b) a polypeptide having an amino acid sequence of SEQ ID NOs: 29-32. In certain embodiments, the polypeptide is an antibody, and / or the polypeptide specifically binds to FOLR1. In certain embodiments, the polypeptide is a murine, chimeric, or humanized antibody that specifically binds to FOLR1. In certain embodiments, the polypeptides having a certain percentage of sequence identity to SEQ ID NOs:25-32 differ from SEQ ID NOs:25-32 only by conservative amino acid substitutions.

[0087] The polypeptides may comprise one of the individual light or heavy chains described herein. The antibodies and polypeptides may also comprise both light and heavy chains. The light and variable chain sequences of murine muFR1-9, muFR1-13, muFR1-53, muFR1-62, and FOLR1-2.1 (also referred to as "IMGN 353.2-1", "353.2-1", "2.1", or "muFRIHC2-1") antibodies are provided in Tables 5 and 6 below. In a particular embodiment, the anti-FOLR1 antibody is produced by a hybridoma deposited with the ATCC on April 16, 2013, and has ATCC deposit number PTA-120197 ("FOLR1-2.1", also referred to as "IMGN 353.2-1", "353.2-1", "2.1", or "muFRIHC2-1"). [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 6-1] [Table 6-2]

[0088] Further provided are polypeptides comprising (a) a polypeptide having at least about 90% sequence identity to SEQ ID NOs: 33-36, and / or (b) a polypeptide having at least about 90% sequence identity to SEQ ID NOs: 37-40. In certain embodiments, the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 33-40. Thus, in certain embodiments, the polypeptide comprises (a) a polypeptide having at least about 95% sequence identity to SEQ ID NOs: 33-36, and / or (b) a polypeptide having at least about 95% sequence identity to SEQ ID NOs: 37-40. In certain embodiments, the polypeptide comprises (a) a polypeptide having an amino acid sequence of SEQ ID NOs: 33-36, and / or (b) a polypeptide having an amino acid sequence of SEQ ID NOs: 37-40. In certain embodiments, the polypeptide is an antibody, and / or the polypeptide specifically binds to FOLR1. In certain embodiments, the polypeptide is a murine, chimeric, or humanized antibody that specifically binds to FOLR1. In certain embodiments, the polypeptides having a certain percentage of sequence identity to SEQ ID NOs: 33-40 differ from SEQ ID NOs: 30-40 only by conservative amino acid substitutions.

[0089] In certain embodiments, the polypeptide is not competitively inhibited by the binding of huMov19 to FOLR1. In certain embodiments, the polypeptide is not competitively inhibited by the binding of IMGN853 to FOLR1. In certain embodiments, the polypeptide is not competitively inhibited by the binding of huMov19 to FOLR1. In certain embodiments, the polypeptide is not competitively inhibited by the binding of an antibody or antigen-binding fragment to FOLR1, the antibody or antigen-binding fragment comprising a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO:59, a VL CDR-2 of SEQ ID NO:60, a VL CDR-3 of SEQ ID NO:61, a variable heavy chain (VH) CDR-1 of SEQ ID NO:62, a VH CDR-2 of SEQ ID NO:64, and a VH CDR-3 of SEQ ID NO:65. In certain embodiments, the polypeptide is not competitively inhibited by the binding of an antibody or antigen-binding fragment to FOLR1, the antibody or antigen-binding fragment comprising a variable heavy chain (VH) having the sequence of SEQ ID NO: 56, and a variable light chain (VL) having the sequence of SEQ ID NO: 57 or SEQ ID NO: 58. In certain embodiments, the polypeptide is not competitively inhibited by the binding of an antibody or antigen-binding fragment to FOLR1, the antibody or antigen-binding fragment comprising (i) a heavy chain comprising the same amino acid sequence as the amino acid sequence of the heavy chain encoded by the plasmid deposited with the American Type Culture Collection (ATCC) as PTA-10772, and (ii) a light chain comprising the same amino acid sequence as the amino acid sequence of the light chain encoded by the plasmid deposited with the ATCC as PTA-10774. In certain embodiments, the polypeptide is not inhibited by the binding of folic acid to FOLR1.

[0090] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method known in the art, such as cytometry (including flow cytometry), enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) or kinetics (e.g., surface plasmon resonance spectroscopy (BIACORE™) analysis). Direct binding assays as well as competitive binding assay formats can be readily employed (see, e.g., Berzofsky, et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992), and methods described herein. The measured affinity of a particular antibody-antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH, temperature). Thus, affinity and other antigen-binding parameters (e.g., KD or Kd, K on , K off Measurements of ) are performed in standardized solutions of antibody and antigen and standardized buffers as known in the art, such as the buffers described herein.

[0091] In one embodiment, the binding assay can be performed using cytometry (e.g., flow cytometry) on cells expressing the FOLR1 antigen on their surface. For example, 1×10 cells per sample can be cultured in 100 μL of FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum). 5FOLR1 positive cells, such as SKOV3, were incubated with varying concentrations of anti-FOLR1 antibody with the cells. The cells were then pelleted, washed, and incubated with 100 μL of FITC-conjugated goat anti-mouse or goat anti-human IgG antibody (6 μg / mL in FACS buffer, available, for example, from Jackson Laboratory) for 1 hour. The cells were pelleted again, washed with FACS buffer, and resuspended in 200 μL of PBS containing 1% formamide. Samples were captured, for example, using a FACSCalibur flow cytometer with an HTS multiwell sampler, and analyzed using CellQuest Pro (all from BD Biosciences, San Diego, US). For each sample, the mean fluorescence intensity (MFI) of FL1 was taken and plotted against the antibody concentration in a semi-logarithmic plot to generate a binding curve. A sigmoidal dose-response curve was fitted to the binding curve, and EC50 values ​​were calculated using a program such as, for example, GraphPad Prism v4 (GraphPad software, San Diego, CA) with default parameters. The EC50 value can be used as a measure of the apparent dissociation constant "Kd" or "KD" for each antibody.

[0092] Monoclonal antibodies can be prepared using hybridoma techniques such as those described by Kohler and Milstein (1975) Nature 256:495. Using the hybridoma technique, a mouse, hamster, or other suitable host animal is immunized as described above to elicit the production by lymphocytes of antibodies that specifically bind to the immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line, for example, using polyethylene glycol, to form hybridoma cells, which can then be selected from unfused lymphocytes and myeloma cells. Hybridomas producing monoclonal antibodies specifically directed against a selected antigen as determined by immunoprecipitation, immunoblot, or by in vitro binding assays (e.g., radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA)) can then be grown in in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in animals. Monoclonal antibodies can then be purified from the culture medium or ascites fluid as described for polyclonal antibodies.

[0093] Alternatively, monoclonal antibodies can be produced using recombinant DNA methods, as described in U.S. Patent No. 4,816,567. For example, polynucleotides encoding the monoclonal antibodies are isolated from mature B cells or hybridoma cells by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the heavy and light chains of the antibody, and sequenced using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector and transfected into E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, resulting in the production of the monoclonal antibodies by the host cells. Alternatively, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries expressing the CDRs of the desired species as described (McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).

[0094] The polynucleotide(s) encoding the monoclonal antibody can be further modified to generate replacement antibodies in a number of different ways using recombinant DNA techniques. In some embodiments, for example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be 1) substituted for those regions of, for example, a human antibody to generate a chimeric antibody, or 2) substituted for a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate desired antibody fragments of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable regions can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0095] In some embodiments, the monoclonal antibody against human FOLR1 is a humanized antibody. In certain embodiments, such antibodies are used therapeutically to reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to a human subject.

[0096] Methods for engineering, humanizing, or resurfacing non-human or human antibodies can also be used and are well known in the art. Humanized, resurfaced, or similarly engineered antibodies can have one or more amino acid residues that are non-human, but are not limited to, mouse, rat, rabbit, non-human primate, or other mammalian sources. These non-human amino acid residues are replaced by residues that are often referred to as "import" residues, which are typically taken from the variable, constant, or other domains of the "import" of a known human sequence.

[0097] Such imported sequences can be used to reduce immunogenicity, or to reduce, improve or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable properties as known in the art. Generally, CDR residues are directly and most substantially involved in influencing FOLR1 binding. Thus, while some or all of the non-human or human CDR sequences are maintained, the non-human sequences of variable and constant regions can be replaced with human amino acids or other amino acids.

[0098] Antibodies can also be optionally humanized, resurfaced, engineered, or human antibodies engineered, while retaining high affinity for the antigen FOLR1 and other favorable biological properties. To achieve this goal, humanized (human) or engineered anti-FOLR1 antibodies and resurfaced antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanized and engineered products using three-dimensional models of the parental, engineered, and humanized sequences. Three-dimensional immunoglobulin models are commercially available and are familiar to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformations of selected candidate immunoglobulin sequences. Examination of these displays allows the analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind to an antigen such as FOLR1. In this way, framework (FR) residues can be selected and combined with the consensus and import sequences to achieve the desired antibody characteristics, e.g., increased affinity for the target antigen(s).

[0099] Humanization, resurfacing, or engineering of the antibodies disclosed herein may be performed using methods described by Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993); Roguska et al. al., Proc. Natl. Acad. Sci., USA, 91(3):969-973(1994), Roguska et al., Protein Eng.9(10):895-904(1996), U.S. Patent No. 5,639,641, U.S. Patent No. 5,723,323, U.S. Patent No. 5,976,86 No. 2, No. 5,824,514, No. 5,817,483, No. 5,814,476, No. 5,763,192, No. 5,723, No. 323, No. 5,766,886, No. 5,714,352, No. 6,204,023, No. 6,180,370, No. 5,69 No. 3,762, No. 5,530,101, No. 5,585,089, No. 5,225,539, No. 4,816,567, PCT / :U This can be carried out using any known method, such as, but not limited to, those described in US98 / 16280, US96 / 18978, US91 / 09630, US91 / 05939, US94 / 01234, GB89 / 01334, GB91 / 01134, GB92 / 01755, WO90 / 14443, WO90 / 14424, WO90 / 14430, EP229246, 7,557,189, 7,538,195, and 7,342,110 (each of which applications is incorporated herein by reference, including the references cited therein).

[0100] The agents that specifically bind to FOLR1 disclosed herein also encompass antibody fragments. Various techniques are known for the production of antibody fragments. Traditionally, these fragments are derived through proteolytic digestion of intact antibodies (e.g., Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods 24:107-117; Brennan et al., 1985, Science, 229:81). In certain embodiments, antibody fragments are produced recombinantly. Fab, Fv, and scFv antibody fragments are all expressed in and secreted from E. coli or other host cells, allowing for the large scale production of these fragments. Such antibody fragments can also be isolated from the antibody phage libraries discussed above. Antibody fragments can also be linear antibodies as described in U.S. Pat. No. 5,641,870, and can be, for example, monospecific or bispecific. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0101] For the purposes of the present invention, it should be appreciated that the modified antibody can comprise any type of variable region that provides for the association of the antibody with the human FOLR1 polypeptide. In this regard, the variable region can comprise or be derived from any type of mammal that provides humoral immunity against the desired tumor-associated antigen and can be induced to produce immunoglobulins. Thus, the variable region of the modified antibody can be of, for example, human, murine, non-human primate (e.g., cynomolgus monkey, macaque, etc.) or wolf origin. In some embodiments, both the variable region and the constant region of the modified immunoglobulin are human. In other embodiments, the variable region of the corresponding antibody (usually derived from a non-human source) can be engineered or specifically tailored to improve the binding characteristics or reduce the immunogenicity of the molecule. In this regard, the variable region useful in the present invention can be humanized or otherwise altered through the inclusion of imported amino acid sequences.

[0102] In certain embodiments, the variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs, and, if necessary, by replacement and sequence changes of partial framework regions. The CDRs can be derived from antibodies of the same class or even subclass as the antibody from which the framework regions are derived, although it is envisaged that the CDRs are derived from antibodies of different classes, and in certain embodiments, from different species. It may not be necessary to replace all of the CDRs with complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the antigen binding site. Given the descriptions in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it would be well within the capabilities of a person skilled in the art to obtain functional antibodies with reduced immunogenicity by performing routine experimentation or by performing trial and error testing.

[0103] Notwithstanding the changes to the variable regions, one skilled in the art will appreciate that the modified antibodies of the present invention include antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) in which at least a portion of one or more of the constant region domains has been deleted or otherwise altered to provide a desired biochemical characteristic, such as, for example, higher tumor localization or reduced serum half-life, when compared to an antibody of substantially the same immunogenicity that contains a native or unaltered constant region. In some embodiments, the constant region of the modified antibody will comprise a human constant region. Modifications to the constant region that are compatible with the present invention include the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein can include changes or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or the light chain constant domain (CL). In some embodiments, modified constant domains that partially or entirely delete one or more domains are contemplated. In some embodiments, the modified antibodies will include domain deleted constructs or variants in which the entire CH2 domain has been removed (ΔCH2 constructs). In some embodiments, the omitted constant region domain will be replaced by a short amino acid spacer (e.g., 10 residues) that provides some of the molecular flexibility normally conferred by the absent constant region.

[0104] It will be mentioned that in certain embodiments, modified antibodies can be engineered to fuse the CH3 domain directly to the hinge region of the respective modified antibody. In other constructs, it may be desirable to provide a peptide spacer between the hinge region and the modified CH2 and / or CH3 region. For example, compatible constructs can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is linked to the hinge region with a spacer of 5-20 amino acids. Such a spacer can be added to ensure, for example, that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be mentioned that amino acid spacers can clearly prove to be immunogenic in some cases and elicit undesirable immune responses against the construct. Thus, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic or even omitted entirely so as to maintain the desired biochemical qualities of the modified antibody.

[0105] In addition to the deletion of the entire constant region domain, it will be appreciated that the antibodies disclosed herein can be provided by partial deletion or substitution of a few, or even a single amino acid. For example, the mutation of a single amino acid in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding, thereby increasing tumor localization. Similarly, it may be desirable to simply delete that portion of one or more constant region domains that controls the effector function to be regulated (e.g., complement C1Q binding). Such partial deletion of the constant region may improve selected characteristics of the antibody (serum half-life), while other desirable functions associated with the constant region of interest remain intact. Furthermore, as alluded to above, the constant region of the disclosed antibodies can be modified through the mutation or substitution of one or more amino acids that improve the profile of the resulting construct. In this regard, it is possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding), while maintaining the conformational and immunogenic profile of the modified antibody. Certain embodiments may include the addition of one or more amino acids to the constant region to improve desirable characteristics, such as increased or decreased effector function, or to provide for the attachment of additional cytotoxins or carbohydrates. In such embodiments, it may be desirable to insert or repeat particular sequences from a selected constant region domain.

[0106] The antibodies disclosed herein also include variants and equivalents that are substantially homologous to the chimeric, humanized, and human antibodies or antibody fragments thereof described herein. These can contain, for example, conservative substitution mutations, i.e., the replacement of one or more amino acids with similar amino acids. For example, conservative substitution refers to the replacement of an amino acid with another amino acid within the same general class, such as, for example, an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid with another neutral amino acid. What is intended by conservative amino acid substitution is well known in the art.

[0107] The FOLR1 binding agent disclosed herein may be a recombinant, natural or synthetic polypeptide, or a fragment thereof, including an antibody against human FOLR1. It will be recognized in the art that some amino acid sequences of the present invention may be changed without significantly affecting the structure or function of the protein. The FOLR1 binding agent disclosed herein may be a recombinant, natural or synthetic polypeptide, or a fragment thereof, including an antibody against human folate receptor protein. Such variants include deletions, insertions, inversions, repeats and type substitutions.

[0108] Polypeptides and analogs can be further modified to contain additional chemical moieties that are not normally part of the protein. These derivatized moieties can improve the solubility, biological half-life, or absorption of the protein. The moieties can also reduce or eliminate any reasonable side effects of the protein. A review of these moieties can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0109] III. Methods of Detection The method of the present invention provides a multi-step approach to detect FOLR1 in a sample. In particular, an initial immunocapture step is performed to enrich FOLR1 in the sample, followed by digestion of FOLR1 into peptides and analysis by liquid chromatography mass spectrometry (LC / MS). Analysis of peptides by LC / MS further allows quantitative determination of the level of FOLR1 present in the sample, including, for example, the level of FOLR1 in a sample from a patient suffering from a cancer that expresses FOLR1. In some embodiments, a method of detecting human folate receptor 1 (FOLR1) in a sample includes (a) capturing the folate receptor 1 (FOLR1) using an immunocapture reagent bound to a solid support, (b) eluting FOLR1 from the solid support, (c) digesting the eluted FOLR1, and (d) performing liquid chromatography mass spectrometry (LC / MS) analysis on the digested FOLR1, where the FOLR1 is detected by monitoring the chromatographic separation and mass spectrometry response of at least one signature FOLR1 peptide. Compared to other known methods for the detection of FOLR1, the methods of the present invention offer the advantages of enhanced sensitivity and selectivity.

[0110] In some embodiments, the sample containing FOLR1 comprises a bodily fluid. In further embodiments, the bodily fluid may be plasma, serum, or ascites. In certain embodiments, the sample is plasma. In some embodiments, the sample comprises a peripheral blood sample. In some embodiments, the sample is obtained from a patient with cancer. In further embodiments, the cancer is selected from the group consisting of ovarian cancer, lung cancer, colorectal cancer, pancreatic cancer, liver cancer, breast cancer, brain cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, endometrial cancer, peritoneal cancer, and head and neck cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is endometrial cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the lung cancer is non-small cell lung cancer. In certain embodiments, the non-small cell lung cancer is adenocarcinoma of the lung.

[0111] Immunocapture The initial immunocapture step is carried out on the sample using an immunocapture reagent bound to a solid support. The immunocapture reagent may be any immunological reagent that binds to FOLR1, including, for example, an antibody or antigen-binding fragment thereof that binds to FOLR1. When an antibody or antigen-binding fragment is used as an immunocapture reagent, the binding of the antibody or antigen-binding fragment to FOLR1 cannot be competitively inhibited by the binding of an antibody-based active agent such as IMGN853 to FOLR1 in the sample. Furthermore, when an antibody or antigen-binding fragment is used as an immunocapture reagent, the binding of the antibody or antigen-binding fragment to FOLR1 cannot be inhibited by folic acid present in the sample.

[0112] In some embodiments, the immunocapture reagent is antibody muFR1-9. In some embodiments, the immunocapture reagent is antibody muFR1-13. In other embodiments, the immunocapture reagent is antibody muFR1-53. In other embodiments, the immunocapture reagent is antibody muFR1-62.

[0113] In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a variable heavy chain (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 1, a VH CDR-2 of SEQ ID NO: 2, a VH CDR-3 of SEQ ID NO: 3, a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 13, a VL CDR-2 of SEQ ID NO: 14, and a VL CDR-3 of SEQ ID NO: 15. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH CDR-1 of SEQ ID NO: 4, a VH CDR-2 of SEQ ID NO: 5, a VH CDR-3 of SEQ ID NO: 6, a VL CDR-1 of SEQ ID NO: 16, a VL CDR-2 of SEQ ID NO: 17, and a VL CDR-3 of SEQ ID NO: 18. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH CDR-1 of SEQ ID NO: 7, a VH CDR-2 of SEQ ID NO: 8, a VH CDR-3 of SEQ ID NO: 9, a VL CDR-1 of SEQ ID NO: 19, a VL CDR-2 of SEQ ID NO: 20, and a VL CDR-3 of SEQ ID NO: 21. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH CDR-1 of SEQ ID NO: 10, a VH CDR-2 of SEQ ID NO: 11, a VH CDR-3 of SEQ ID NO: 12, a VL CDR-1 of SEQ ID NO: 22, a VL CDR-2 of SEQ ID NO: 23, and a VL CDR-3 of SEQ ID NO: 24.

[0114] In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH of SEQ ID NO:25 and a VL of SEQ ID NO:29. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH of SEQ ID NO:26 and a VL of SEQ ID NO:30. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH of SEQ ID NO:27 and a VL of SEQ ID NO:31. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH of SEQ ID NO:28 and a VL of SEQ ID NO:32. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH having at least 90% identity to the sequence of SEQ ID NO:25 and a VL having at least 90% identity to the sequence of SEQ ID NO:29. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a variable heavy chain (VH) having at least 95% identity to the sequence of SEQ ID NO: 25, and a variable light chain (VL) having at least 95% identity to the sequence of SEQ ID NO: 29. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a variable heavy chain (VH) having at least 90% identity to the sequence of SEQ ID NO: 26, and a variable light chain (VL) having at least 90% identity to the sequence of SEQ ID NO: 30. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a variable heavy chain (VH) having at least 95% identity to the sequence of SEQ ID NO: 26, and a variable light chain (VL) having at least 95% identity to the sequence of SEQ ID NO: 30. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment, including an antibody or antigen-binding fragment, comprising a variable heavy chain (VH) having at least 90% identity to the sequence of SEQ ID NO: 27, and a variable light chain (VL) having at least 90% identity to the sequence of SEQ ID NO: 31. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment, including a variable heavy chain (VH) having at least 95% identity to the sequence of SEQ ID NO: 27, and a variable light chain (VL) having at least 95% identity to the sequence of SEQ ID NO: 31.In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a variable heavy chain (VH) having at least 90% identity to the sequence of SEQ ID NO: 28, and a variable light chain (VL) having at least 90% identity to the sequence of SEQ ID NO: 32. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a variable heavy chain (VH) having at least 95% identity to the sequence of SEQ ID NO: 28, and a variable light chain (VL) having at least 95% identity to the sequence of SEQ ID NO: 32.

[0115] In one embodiment, the immunocapture reagent is an antibody or antigen-binding fragment comprising a heavy chain having the sequence of SEQ ID NO: 33 and a light chain having the sequence of SEQ ID NO: 37. In another embodiment, the immunocapture reagent is an antibody or antigen-binding fragment comprising a heavy chain having the sequence of SEQ ID NO: 34 and a light chain having the sequence of SEQ ID NO: 38. In another embodiment, the immunocapture reagent is an antibody or antigen-binding fragment comprising a heavy chain having the sequence of SEQ ID NO: 35 and a light chain having the sequence of SEQ ID NO: 39. In another embodiment, the immunocapture reagent is an antibody or antigen-binding fragment comprising a heavy chain of SEQ ID NO: 36 and a light chain of SEQ ID NO: 40.

[0116] In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment that binds to human FOLR1 with a Kd of about 1.0 nM to about 10 nM. In other embodiments, the immunocapture reagent is an antibody or antigen-binding fragment that binds to human FOLR1 with a Kd of about 0.5 nM to about 5 nM.

[0117] In some embodiments, the immunocapture reagent is biotinylated. In some embodiments, the immunocapture reagent is bound to a solid support via a biotin-streptavidin interaction. In some embodiments, the solid support is a mass spectrometry immunoassay (MSIA) microcolumn. In other embodiments, the immunocapture reagent comprises a magnetic bead.

[0118] To perform the initial immunocapture step, a sample containing FOLR1 is incubated with an immunocapture reagent bound to a solid support. A washing step can be performed after the immunocapture step to further purify the captured FOLR1. In some embodiments, one or more washing steps are performed after incubation of the sample with the immunocapture reagent and before elution of the captured FOLR1. In some embodiments, two or more washing steps are performed before elution of the captured FOLR1. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten washing steps are performed on the captured FOLR1. In some embodiments, the washing step comprises contacting the captured FOLR1 with a washing buffer. In some embodiments, the washing buffer is a commercially available washing buffer. In some embodiments, the washing step comprises contacting the captured FOLR1 with a salt solution and a detergent. In some embodiments, the salt solution is 400 mM NaCl and the detergent is 0.1% Tween20.

[0119] After immunocapture, FOLR1 is released from the solid support by performing an elution step. In some embodiments, the elution step is performed by contacting the captured FOLR1 with an acidic solution. In some embodiments, the acidic solution is a commercially available solution. In some embodiments, the elution solution is brought to a neutral pH by the addition of a neutralization buffer. In some embodiments, the neutralization buffer is 500 mM ammonium bicarbonate at pH 8. In some embodiments, the neutralization buffer is a commercially available buffer.

[0120] b. Liquid chromatography-mass spectrometry After immunocapture and elution, the resulting FOLR1 protein is digested into peptides and analyzed by liquid chromatography-mass spectrometry (LC / MS). In some embodiments, a solution containing FOLR1 is reduced and alkylated before analysis by LC / MS. In some embodiments, FOLR1 is alkylated with methanol. In some embodiments, FOLR1 is reduced by contacting FOLR1 with a solution containing tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, a solution containing 100 mM TCEP is used to reduce FOLR1. In some embodiments, a cysteine ​​blocking reagent is added to the solution containing FOLR1 after alkylation and reduction. In some embodiments, the cysteine ​​blocking reagent is iodoacetamide (IAM). In some embodiments, a solution containing 100 mM IAM is added to the solution containing FOLR1.

[0121] After alkylation and reduction, FOLR1 is digested into peptides. In some embodiments, FOLR1 is digested with trypsin. In some embodiments, FOLR1 is digested with Lys-C. In some embodiments, FOLR1 is digested with a mixture of trypsin and Lys-C. In some embodiments, FOLR1 is digested by contacting FOLR1 with a 50 mM ammonium bicarbonate solution containing 30 ng / μL trypsin / Lys-C.

[0122] Digestion of FOLR1 with trypsin / Lys-C produces peptides useful in quantitative analysis of samples by LC / MS. In some embodiments, the isolated signature peptides of the present invention are provided in the digestion products of FOLR1. The signature peptides produced by digestion of FOLR1 are shown in Table 7 below. [Table 7]

[0123] After digestion of FOLR1 into peptides, the solution containing the peptides is prepared for LC / MS analysis. In some embodiments, a detergent is added to the solution containing the peptides before LC / MS analysis. In some embodiments, the detergent is a commercially available reagent formulated for mass spectrometry analysis. In some embodiments, the detergent reaction is quenched by adding 10% formic acid.

[0124] After digestion and preparation of the sample for LC / MS analysis, the sample is injected into an LC / MS instrument and analyzed. In some embodiments, at least two signature peptides of FOLR1 are selected and monitored in the LC / MS analysis step. In some embodiments, at least three signature peptides of FOLR1 are selected and monitored in the LC / MS analysis step. In some embodiments, at least four signature peptides of FOLR1 are selected and monitored in the LC / MS analysis step. In some embodiments, the at least four signature peptides selected and monitored in the LC / MS step include a peptide having an amino acid sequence of SEQ ID NO: 42, a peptide having an amino acid sequence of SEQ ID NO: 43, a peptide having an amino acid sequence of SEQ ID NO: 44, and a peptide having an amino acid sequence of SEQ ID NO: 45. In some embodiments, a quantitative measurement of FOLR1 levels is provided by LC / MS analysis. In further embodiments, the level of FOLR1 in the sample is quantified by comparing the level of FOLR1 in the sample to a reference level of FOLR1. Methods for analysis by LC / MS analysis are known in the art and are described, for example, in Yang et al. Scientific Reports. 2015 November 17;5:16733, doi10.1038 / srep16733.

[0125] In some embodiments, detection of FOLR1 in a sample is not inhibited by the presence of IMGN853 in the sample. In some embodiments, detection of FOLR1 in a sample is not inhibited by the presence of huMov19 in the sample. In some embodiments, detection of FOLR1 in a sample is not inhibited by the presence of an antibody or antigen-binding fragment present in the sample, the antibody or antigen-binding fragment comprising a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO:59, a VL CDR-2 of SEQ ID NO:60, a VL CDR-3 of SEQ ID NO:61, a variable heavy chain (VH) CDR-1 of SEQ ID NO:62, a VH CDR-2 of SEQ ID NO:64, and a VH CDR-3 of SEQ ID NO:65. In some embodiments, detection of FOLR1 in a sample is not inhibited by the presence of an antibody or antigen-binding fragment present in the sample, the antibody or antigen-binding fragment comprising a VH having the sequence of SEQ ID NO:56, and a VL having the sequence of SEQ ID NO:57 or SEQ ID NO:58. In some embodiments, detection of FOLR1 is not inhibited by the presence of an antibody or antigen-binding fragment present in the sample, the antibody or antigen-binding fragment comprising (i) a heavy chain that comprises the same amino acid sequence as the amino acid sequence of the heavy chain encoded by the plasmid deposited with the American Type Culture Collection (ATCC) under PTA-10772, and (ii) a light chain that comprises the same amino acid sequence as the amino acid sequence of the light chain encoded by the plasmid deposited with the ATCC under PTA-10774. In some embodiments, detection of FOLR1 in the sample is not inhibited by the presence of folic acid in the sample.

[0126] In some embodiments, at least 0.5 ng / mL of FOLR1 may be detected in a sample by performing a method of the present invention. In some embodiments, at least 0.3 ng / mL of FOLR1 may be detected in a sample by performing a method of the present invention. In some embodiments, at least 0.25 ng / mL of FOLR1 may be detected in a sample by performing a method of the present invention. In some embodiments, at least 0.2 ng / mL of FOLR1 may be detected in a sample by performing a method of the present invention. In some embodiments, at least 0.15 ng / mL of FOLR1 may be detected in a sample by performing a method of the present invention. In some embodiments, a signal to noise ratio of at least 5 is observed by performing a method of the present invention. In some embodiments, a signal to noise ratio of at least 6 is observed by performing a method of the present invention. In some embodiments, a signal to noise ratio of at least 7 is observed by performing a method of the present invention. In some embodiments, a signal to noise ratio of at least 8 is observed by performing a method of the present invention. In some embodiments, a signal to noise ratio of at least 9 is observed by performing a method of the present invention. In some embodiments, a signal to noise ratio of at least 10 is observed by performing a method of the present invention.

[0127] IV. Kit For convenience, the assay method of the present invention can be provided in the form of a kit. Such a kit is a packaged combination that includes the basic elements of (a) a first reagent that can be an immunocapture reagent that binds to FOLR1, and (b) a digestion reagent that can digest the captured FOLR1 into peptides. The kit can further include at least one peptide derived from FOLR1. These basic elements are defined above and in the following examples.

[0128] In some embodiments, the immunocapture reagent comprises an antibody or antigen-binding fragment that binds to FOLR1. In further embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a variable heavy chain (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 1, a VH CDR-2 of SEQ ID NO: 2, a VH CDR-3 of SEQ ID NO: 3, a variable light chain (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 13, a VL CDR-2 of SEQ ID NO: 14, and a VL CDR-3 of SEQ ID NO: 15. In other embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH CDR-1 of SEQ ID NO: 4, a VH CDR-2 of SEQ ID NO: 5, a VH CDR-3 of SEQ ID NO: 6, a VL CDR-1 of SEQ ID NO: 16, a VL CDR-2 of SEQ ID NO: 17, and a VL CDR-3 of SEQ ID NO: 18. In another embodiment, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH CDR-1 of SEQ ID NO: 7, a VH CDR-2 of SEQ ID NO: 8, a VH CDR-3 of SEQ ID NO: 9, a VL CDR-1 of SEQ ID NO: 19, a VL CDR-2 of SEQ ID NO: 20, and a VL CDR-3 of SEQ ID NO: 21. In another embodiment, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH CDR-1 of SEQ ID NO: 10, a VH CDR-2 of SEQ ID NO: 11, a VH CDR-3 of SEQ ID NO: 12, a VL CDR-1 of SEQ ID NO: 22, a VL CDR-2 of SEQ ID NO: 23, and a VL CDR-3 of SEQ ID NO: 24.

[0129] In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH of SEQ ID NO:25 and a VL of SEQ ID NO:29. In other embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH of SEQ ID NO:26 and a VL of SEQ ID NO:30. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH of SEQ ID NO:27 and a VL of SEQ ID NO:31. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a VH of SEQ ID NO:28 and a VL of SEQ ID NO:32. In some embodiments, the immunocapture reagent comprises an antibody comprising a VH having at least 90% identity to any one of SEQ ID NOs:25-28. In some embodiments, the immunocapture reagent comprises an antibody comprising a VH having at least 95% identity to any one of SEQ ID NOs:25-28. In some embodiments, the immunocapture reagent comprises an antibody comprising a VL having at least 90% identity to any one of SEQ ID NOs:29-32. In some embodiments, the immunocapture reagent comprises an antibody comprising a VL having at least 95% identity to any one of SEQ ID NOs:29-32.

[0130] In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a heavy chain having the sequence of SEQ ID NO: 33 and a light chain having the sequence of SEQ ID NO: 37. In other embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a heavy chain having the sequence of SEQ ID NO: 34 and a light chain having the sequence of SEQ ID NO: 38. In other embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a heavy chain having the sequence of SEQ ID NO: 35 and a light chain having the sequence of SEQ ID NO: 39. In other embodiments, the immunocapture reagent is an antibody or antigen-binding fragment comprising a heavy chain of SEQ ID NO: 36 and a light chain of SEQ ID NO: 40.

[0131] In some embodiments, the immunocapture reagent is muFR1-9. In other embodiments, the immunocapture reagent is muFR1-13. In some embodiments, the immunocapture reagent is an antibody or antigen-binding fragment that binds to human FOLR1 with a Kd of about 1.0 nM to about 10 nM. In other embodiments, the immunocapture reagent is an antibody or antigen-binding fragment that binds to human FOLR1 with a Kd of about 0.5 nM to about 5 nM. In some embodiments, the immunocapture reagent is biotinylated.

[0132] In some embodiments, the kit further comprises a solid support for the capture reagent, which can be provided as a separate element or on which the capture reagent is already immobilized. Thus, the capture antibody in the kit can be immobilized on a solid support, or they can be immobilized on such a support that is included with the kit or provided separately from the kit. In some embodiments, the solid support comprises a mass spectrometry immunoassay (MSIA) microcolumn. In other embodiments, the solid support comprises a magnetic bead. In some embodiments, the solid support is coated with streptavidin. In some embodiments, the immunocapture reagent is bound to the solid support via biotin-streptavidin interaction.

[0133] In some embodiments, the kit further comprises at least one FOLR1 peptide that can be used as a standard in liquid chromatography-mass spectrometry analysis. In some embodiments, the kit comprises a peptide comprising the sequence of SEQ ID NO: 42. In some embodiments, the kit comprises a peptide comprising the sequence of SEQ ID NO: 43. In some embodiments, the kit comprises a peptide comprising the sequence of SEQ ID NO: 44. In some embodiments, the kit comprises a peptide comprising the sequence of SEQ ID NO: 45. In one embodiment, the kit comprises four signature peptides that can be used as a standard in liquid chromatography-mass spectrometry analysis. In one embodiment, the four signature peptides consist of 1) a peptide comprising the sequence of SEQ ID NO: 42, 2) a peptide comprising the sequence of SEQ ID NO: 43, 3) a peptide comprising the sequence of SEQ ID NO: 44, and 4) a peptide comprising the sequence of SEQ ID NO: 45.

[0134] The kit will also typically contain instructions for performing the assay, and / or FOLR1 protein or FOLR1 peptides that act as standards, as well as other additives such as stabilizers, washing and incubation buffers, etc. The kit can also include instructions for the quantitative measurement of FOLR1 levels in a sample.

[0135] The components of the kit are provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide concentrations in solution of the reagents that substantially maximize the sensitivity of the assay. In particular, the reagents may be provided as dry powders, usually lyophilized, that include excipients, which upon dissolution provide a reagent solution having the appropriate concentration for combination with the sample to be tested.

[0136]

[0041] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which illustrate in detail the methods of the present disclosure. It will be apparent to those skilled in the art that numerous modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure. EXAMPLES

[0137] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in this respect may be suggested to those skilled in the art and are to be included within the spirit and scope of the present application.

[0138] Example 1 sFRα immunocapture-LC / MS assay An ELISA-based assay was developed and described in WO2014 / 036495. The assay was performed using an ELISA plate coated with FOLR1-Fc (a fusion peptide of huFOLR1 and mouse IgG2A hinge, CH2, CH3), muFR1-9 as a capture antibody, and biotinylated muFR1-13. The assay was optimized by a systematic approach, where the criteria were reproducible signal with high signal-to-noise ratio, minimal matrix effect in human plasma samples, high reproducibility and precision, and minimal detection limit. The optimal ELISA conditions selected resulted in detection of as little as 25 ng / mL of sFRα with neglibible / low noise. Despite the optimized conditions, the ELISA assay did not provide sufficient sensitivity to distinguish the range of distribution of sFRα levels in healthy subjects and ovarian cancer patients, allowing correlation analysis of sFRα levels and pathology.

[0139] A more recent ELISA-based assay using two different human FRα-specific mouse monoclonal antibodies reported in Kurosaki et al., Int. J. Cancer. 2016 April 15;138(8):1994-2002, was developed to investigate the levels of sFRα in the serum of patients clinically suspected of having malignant ovarian tumors and to evaluate sFRα as a diagnostic marker for ovarian cancer. This ELISA assay had a dynamic range of 250-8000 pg / mL and a signal-to-noise ratio of 3.4 for the lowest calibrator (250 pg / mL). Although this ELISA assay offered a more sensitive dynamic range of sFRα detection, the ELISA readout is generally measured spectrophotometrically at 450 nm and corrected for absorbance at 620 nm, and is therefore less selective and may generate false positive results.

[0140] An assay of the present invention has been developed that meets both the criteria of sensitivity and selectivity. The accurate measurement of the signature peptide by the LC / MS step provides a second level of selectivity that is not provided by the common ELISA method. Furthermore, the sensitivity and selectivity of the detection of sFRα obtained by the immunocapture-LC / MS assay also makes this assay useful in exploring sFRα as a predictive and / or prognostic biomarker for ovarian cancer patients.

[0141] As shown in FIG. 1, the assay includes the steps of incubating a biotinylated capture antibody (e.g., an antibody of the present invention, such as muFR1-9 or muFR1-13 antibody) solution with streptavidin-coated immunocapture medium (MSIA microcolumn or magnetic beads), incubating the sample with the immunocapture medium, washing the immunocapture medium with wash buffer, salt solution, and detergent in multiple washes, discarding the supernatant, releasing sFRα from the immunocapture medium with an acidic solution, and neutralizing the sFRα solution, followed by reduction and alkylation. The sample is then digested with trypsin / Lys-C and injected into LC / MS for analysis. Four distinct signature peptides of sFRα were selected and monitored in the LC / MS analysis step. Specifically, the peptide of SEQ ID NO: 42 was used to report protein concentration, and the peptides of SEQ ID NO: 43-45 were used for confirmation.

[0142] Optimization steps to improve assay performance during sample preparation included modifying conditions to improve immunocapture efficiency, washing procedures to remove potential interference from the plasma matrix, and modifying conditions to improve enzymatic digestion efficiency. Optimization steps during LC / MS included identifying signature peptide sequences that provide good chromatographic separation and mass spectrometric response. Automation options for immunocapture were evaluated with MSIA microcolumn technology and magnetic bead processing. Both options were compatible with the use of 96-well plates, significantly improving sample throughput, and similar sensitivity was observed with both automation platforms. Evaluations were also performed with assays using either antigen-depleted plasma or surrogate matrices.

[0143] The working assay parameters were as follows: sample processing including immunocapture and enzymatic digestion for 8.5 h, LC / MS analysis for 10 min per sample, 5% BSA, PBS (alternative matrix, 0.65 ng / mL to 40.63 ng / mL), sample volume of 0.3 mL, S / N of >10 for the four signature peptides at the LLOQ level, STD acceptance criteria of ±20% of the nominal value excluding the LLOQ (±25%), and QC acceptance criteria of ±20% of the nominal value.

[0144] Example 2 Data analysis of human plasma samples using the sFRα immunocapture-LC / MS assay Experiments were designed to measure the levels of sFRα in samples containing low levels of sFRα in a surrogate matrix of 0.3 ng / mL using the parameters described above. As shown in Figure 2, the results of these experiments show that very low levels of sFRα can be measured by immunocapture-LC / MS assay. Experiments were also designed to measure the levels of sFRα in normal human plasma samples. As shown in Figure 3, endogenous levels of sFRα found at approximately 0.5 ng / mL can be measured by immunocapture-LC / MS assay. Figure 4 shows an ion chromatogram extracted from a pre-dose sample of a patient with elevated sFRα. The results of this experiment show that patient plasma samples with elevated levels of sFRα can be clearly distinguished from samples with endogenous levels of normal human plasma samples.

[0145] The experiment was designed to test assay tolerance in the presence of IMGN853, an immune complex that targets FRα, in plasma samples. sFRα QC samples were prepared in plasma at low, medium, and high levels and spiked with IMGN853 at the expected Cmax concentration and compared to sFRα QC samples prepared in plasma without IMGN853. No interference was observed with either set of QC samples, and both sets of samples have similar responses.

[0146] The results of these experiments demonstrate that the immunocapture-LC / MS assay of the present invention has increased sensitivity (0.3 ng / mL, signal-to-noise ratio of at least 10, i.e., twice as sensitive as ELISA assays in the art) and improved selectivity for measuring sFRα in human plasma samples in the presence and absence of immune complexes targeting FRα.

[0147] All publications, patents, patent applications, internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number / database sequence was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method for detecting shed human folate receptor 1 (FOLR1) in a body fluid sample, comprising: (a) capturing the shed human FOLR1 with an immunocapture reagent bound to a solid support, the immunocapture reagent comprising an antibody or antigen-binding fragment thereof that binds to FOLR1, the antibody or antigen-binding fragment comprising: (i) variable heavy (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 1; VH CDR-2 of SEQ ID NO: 2; VH CDR-3 of SEQ ID NO: 3; variable light (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 13; VL CDR-2 of SEQ ID NO: 14 and VL CDR-3 of SEQ ID NO:

15. CDR-3; or (ii) variable heavy (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 4; VH CDR-2 of SEQ ID NO: 5; VH CDR-3 of SEQ ID NO: 6; variable light (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 16; VL CDR-2 of SEQ ID NO: 17 and VL CDR-3 of SEQ ID NO:

18. and capturing (b) eluting the captured FOLR1 from the solid support; and (c) digesting the eluted FOLR1 to generate at least one signature FOLR1 peptide; (d) performing liquid chromatography mass spectrometry (LC / MS) analysis on the digested FOLR1; wherein the shed FOLR1 is a soluble FOLR1 protein detected by chromatographic separation and monitoring the mass spectrometry response of the at least one signature FOLR1 peptide, the method is capable of detecting 0.5 ng / mL of FOLR1 in the sample, and the body fluid sample is plasma, serum or ascites.

2. The method of claim 1 , wherein the level of FOLR1 in the sample is quantified by LC / MS analysis.

3. The method of claim 2, wherein the level of FOLR1 in the sample is quantified by comparing the level of FOLR1 in the sample to a reference level of FOLR1.

4. 4. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a variable heavy (VH) complementarity determining region (CDR)-1 of SEQ ID NO: 1, a VH CDR-2 of SEQ ID NO: 2, a VH CDR-3 of SEQ ID NO: 3, a variable light (VL) complementarity determining region (CDR)-1 of SEQ ID NO: 13, a VL CDR-2 of SEQ ID NO: 14, and a VL CDR-3 of SEQ ID NO:

15.

5. 4. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a variable heavy (VH) complementarity determining region (CDR)-1 of SEQ ID NO:4, a VH CDR-2 of SEQ ID NO:5, a VH CDR-3 of SEQ ID NO:6, a variable light (VL) complementarity determining region (CDR)-1 of SEQ ID NO:16, a VL CDR-2 of SEQ ID NO:17, and a VL CDR-3 of SEQ ID NO:

18.

6. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO:25, and a variable light chain (VL) having the sequence of SEQ ID NO:

29.

7. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) having the sequence of SEQ ID NO:26, and a variable light chain (VL) having the sequence of SEQ ID NO:

30.

8. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a heavy chain having the sequence of SEQ ID NO:33 and a light chain having the sequence of SEQ ID NO:

37.

9. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a heavy chain having the sequence of SEQ ID NO:34 and a light chain having the sequence of SEQ ID NO:

38.

10. The method of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment binds to human FOLR1 with a Kd of about 1.0 nM to about 10 nM.

11. The method of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment binds to human FOLR1 with a Kd of about 0.5 nM to about 5 nM.

12. The method of any one of claims 1 to 11, wherein the solid support comprises a mass spectrometry immunoassay (MSIA) microcolumn.

13. The method of any one of claims 1 to 11, wherein the solid support comprises magnetic beads.

14. The method according to any one of claims 1 to 13, wherein at least one washing step is carried out before eluting FOLR1 from the solid support.

15. The method of claim 14, wherein two or more washing steps are performed prior to eluting FOLR1 from the solid support.

16. The method of claim 14 or 15, wherein the washing step comprises contacting the FOLR1 bound to the solid support with a wash buffer, a salt solution, and a detergent.

17. The method of any one of claims 1 to 16, wherein FOLR1 is eluted from the solid support using an acidic solution.

18. The FOLR1 is reduced and alkylated prior to digestion of the FOLR1. Item 18. The method according to item 17.

19. The method according to any one of claims 1 to 18, wherein FOLR1 is digested with trypsin and / or Lys-C.

20. The method according to any one of claims 1 to 19, wherein a peptide comprising the sequence of SEQ ID NO: 42 is generated by digesting the FOLR1.

21. The method according to any one of claims 1 to 20, wherein a peptide comprising the sequence of SEQ ID NO: 43 is generated by digesting the FOLR1.

22. The method according to any one of claims 1 to 21, wherein a peptide comprising the sequence of SEQ ID NO: 44 is generated by digesting the FOLR1.

23. The method according to any one of claims 1 to 22, wherein a peptide comprising the sequence of SEQ ID NO: 45 is generated by digesting the FOLR1.

24. The method according to any one of claims 1 to 23, wherein at least two signature peptides of FOLR1 are selected and monitored in the LC / MS analysis step.

25. The method according to any one of claims 1 to 24, wherein at least three signature peptides of FOLR1 are selected and monitored in the LC / MS analytical step.

26. The method of any one of claims 1 to 25, wherein at least four signature peptides of FOLR1 are selected and monitored in the LC / MS analysis step.

27. The signature peptide is (a) a peptide comprising the sequence of SEQ ID NO: 42; (b) a peptide comprising the sequence of SEQ ID NO: 43; (c) a peptide comprising the sequence of SEQ ID NO: 44, and (d) the method of claim 26, comprising a peptide comprising the sequence of SEQ ID NO:

45.

28. The method according to any one of claims 1 to 27, wherein the body fluid is plasma.

29. The method according to any one of claims 1 to 27, wherein the body fluid is serum.

30. The method according to any one of claims 1 to 27, wherein the body fluid is ascites.

31. The method of any one of claims 1 to 30, wherein the sample comprises a peripheral blood sample.

32. The method of any one of claims 1 to 31, wherein the sample is a sample obtained from a patient with cancer.

33. 33. The method of claim 32, wherein the cancer is selected from the group consisting of ovarian, brain, breast, uterine, endometrial, pancreatic, renal, lung, and peritoneal cancer.

34. 34. The method of claim 33, wherein the cancer is ovarian cancer.

35. The method according to any one of claims 1 to 34, capable of detecting 0.3 ng / mL of FOLR1 in a sample.

36. The method according to any one of claims 1 to 34, capable of detecting 0.25 ng / mL of FOLR1 in a sample.

37. 1. A method for detecting shed human folate receptor 1 (FOLR1) in a human plasma sample, comprising: (a) capturing the shed human FOLR1 with an immunocapture reagent bound to a solid support, the immunocapture reagent comprising an antibody or antigen-binding fragment comprising a variable heavy chain (VH) having the sequence of SEQ ID NO:25 and a variable light chain (VL) having the sequence of SEQ ID NO:29; (b) eluting the captured FOLR1 from the solid support; and (c) digesting the eluted FOLR1 with trypsin and / or Lys-C to generate at least one signature FOLR1 peptide; (d) performing liquid chromatography mass spectrometry (LC / MS) analysis on the digested FOLR1, wherein the shed FOLR1 is soluble FOLR1 protein detected by monitoring the chromatographic separation and mass spectrometry response of at least one signature FOLR1 peptide comprising the amino acid sequence of SEQ ID NO:

44.

38. 1. A method for detecting shed human folate receptor 1 (FOLR1) in a human plasma sample, comprising: (a) capturing the shed human FOLR1 with an immunocapture reagent bound to a solid support, the immunocapture reagent comprising an antibody or antigen-binding fragment comprising a variable heavy chain (VH) having the sequence of SEQ ID NO:26 and a variable light chain (VL) having the sequence of SEQ ID NO:30; (b) eluting the captured FOLR1 from the solid support; and (c) digesting the eluted FOLR1 with trypsin and / or Lys-C to generate at least one signature FOLR1 peptide; (d) performing liquid chromatography mass spectrometry (LC / MS) analysis on the digested FOLR1, wherein the shed FOLR1 is soluble FOLR1 protein detected by monitoring the chromatographic separation and mass spectrometry response of at least one signature FOLR1 peptide comprising the amino acid sequence of SEQ ID NO:

44.

39. At least four signature peptides of FOLR1 are selected and monitored in the LC / MS analysis step, and the signature peptides are (a) a peptide comprising the sequence of SEQ ID NO: 42; (b) a peptide comprising the sequence of SEQ ID NO: 43; (c) a peptide comprising the sequence of SEQ ID NO: 44; and (d) a peptide comprising the sequence of SEQ ID NO: 45 39. The method of claim 37 or claim 38, comprising:

40. The method according to any one of claims 37 to 39, capable of detecting 0.5 ng / mL of FOLR1 in a sample.

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