Antibodies and assays for detecting folate receptor 1

Abstract

The present invention generally relates to antibodies that bind to human folate receptor and diagnostic assays for folate receptor 1 based therapies. Methods of monitoring therapy using the antibodies are also provided.

Description

[0001] This application is a divisional application of an invention patent application with a filing date of August 29, 2014, Chinese patent application number 201480054040.2 (international application number PCT / US2014 / 053512), and invention name “Antibodies and assays for detecting folate receptor 1”. Field of the Invention

[0002] The field of the invention relates generally to diagnostic assays and kits for folate receptor 1 based therapy and antibodies that bind human folate receptor 1. Background of the Invention

[0003] Cancer is one of the leading causes of death in developed countries, with more than one million people diagnosed with cancer and 500,000 deaths each year in the United States alone. Overall, it is estimated that more than one-third of people will develop some form of cancer in their lifetime. There are more than 200 different types of cancer, of which four - breast cancer, lung cancer, colorectal cancer and prostate cancer - account for more than half of all new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26). 53:5-26).

[0004] Folate receptor 1 (FOLR1) (also known as folate receptor-α or folate binding protein) is an N-glycosylated protein expressed on the plasma membrane of cells. FOLR1 has a high affinity for folic acid and several reduced folic acid derivatives. FOLR1 mediates the delivery of physiological folate (5-methyltetrahydrofolate) to the interior of cells.

[0005] FOLR1 is overexpressed in the vast majority of ovarian cancers and in many uterine cancers, endometrial cancers, pancreatic cancers, renal cancers, lung cancers, and breast cancers, while FOLR1 expression in normal tissues is limited to the apical membrane of epithelial cells in the renal proximal tubules, alveolar pneumocytes of the lungs, bladder, testis, choroid plexus, and thyroid gland (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 an ideal target for FOLR1-guided cancer therapy.

[0006] Because ovarian cancer is often asymptomatic until advanced stages, it is often diagnosed at an advanced stage and has a poor prognosis when treated with currently available procedures (usually surgical debulking followed by chemotherapeutic drugs) (vonGruenigen V et al., Cancer 112:2221-2227 (2008); Ayhan A et al., Am J Obstet Gynecol 196:81e81-86 (2007); Harry VN et al., Obstet Gynecol Surv 64:548-560 (2009)). Therefore, there is a clear unmet medical need for more effective diagnostics for ovarian cancer.

[0007] Some previous assays for detecting shed FOLR1 are not specific enough for FOLR1. For example, some assays cannot distinguish FOLR1 from other folate receptor family members (FOLR2, 3 and 4) or report values ​​for total FBP (folate binding protein). In addition, some assays require pretreatment of human samples (e.g., plasma) with a light acid wash step to dissociate folate from the receptor. Some assay results may also be inaccurate due to competitive effects between antibody therapy and diagnostic antibodies. In addition, many commercially available kits are traditionally unreliable in both their reagents and their batch stability. Evaluations of these kits give very mixed results and are intended for research use only. Many require human samples to be pre-diluted prior to analysis to reduce the possibility of false positives due to "matrix effects". Therefore, there is a clear need for highly sensitive and accurate diagnostic assays that can detect a clinically relevant dynamic range of FOLR1 as a concomitant of FOLR1-based therapies. SUMMARY OF THE INVENTION

[0008] Anti-FOLR1 antibodies and antigen-binding fragments thereof, as well as methods for detecting FOLR1, diagnosing FOLR1-mediated diseases and conditions (such as cancer), monitoring the efficacy of anti-FOLR1 therapy, optimizing anti-FOLR1 therapy, and stratifying patients are all provided herein.

[0009] The anti-FOLR1 antibodies provided herein can have a diagnostic effect. For example, the anti-FOLR1 antibodies provided herein are used to distinguish between tumors and non-tumor cells or tissues or to identify tumor types, subtypes or grades. In one embodiment, the anti-FOLR1 antibodies provided herein and / or the FOLR1 detection assays provided herein can be used to distinguish subtypes of non-small cell lung cancer (NSCLC), including adenocarcinoma and squamous cell carcinoma as described herein. In another embodiment, the anti-FOLR1 antibodies provided herein and / or the FOLR1 detection assays provided herein can be used to exclude a certain type of cancer (e.g., for determining that a cell or tissue is not a certain type of cancer), such as a sarcoma.

[0010] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can specifically bind to an epitope of FOLR1, wherein the epitope comprises at least one, at least two, or three N-glycosylated amino acids. Glycosylation may be critical for membrane localization. See, e.g., Yan et al., J. Am. Soc. Nephol. 13: 1385-1389 (2002). Advantageously, the antibodies and antigen-binding fragments herein can detect FOLR1 expression on cell membranes and detect a clinically relevant dynamic range of FOLR1. The more cautious staining obtained with the antibodies and antigen-binding fragments provided herein allows the use of antibodies that bind to different FOLR1 epitopes, lack sufficient specificity, and / or lack sufficient sensitivity to distinguish samples that are all grouped together as high expression levels (having a score of 3).

[0011] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can specifically bind to the same FOLR1 epitope as an antibody selected from the group consisting of: (a) an antibody comprising a polypeptide of SEQ ID NO: 27 and a polypeptide of SEQ ID NO: 28; (b) an antibody comprising a polypeptide of SEQ ID NO: 29 and a polypeptide of SEQ ID NO: 30; (c) an antibody comprising a polypeptide of SEQ ID NO: 31 and a polypeptide of SEQ ID NO: 32; (d) an antibody comprising a polypeptide of SEQ ID NO: 62 and a polypeptide of SEQ ID NO: 63 or SEQ ID NO: 64; and (e) an antibody comprising a polypeptide of SEQ ID NO: 65 and a polypeptide of SEQ ID NO: 66 or SEQ ID NO: 67. In some embodiments, the epitope comprises an N-glycosylated amino acid.

[0012] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can specifically bind to FOLR1, wherein the antibodies or fragments thereof competitively inhibit the binding of an antibody selected from the group consisting of: (a) an antibody comprising a polypeptide of SEQ ID NO:27 and a polypeptide of SEQ ID NO:28; (b) an antibody comprising a polypeptide of SEQ ID NO:29 and a polypeptide of SEQ ID NO:30; (c) an antibody comprising a polypeptide of SEQ ID NO:31 and a polypeptide of SEQ ID NO:32; (d) an antibody comprising a polypeptide of SEQ ID NO:62 and a polypeptide of SEQ ID NO:63 or SEQ ID NO:64; and (e) an antibody comprising a polypeptide of SEQ ID NO:65 and a polypeptide of SEQ ID NO:66 or SEQ ID NO:67.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VHCDR1-3 and a VL CDR1-3 polypeptide sequence selected from the group consisting of: (a) SEQ ID NOs: 3-8, respectively; (b) SEQ ID NOs: 9-14, respectively; (c) SEQ ID NOs: 15-20, respectively; (d) SEQ ID NOs: 21-26, respectively; (e) SEQ ID NOs: 3-5 and SEQ ID NOs: 59, 7, and 8, respectively; (f) SEQ ID NOs: 3, 60, and 5, and SEQ ID NOs: 6-8, respectively; (g) SEQ ID NOs: 3, 61, and 5, and SEQ ID NOs: 6-8, respectively; (h) SEQ ID NOs: 3, 60, and 5, and SEQ ID NOs: 59, 7, and 8, respectively; and (i) SEQ ID NOs: 3, 61, and 5, and SEQ ID NOs: 59, 7, and 8, respectively.

[0014] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to FOLR1, wherein the antibodies or fragments thereof comprise a VH CDR1-3 and a VL CDR1-3 polypeptide sequence selected from the group consisting of: (a) SEQ ID NOs: 3-8, respectively; (b) SEQ ID NOs: 9-14, respectively; (c) SEQ ID NOs: 15-20, respectively; (d) SEQ ID NOs: 21-26, respectively; (e) SEQ ID NOs: 3-5 and SEQ ID NOs: 59, 7, and 8, respectively; (f) SEQ ID NOs: 3, 60, and 5, and SEQ ID NOs: 6-8, respectively; (g) SEQ ID NOs: 3, 61, and 5, and SEQ ID NOs: 6-8, respectively; (h) SEQ ID NOs: 3, 60, and 5, and SEQ ID NOs: 59, 7, and 8, respectively; (i) SEQ ID NOs: 3, 61, and 5, and ... NO: 59, 7 and 8; and (j) variants of (a) to (i) comprising 1, 2, 3 or 4 conservative amino acid substitutions.

[0015] In some embodiments, the antibody or its antigen-binding fragment comprises a polypeptide sequence that is at least 90%, at least 95% or at least 99% identical to a polypeptide sequence selected from the group consisting of: (a) SEQ ID NO:27 and SEQ ID NO:28; (b) SEQ ID NO:29 and SEQ ID NO:30; (c) SEQ ID NO:31 and SEQ ID NO:32; (d) SEQ ID NO:62 and SEQ ID NO:63 or SEQ ID NO:64; (e) SEQ ID NO:65 and SEQ ID NO:66 or SEQ ID NO:67; (f) SEQ ID NO:68 and SEQ ID NO:69. In some embodiments, the polypeptide sequence comprises or consists essentially of or consists of amino acids of a sequence selected from the group consisting of: (a) SEQ ID NO:27 and SEQ ID NO:28; (b) SEQ ID NO:29 and SEQ ID NO:30; (c) SEQ ID NO:31 and SEQ ID NO:32; (d) SEQ ID NO:62 and SEQ ID NO:63 or SEQ ID NO:64; (e) SEQ ID NO:65 and SEQ ID NO:66 or SEQ ID NO:67; (f) SEQ ID NO:68 and SEQ ID NO:69.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can specifically bind to FOLR1, wherein the antibodies or fragments thereof comprise a humanized heavy chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3 regions, the CDR1, CDR2 and CDR3 regions comprising amino acids of SEQ ID NO: 51, SEQ ID NO: 52 or 53 and SEQ ID NO: 54, respectively; a humanized light chain variable region, the light chain variable region comprising CDR1, CDR2 and CDR3 regions, the CDR1, CDR2 and CDR3 regions comprising amino acids of SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, respectively; and a murine constant region. In some embodiments, the humanized heavy chain variable region comprises amino acids of SEQ ID NO: 45, and the humanized light chain variable region comprises amino acids of SEQ ID NO: 47.

[0017] In some embodiments, the antibody or antigen binding fragment thereof is recombinantly produced. In some embodiments, the antibody or antigen binding fragment thereof is mouse, non-human, humanized, chimeric, surface reconstructed or human. In some embodiments, the antibody or antigen binding fragment thereof binds to human FOLR1, but does not bind to FOLR2 or FOLR3. In some embodiments, the antibody or antigen binding fragment thereof is a full-length antibody. In some embodiments, the antibody or antigen binding fragment thereof is an antigen binding fragment. In some embodiments, the antibody or antigen binding fragment thereof comprises, consists essentially of, or consists of the following: Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, mini antibody, F(ab')3, tetrabodies, triabodies, diabodies, single-domain antibodies, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0018] In some embodiments, the polypeptide provided herein can specifically bind to FOLR1, wherein the polypeptide comprises a sequence selected from the group consisting of: (a) SEQ ID NOs: 3-8, respectively; (b) SEQ ID NOs: 9-14, respectively; (c) SEQ ID NOs: 15-20, respectively; (d) SEQ ID NOs: 21-26, respectively; (e) SEQ ID NOs: 3-5 and SEQ ID NOs: 59, 7, and 8, respectively; (f) SEQ ID NOs: 3, 60, and 5, respectively, and SEQ ID NOs: 6-8; (g) SEQ ID NOs: 3, 61, and 5, respectively, and SEQ ID NOs: 6-8; (h) SEQ ID NOs: 3, 60, and 5, respectively, and SEQ ID NOs: 59, 7, and 8; (i) SEQ ID NOs: 3, 61, and 5, respectively, and SEQ ID NOs: 59, 7, and 8; and (j) variants of (a) to (i) comprising 1, 2, 3, or 4 conservative amino acid substitutions. In some embodiments, the polypeptide comprises a sequence that is at least 90%, at least 95%, or at least 99% identical to a sequence selected from the group consisting of: (a) SEQ ID NO:27 and SEQ ID NO:28; (b) SEQ ID NO:29 and SEQ ID NO:30; (c) SEQ ID NO:31 and SEQ ID NO:32; (d) SEQ ID NO:62 and SEQ ID NO:63 or SEQ ID NO:64; (e) SEQ ID NO:65 and SEQ ID NO:66 or SEQ ID NO:67; and (f) SEQ ID NO:68 and SEQ ID NO:69. In some embodiments, the polypeptide comprises amino acids of: (a) SEQ ID NO:27 and SEQ ID NO:28; (b) SEQ ID NO:29 and SEQ ID NO:30; (c) SEQ ID NO:31 and SEQ ID NO:32; (d) SEQ ID NO:62 and SEQ ID NO:63 or SEQ ID NO:64; (e) SEQ ID NO:65 and SEQ ID NO:66 or SEQ ID NO:67; or (f) SEQ ID NO:68 and SEQ ID NO:69.

[0019] In some embodiments, the antibody, antigen-binding fragment thereof, or polypeptide binds to FOLR1 with a Kd of about 0.5 to about 10 nM. In some embodiments, the antibody, antigen-binding fragment thereof, or polypeptide binds to human FOLR1 with a Kd of about 1.0 nM or better. In some embodiments, binding affinity is measured by flow cytometry, Biacore, ELISA, or radioimmunoassay.

[0020] In some embodiments, the antibody, antigen-binding fragment thereof, or polypeptide binds to an epitope of FOLR1 that comprises an amino acid that is N-glycosylated.

[0021] In some embodiments, the antibody, antigen-binding fragment thereof, or polypeptide is detectably labeled.

[0022] In some embodiments, the cells provided herein produce the antibodies, antigen-binding fragments thereof, or polypeptides. In some embodiments, the cells are isolated.

[0023] Also provided is a method for preparing the antibody, its antigen-binding fragment or polypeptide. The method may include (a) culturing the cells provided herein; and (b) isolating the antibody, its antigen-binding fragment or polypeptide from the cultured cells.

[0024] Also provided are compositions comprising the antibody, antigen-binding fragment thereof, or polypeptide.In some embodiments, the composition comprises the antibody, antigen-binding fragment thereof, or polypeptide and a buffer selected from the group consisting of: FACS buffer, IHC buffer, and ELISA buffer.

[0025] Also provided are methods of using the antibodies, antigen-binding fragments thereof, or polypeptides.

[0026] In some embodiments, the method of detecting FOLR1 expression in a sample comprises contacting the sample with an antibody, an antigen-binding fragment thereof, a polypeptide or a composition provided herein. In some embodiments, the antibody or antigen-binding fragment thereof is detectably labeled. In some embodiments, the label is selected from the group consisting of: immunofluorescence labeling, chemiluminescent labeling, phosphorescent labeling, enzyme labeling, radioactive labeling, avidin / biotin, colloidal gold particles, colored particles and magnetic particles. In some embodiments, FOLR1 expression is determined by radioimmunoassay, Western blot assay, cell counting, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescent assay or immunohistochemical assay. In some embodiments, cell counting is flow cytometry. In some embodiments, FOLR1 expression is determined by IHC.

[0027] In some embodiments, a method of increasing the efficacy of a cancer therapy using an agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof comprises administering the agent to a subject having cancer, wherein increased FOLR1 expression has been detected in a cancerous sample from the subject using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein.

[0028] In some embodiments, a method for identifying a cancer that may be responsive to an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof comprises: (a) contacting a biological sample comprising cells from the cancer with an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; (b) detecting binding of the antibody, antibody fragment, or polypeptide to FOLR1 in the biological sample of (a); (c) assigning a score to the binding of step (b), wherein the score is assigned based on a comparison with one or more reference samples; and (d) comparing the score in step (c) to a score of a reference tissue or cell, wherein the score for the cancer FOLR1 level is greater than the score for a reference sample expressing normal or low FOLR1 or the score for the cancer FOLR1 level is equal to or greater than the score for a reference sample expressing high FOLR1, identifying the cancer as being likely to be responsive to an anti-FOLR1 antibody.

[0029] In some embodiments, a method of treating a patient suffering from cancer comprises: (a) determining a FOLR1 expression score from detection of FOLR1 expression in a cancerous sample obtained from the patient, wherein the detection is performed using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (b) if the score indicates that the patient would benefit from administration of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, administering the active agent to the patient.

[0030] In some embodiments, a method of treating a patient having cancer comprises: (a) determining a FOLR1 expression score from detection of FOLR1 expression in a cancerous sample obtained from the patient, wherein the detection is performed using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (b) if the score indicates that the patient would benefit from administration of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, notifying a healthcare provider to administer the active agent to the patient.

[0031] In some embodiments, a method of treating a patient having cancer comprises: (a) submitting a cancerous sample obtained from a patient having cancer for determination of a FOLR1 expression score from detection of FOLR1 expression using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (b) if the score indicates that the patient would benefit from administration of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, administering the active agent to the subject.

[0032] In some embodiments, a method of treating a patient suffering from cancer comprises: (a) detecting FOLR1 expression in a cancerous sample obtained from the patient, wherein the detecting is performed using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; (b) determining a FOLR1 expression score for the cancerous sample; and (c) if the score indicates that the patient would benefit from administration of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, administering the active agent to the patient.

[0033] In some embodiments, a method of treating a patient suffering from cancer comprises: (a) administering to the patient a fixed dose of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof; (b) detecting FOLR1 in the patient relative to the level of FOLR1 in a reference sample, wherein the detecting is performed using an antibody, antigen-binding fragment thereof, polypeptide or composition provided herein; and (c) if the patient's FOLR1 level is elevated, increasing the amount or frequency of subsequent fixed doses.

[0034] In some embodiments, a method of optimizing a treatment regimen for a subject having cancer using an agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof comprises: (a) administering an increasing dose of an agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof to a subject having cancer, wherein increased FOLR1 expression in the subject has been detected using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; or (b) administering a decreasing dose of the agent to a subject having cancer, wherein decreased FOLR1 expression in the subject has been detected.

[0035] In some embodiments, a method for optimizing a treatment regimen for a subject having cancer using an agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof comprises: (a) detecting the level of FOLR1 expression in a cancerous sample from the subject using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; (b) determining the FOLR1 expression score of the cancerous sample; and (c) administering to the subject an increased dose of the agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof if the score is low, or administering to the subject a decreased dose of the agent if the score is high.

[0036] In some embodiments, a method of reducing FOLR1-expressing cancer cells in a cancer patient comprises: (a) using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein, detecting the level of FOLR1 in a cancerous sample taken from a patient compared to the level of FOLR1 in a reference sample; and (b) if the patient's FOLR1 level is elevated compared to the reference sample, administering to the patient a fixed dose of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof; wherein the administration of the active agent reduces the number of FOLR1-expressing cancer cells in the patient. In some embodiments, a method of treating cancer in a patient comprises: (a) using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein, detecting the level of FOLR1 in a cancerous sample taken from a patient compared to the level of FOLR1 in a reference sample; and (b) if the patient's FOLR1 level is elevated compared to the reference sample, administering to the patient a fixed dose of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof; wherein the administration of the active agent reduces the size of a tumor expressing FOLR1 or reduces CA125 levels.

[0037] In some embodiments, a method of reducing FOLR1-expressing cancer cells in a cancer patient comprises: (a) administering a fixed dose of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof to a patient having cancer; (b) detecting the patient's FOLR1 level relative to the FOLR1 level in a reference sample using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (c) increasing the amount or frequency of subsequent fixed doses if the patient's FOLR1 level is elevated compared to the reference sample; wherein the administration of the active agent reduces the number of FOLR1-expressing cancer cells in the patient. In some embodiments, a method of treating cancer in a patient comprises: (a) administering a fixed dose of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof to a patient having cancer; (b) detecting the patient's FOLR1 level relative to the FOLR1 level in a reference sample using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (c) increasing the amount or frequency of subsequent fixed doses if the patient's FOLR1 level is elevated compared to the reference sample; wherein the administration of the active agent reduces the size of a tumor expressing FOLR1 or reduces CA125 levels.

[0038] In some embodiments, a method for monitoring the therapeutic efficacy of a fixed dose of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof in a patient comprises: (a) detecting a first FOLR1 level in a biological sample from a patient suffering from cancer using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; (b) administering a fixed dose of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof to the patient; (c) detecting a second FOLR1 level in a biological sample from the patient after administration of the active agent, wherein the detecting is performed using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (d) comparing the second FOLR1 level to the first FOLR1 level; wherein a decrease between the first and second FOLR1 levels indicates therapeutic efficacy.

[0039] In some embodiments, a method of identifying a subject having cancer as likely to respond to a low-dose anti-FOLR1 treatment regimen comprises: (a) contacting a biological sample comprising cells from the cancer with an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; (b) detecting binding of the antibody, antigen-binding fragment, or polypeptide to the biological sample of (a); (c) assigning a score to the binding of step (b), wherein the score is assigned based on a comparison with one or more reference samples; and (d) comparing the score in step (c) to a score of a reference tissue or cell, wherein the score for the cancer FOLR1 level is greater than the score for a reference sample expressing normal or low FOLR1 or the score for the cancer FOLR1 level is equal to or greater than the score for a reference sample expressing high FOLR1, identifying the cancer as likely to respond to a low-dose anti-FOLR1 treatment.

[0040] In some embodiments, a method of identifying a cancer as sensitive to treatment with an anti-FOLR1 active agent comprises: (a) detecting the level of FOLR1 expression in a cancerous sample from the cancer using an antibody, antigen-binding fragment thereof, polypeptide or composition provided herein, wherein the detecting comprises using a method for distinguishing the staining intensity or staining evenness in a cancerous sample expressing FOLR1 compared to the staining intensity or staining evenness in one or more reference samples; (b) determining the FOLR1 staining intensity or staining evenness score of the cancerous sample; and (c) comparing the FOLR1 staining intensity or staining evenness score determined in step (b) with a relative value determined by measuring FOLR1 protein expression in at least one reference sample, wherein the at least one reference sample is a tissue, cell or cell mass sample that is not sensitive to treatment with an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, and wherein the FOLR1 staining intensity score of the cancerous sample determined in step (b) is higher than the relative value to identify the cancer as sensitive to treatment with the active agent.

[0041] In some embodiments, a method of identifying a cancer as sensitive to treatment with an anti-FOLR1 agent comprises: (a) detecting the level of FOLR1 expression in a cancerous sample from the cancer using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein, wherein the detecting comprises using a method of staining membrane FOLR1 in a cancerous sample that specifically expresses FOLR1 compared to membrane FOLR1 in one or more reference samples; (b) determining a FOLR1 score for the cancerous sample; and (c) comparing the FOLR1 score determined in step (b) with a relative value determined by measuring FOLR1 in at least one reference sample, wherein the at least one reference sample is a tissue, cell, or cell mass sample that is not sensitive to treatment with an agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, and wherein a FOLR1 score for the cancerous sample determined in step (b) that is higher than the relative value identifies the cancer as sensitive to treatment with the agent.

[0042] In some embodiments, a method of identifying a cancer as sensitive to treatment with an anti-FOLR1 agent comprises: (a) detecting the level of FOLR1 expression in a cancerous sample from the cancer using an antibody, antigen-binding fragment thereof, polypeptide or composition provided herein, wherein the detecting comprises using a method for distinguishing the staining intensity or staining evenness in a cancerous sample expressing FOLR1 compared to the staining intensity or staining evenness in one or more reference samples; (b) determining the FOLR1 staining intensity or staining evenness score of the cancerous sample; and (c) comparing the FOLR1 staining intensity or staining evenness score determined in step (b) with a relative value determined by measuring FOLR1 protein expression in at least one reference sample, wherein the at least one reference sample is a tissue, cell or cell mass sample that is sensitive to treatment with an agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, and wherein a FOLR1 staining intensity score of the cancerous sample determined in step (b) that is greater than or equal to the relative value identifies the cancer as sensitive to treatment with the agent.

[0043] In some embodiments, a method of identifying a cancer as sensitive to treatment with an anti-FOLR1 agent comprises: (a) detecting the level of FOLR1 expression in a cancerous sample from the cancer using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein, wherein the detecting comprises using a method to specifically stain membrane FOLR1 in a cancerous sample expressing FOLR1 compared to membrane FOLR1 in one or more reference samples; (b) determining a FOLR1 score for the cancerous sample; and (c) comparing the FOLR1 score determined in step (b) with a relative value determined by measuring FOLR1 in at least one reference sample, wherein the at least one reference sample is a tissue, cell, or cell mass sample that is sensitive to treatment with an agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, and wherein a FOLR1 score for the cancerous sample determined in step (b) that is greater than or equal to the relative value identifies the cancer as sensitive to treatment with the agent.

[0044] In some embodiments, the method further comprises administering an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof to the subject from which the cancerous sample or biological sample was obtained.

[0045] In some embodiments, the FOLR1 level of the patient is detected in a cancerous sample or biological sample obtained from the patient. In some embodiments, the cancerous sample or biological sample is a body fluid, cell or tissue sample. In some embodiments, the cell is a circulating tumor cell. In some embodiments, the body fluid is blood, ascites, urine, plasma, serum or peripheral blood.

[0046] In some embodiments, the FOLR1 is membrane-localized FOLR1.

[0047] In some embodiments, the FOLR1 is shedding FOLR1.

[0048] In some embodiments, the detecting is performed by enzyme-linked immunosorbent assay (ELISA).

[0049] In some embodiments, the detection is performed by immunohistochemistry (IHC). In some embodiments, the IHC is a calibrated IHC that can distinguish different levels of FOLR1 expression. In some embodiments, the IHC produces a range of staining intensities for samples with low cell surface FOLR1 expression, medium FOLR1 cell surface expression, or high FOLR1 cell surface expression. In some embodiments, the IHC distinguishes staining intensity and uniformity in cancerous samples or biological samples expressing FOLR1 compared to reference samples. In some embodiments, the IHC detects membrane FOLR1. In some embodiments, the IHC is performed manually. In some embodiments, the IHC is performed using an automated system.

[0050] In some embodiments, the FOLR1 score is determined from the IHC.

[0051] In some embodiments, a score of at least 1 indicates increased FOLR1 expression and identifies the cancer as likely to be responsive to an agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof.

[0052] In some embodiments, a score of at least 2, at least 2 homogeneous (>75% uniformity), or at least 2 heterogeneous (25%-75% uniformity) identifies the cancer as likely to respond to an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer or endometrial cancer. In some embodiments, a score of at least 3, at least 3 homogeneous (>75% uniformity), or at least 3 heterogeneous (25%-75% uniformity) identifies the cancer as likely to respond to an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0053] In some embodiments, an H-score of at least 50 identifies a cancer as likely to respond to an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, an H-score of at least 75 identifies an ovarian cancer as likely to respond to an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, an H-score of at least 50 identifies NSCLC as likely to respond to an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, an H-score of at least 50 identifies an endometrial cancer as likely to respond to an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In one embodiment, the H-score is determined using a FOLR1-2.1 antibody.

[0054] In some embodiments, at least 25% of FOLR1 membrane expression with an intensity of at least 3 in ovarian tumor samples identifies ovarian cancer as likely to respond to an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, at least 25% of FOLR1 membrane expression with an intensity of at least 2 in NSCLC samples identifies NSCLC as likely to respond to an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, at least 25% of FOLR1 membrane expression with an intensity of at least 2 in endometrial tumor samples identifies endometrial cancer as likely to respond to an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In one embodiment, the expression score is determined using the FOLR1-2.1 antibody.

[0055] In some embodiments, a score of at least 1 indicates increased FOLR1 expression and indicates that the patient will benefit from the administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, a score of at least 2, at least 2 homogeneous (>75% uniformity), or at least 2 heterogeneous (25%-75% uniformity) indicates that the patient will benefit from the administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer or endometrial cancer. In some embodiments, a score of at least 3, at least 3 homogeneous (>75% uniformity), or at least 3 heterogeneous (25%-75% uniformity) indicates that the patient will benefit from the administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0056] In some embodiments, an H-score of at least 50 indicates that a patient will benefit from administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, an H-score of at least 75 indicates that a patient with ovarian cancer will benefit from administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, an H-score of at least 50 indicates that a patient with NSCLC will benefit from administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, an H-score of at least 50 indicates that a patient with endometrial cancer will benefit from administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In one embodiment, the H-score is determined using a FOLR1-2.1 antibody.

[0057] In some embodiments, at least 25% of FOLR1 membrane expression with an intensity of at least 3 in an ovarian tumor sample indicates that the patient will benefit from administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, at least 25% of FOLR1 membrane expression with an intensity of at least 2 in an NSCLC sample indicates that the patient will benefit from administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, at least 25% of FOLR1 membrane expression with an intensity of at least 2 in an endometrial tumor sample indicates that the patient will benefit from administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In one embodiment, the expression score is determined using the FOLR1-2.1 antibody.

[0058] In some embodiments, a score of at least 1 indicates increased FOLR1 expression. In some embodiments, a score of at least 2, at least 2 homogeneous (>75% uniformity), or at least 2 heterogeneous (25%-75% uniformity) indicates that a reduced dose of the active agent should be administered. In some embodiments, the cancer is lung cancer or endometrial cancer. In some embodiments, a score of at least 3, at least 3 homogeneous (>75% uniformity), or at least 3 heterogeneous (25%-75% uniformity) indicates that a reduced dose of the active agent should be administered. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0059] In some embodiments, a score of at least 1 indicates increased FOLR1 expression. In some embodiments, a score of at least 2, at least 2 homogeneous (>75% uniformity), or at least 2 heterogeneous (25%-75% uniformity) identifies the cancer as likely to respond to low-dose anti-FOLR1 treatment. In some embodiments, the cancer is lung cancer or endometrial cancer. In some embodiments, a score of at least 3, at least 3 homogeneous (>75% uniformity), or at least 3 heterogeneous (25%-75% uniformity) identifies the cancer as likely to respond to low-dose anti-FOLR1 treatment. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0060] In some embodiments, a score of at least 2, at least 2 homogeneous (>75% uniformity), or at least 2 heterogeneous (25%-75% uniformity) identifies the cancer as sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer or endometrial cancer. In some embodiments, a score of at least 3, at least 3 homogeneous (>75% uniformity), or at least 3 heterogeneous (25%-75% uniformity) identifies the cancer as sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0061] In some embodiments, an H-score of at least 50 identifies a cancer as being sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, an H-score of at least 75 identifies an ovarian cancer as being sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, an H-score of at least 50 identifies NSCLC as being sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, an H-score of at least 50 identifies an endometrial cancer as being sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In one embodiment, the H-score is determined using a FOLR1-2.1 antibody.

[0062] In some embodiments, at least 25% membrane expression of FOLR1 with an intensity of at least 3 in ovarian tumor samples identifies the cancer as sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, at least 25% membrane expression of FOLR1 with an intensity of at least 2 in NSCLC samples identifies the cancer as sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, at least 25% membrane expression of FOLR1 with an intensity of at least 2 in endometrial tumor samples identifies the cancer as sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In one embodiment, the expression score is determined using the FOLR1-2.1 antibody.

[0063] In some embodiments, the reference sample is a positive reference sample or a negative reference sample. In some embodiments, the reference sample comprises a cell, a cell mass or a tissue.

[0064] In some embodiments, the antibody, antigen-binding fragment thereof, or polypeptide comprises a detection agent selected from the group consisting of an enzyme, a fluorophore, a radiolabel, and a luminophore. In some embodiments, the detection agent is selected from the group consisting of biotin, digoxigenin, fluorescein, tritium, and rhodamine.

[0065] In some embodiments, the cancer is a FOLR1 positive cancer. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, brain cancer, breast cancer, uterine cancer, endometrial cancer, pancreatic cancer, kidney cancer, and lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer or bronchioloalveolar carcinoma. In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, the ovarian cancer is platinum-resistant, relapsed, or refractory.

[0066] In some embodiments, FOLR1 expression is detected using at least one additional anti-FOLR1 antibody or antigen-binding fragment thereof. In some embodiments, FOLR1 expression is measured using two anti-FOLR1 antibodies or antigen-binding fragments thereof. In some embodiments, at least one antibody or antigen-binding fragment thereof is bound to a solid support. In some embodiments, at least one antibody or antigen-binding fragment thereof is bound to a microtiter plate.

[0067] In some embodiments, at least one additional antibody or antigen-binding fragment thereof comprises a detection agent. In some embodiments, the detection agent is a chromogenic detection agent, a fluorescent detection agent, an enzyme detection agent, or an electrochemiluminescent detection agent. In some embodiments, the detection agent is horseradish peroxidase (HRP).

[0068] In some embodiments, the ELISA is a sandwich ELISA.

[0069] In some embodiments, the active agent comprises the FOLR1 antibody huMov19. In some embodiments, the active agent is an antibody maytansinoid conjugate comprising the FOLR1 antibody huMov19 (comprising a heavy chain variable region of SEQ ID NO: 45 and a light chain variable region of SEQ ID NO: 47), maytansinoid DM4, and a cleavable sulfo-SPDB linker (IMGN853).

[0070] In some embodiments, a method of identifying a cancer as likely to respond to treatment with an antibody maytansinoid conjugate comprising the FOLR1 antibody huMov19, maytansinoid DM4, and a sulfo-SPDB linker (IMGN853) comprises measuring FOLR1 in an IHC assay using an antibody comprising a heavy chain comprising amino acids of SEQ ID NO:27 and a light chain comprising amino acids of SEQ ID NO:28, wherein a heterogeneous score of at least 2 indicates that the cancer is likely to respond to the treatment.

[0071] In some embodiments, a method of identifying a cancer as likely to respond to treatment with an antibody maytansinoid conjugate comprising the FOLR1 antibody huMov19, maytansinoid DM4, and a sulfo-SPDB linker (IMGN853) comprises measuring FOLR1 in an IHC assay using an antibody comprising a heavy chain comprising amino acids of SEQ ID NO:27 and a light chain comprising amino acids of SEQ ID NO:28, wherein a heterogeneous score of at least 1 indicates that the cancer is likely to respond to the treatment.

[0072] In some embodiments, the articles provided herein include a therapeutic agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof as described herein, a container, and a package insert or label indicating that the agent can be used to treat a cancer characterized by increased expression of FOLR1. In some embodiments, the articles provided herein include a therapeutic agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof as described herein, a container, and a package insert or label indicating that the agent can be used to treat a cancer characterized by expression of FOLR1 at a level of 2 or 3 measured using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein. In some embodiments, the anti-FOLR1 antibody of the agent is coupled to a cytotoxin. In some embodiments, the package insert or label indicates that the agent can be used to treat a cancer characterized by expression of FOLR1 at a level of at least 1. In some embodiments, the package insert or label indicates that the agent can be used to treat a cancer characterized by expression of FOLR1 at a level of at least 2, at least 2 homogeneous (>75% uniformity), or at least 2 heterogeneous (25%-75% uniformity). In some embodiments, the cancer is lung cancer or endometrial cancer. In some embodiments, the package insert or label indicates that the active agent can be used to treat a cancer characterized by expression of FOLR1 at a level of at least 3, at least 3 homogeneous (>75% uniformity), or at least 3 heterogeneous (25%-75% uniformity). In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0073] In some embodiments, the combined diagnostic and pharmaceutical kits provided herein include an antibody, antigen-binding fragment thereof, polypeptide or composition provided herein for use in diagnosis, and an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof for use in treatment. In some embodiments, the detection antibody is capable of detecting FOLR1 expression by IHC. In some embodiments, the detection antibody is capable of detecting FOLR1 expression by ELISA. In some embodiments, the anti-FOLR1 antibody in the active agent is coupled to a cytotoxin.

[0074] In some embodiments, the diagnostic kit provided herein includes an antibody, an antigen-binding fragment thereof, or a polypeptide provided herein, a reagent for immunohistochemistry (IHC), and one or more standardized reference samples, wherein the standardized reference samples include cells, cell aggregates, or formalin-fixed paraffin-embedded tissue samples, and wherein the one or more standardized reference samples are from cells, cell aggregates, or tissues with no FOLR1 expression, low FOLR1 expression, or high FOLR1 expression.

[0075] In some embodiments, an immunoassay kit for detecting shed FOLR1 in a sample comprises: (a) an antibody, an antigen-binding fragment thereof, a polypeptide or a composition provided herein, and (b) a detection reagent. In some embodiments, the kit further comprises a solid support for a capture reagent. In some embodiments, the capture reagent is immobilized on a solid support. In some embodiments, the capture reagent is coated on a microtiter plate. In some embodiments, the detection reagent is a second FOLR1 antibody. In some embodiments, the detection reagent is detected using a species-specific antibody. In some embodiments, the kit further comprises a detection device for the detection reagent. In some embodiments, the detection device is colorimetric. In some embodiments, the kit further comprises a FOLR1 polypeptide as an antigen standard. In some embodiments, the FOLR1 polypeptide is FOLR1-Fc.

[0076] Active agents are also provided herein. In some embodiments, the active agent comprises an anti-FOLR1 antibody or antigen-binding fragment thereof for use in a method of treating cancer, wherein the active agent is administered to a subject having cancer, wherein increased FOLR1 expression has been detected in a cancerous sample obtained from the subject using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein.

[0077] In some embodiments, the active agent comprises an anti-FOLR1 antibody or antigen-binding fragment thereof for use in a method of treating cancer, the method comprising: (a) determining a FOLR1 expression score from detection of FOLR1 expression in a cancerous sample obtained from the patient, wherein the detection is performed using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (b) administering the active agent to the patient if the score indicates that the patient would benefit from administration of the active agent comprising the anti-FOLR1 antibody or antigen-binding fragment thereof.

[0078] In some embodiments, the active agent comprises an anti-FOLR1 antibody or antigen-binding fragment thereof for use in a method of treating cancer, the method comprising: (a) determining a FOLR1 expression score from detection of FOLR1 expression in a cancerous sample obtained from the patient, wherein the detection is performed using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (b) if the score indicates that the patient would benefit from administration of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, notifying a healthcare provider to administer the active agent to the patient.

[0079] In some embodiments, the active agent comprises an anti-FOLR1 antibody or antigen-binding fragment thereof for use in a method of treating cancer, the method comprising: (a) submitting a cancerous sample obtained from a patient suffering from cancer for determination of a FOLR1 expression score from detection of FOLR1 expression using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (b) administering the active agent to the patient if the score indicates that the patient would benefit from administration of the active agent comprising the anti-FOLR1 antibody or antigen-binding fragment thereof.

[0080] In some embodiments, the active agent comprises an anti-FOLR1 antibody or antigen-binding fragment thereof for use in a method of treating cancer, the method comprising: (a) detecting FOLR1 expression in a cancerous sample obtained from the patient, wherein the detecting is performed using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; (b) determining a FOLR1 expression score for the cancerous sample; and (c) administering the active agent to the patient if the score indicates that the patient would benefit from administration of the active agent comprising the anti-FOLR1 antibody or antigen-binding fragment thereof.

[0081] In some embodiments, the active agent comprises an anti-FOLR1 antibody or an antigen-binding fragment thereof for use in a method of treating cancer, the method comprising: (a) administering a fixed dose of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof to a patient; (b) detecting the level of FOLR1 expression in a cancerous sample obtained from the patient relative to the level of FOLR1 in a reference sample, wherein the detecting is performed using an antibody, antigen-binding fragment thereof, polypeptide or composition provided herein; and (c) if the patient's FOLR1 level is elevated, increasing the amount or frequency of subsequent fixed doses.

[0082] In some embodiments, an active agent comprises an anti-FOLR1 antibody or antigen-binding fragment thereof for use in a method of treating cancer, the method comprising the step of optimizing a treatment regimen of the active agent, the step comprising: (a) administering an increasing dose of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof to a subject having cancer, wherein increased FOLR1 expression has been detected in a cancerous sample from the subject using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; or (b) administering a decreasing dose of the active agent to a subject having cancer, wherein decreased FOLR1 expression has been detected in a cancerous sample from the subject.

[0083] In some embodiments, an active agent comprises an anti-FOLR1 antibody or antigen-binding fragment thereof for use in a method of treating cancer, the method comprising the step of optimizing a treatment regimen of the active agent, the steps comprising: (a) detecting the level of FOLR1 expression in a cancerous sample from the subject using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; (b) determining the FOLR1 expression score of the cancerous sample; and (c) administering to the subject an increased dose of the active agent comprising the anti-FOLR1 antibody or antigen-binding fragment thereof if the score is low, or administering to the subject a decreased dose of the active agent if the score is high.

[0084] In some embodiments, the active agent comprises an anti-FOLR1 antibody or an antigen-binding fragment thereof for use in a method of treating cancer, wherein FOLR1-expressing cancer cells in a cancer patient are reduced, wherein (a) the level of FOLR1 in a cancerous sample obtained from the patient is detected by comparing the level of FOLR1 in the cancerous sample obtained from the patient with the level of FOLR1 in a reference sample using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (b) if the patient's FOLR1 level is elevated, a fixed dose of the active agent is administered to the patient; wherein administration of the active agent reduces the number of FOLR1-expressing cancer cells in the patient.

[0085] In some embodiments, the active agent comprises an anti-FOLR1 antibody or antigen-binding fragment thereof for use in a method of treating cancer, wherein FOLR1-expressing cancer cells in a cancer patient are reduced, wherein: (a) a fixed dose of the active agent is administered to a patient suffering from cancer; (b) the level of FOLR1 in a cancerous sample obtained from the patient is detected relative to the level of FOLR1 in a reference sample using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (c) if the patient's FOLR1 level is elevated compared to the reference sample, the amount or frequency of subsequent fixed doses is increased; wherein administration of the active agent reduces the number of FOLR1-expressing cancer cells in the patient.

[0086] Also provided herein are anti-FOLR1 antibodies and antigen-binding fragments thereof for use in monitoring methods and diagnostic methods. In some embodiments, the anti-FOLR1 antibody or antigen-binding fragment thereof is used in a method for monitoring the therapeutic efficacy of a fixed dose of an active agent in a patient, the method comprising: (a) detecting a first FOLR1 level in a biological sample from a patient suffering from cancer using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; (b) administering a fixed dose of the active agent to the patient; (c) detecting a second FOLR1 level in a biological sample from the patient after administration of the active agent, wherein the detecting is performed using an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; and (d) comparing the second FOLR1 level to the first FOLR1 level; wherein a decrease between the first and second FOLR1 levels indicates therapeutic efficacy.

[0087] In some embodiments, an anti-FOLR1 antibody or antigen-binding fragment thereof is used in a method for diagnosing whether a subject with cancer is likely to respond to a low-dose anti-FOLR1 treatment regimen, the method comprising: (a) contacting a biological sample containing cells from the cancer with an antibody, antigen-binding fragment thereof, polypeptide, or composition provided herein; (b) detecting binding of the antibody, antigen-binding fragment, or polypeptide to the biological sample of (a); (c) assigning a score to the binding of step (b), wherein the score is assigned based on a comparison with one or more reference samples; and (d) comparing the score in step (c) to a score of a reference tissue or cell, wherein the score for the cancer FOLR1 level is greater than the score for a reference sample expressing normal or low FOLR1 or the score for the cancer FOLR1 level is equal to or greater than the score for a reference sample expressing high FOLR1, identifying the cancer as likely to respond to a low dose of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof.

[0088] In some embodiments, an anti-FOLR1 antibody or an antigen-binding fragment thereof is used in a method for diagnosing whether a cancer is sensitive to treatment with an anti-FOLR1 antibody, the method comprising: (a) using an antibody, an antigen-binding fragment thereof, a polypeptide or a composition provided herein to detect the expression level of FOLR1 in a cancerous sample from the cancer, wherein the detection comprises a method for distinguishing the staining intensity or staining uniformity in a cancerous sample expressing FOLR1 compared to the staining intensity or staining uniformity in one or more reference samples; (b)

[0089] determining the FOLR1 staining intensity or staining evenness score of the cancerous sample; and (c) comparing the FOLR1 staining intensity or staining evenness score determined in step (b) with a relative value determined by measuring FOLR1 protein expression in at least one reference sample, wherein the at least one reference sample is a tissue, cell or cell mass sample that is not sensitive to treatment with an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof, and wherein the FOLR1 staining intensity score of the cancerous sample determined in step (b) is higher than the relative value to identify the cancer as being sensitive to treatment with the active agent.

[0090] In some embodiments, an anti-FOLR1 antibody or antigen-binding fragment thereof is used in a method for diagnosing whether a cancer is sensitive to treatment with an anti-FOLR1 antibody, the method comprising: (a) detecting the expression level of FOLR1 in a cancerous sample from the cancer using an antibody, antigen-binding fragment thereof, polypeptide or composition provided herein, wherein the detecting comprises using a method for distinguishing the staining intensity or staining uniformity in a cancerous sample expressing FOLR1 compared to the staining intensity or staining uniformity in one or more reference samples; (b) determining the FOLR1 staining intensity or staining uniformity score of the cancerous sample; and (c) comparing the FOLR1 staining intensity or staining uniformity score determined in step (b) with a relative value determined by measuring FOLR1 protein expression in at least one reference sample, wherein the at least one reference sample is a tissue, cell or cell mass sample that is sensitive to treatment with an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, and wherein a FOLR1 staining intensity score of the cancerous sample determined in step (b) that is higher than the relative value identifies the cancer as being sensitive to treatment with the active agent.

[0091] In some embodiments, the use of the active agent or anti-FOLR1 antibody or antigen-binding fragment thereof further comprises administering the active agent comprising the anti-FOLR1 antibody or antigen-binding fragment thereof to the subject from whom the cancerous sample or biological sample was obtained.

[0092] In some embodiments, the cancerous sample or biological sample is a body fluid, cell or tissue sample. In some embodiments, the cell is a circulating tumor cell. In some embodiments, the body fluid is blood, ascites, urine, plasma, serum or peripheral blood.

[0093] In some embodiments of the active agent or anti-FOLR1 antibody or antigen-binding fragment thereof, the detection is performed by enzyme-linked immunosorbent assay (ELISA) and / or by immunohistochemistry (IHC). In some embodiments, the IHC is a calibrated IHC that can distinguish different levels of FOLR1 expression. In some embodiments, the IHC produces a range of staining intensities for samples with low cell surface FOLR1 expression, medium FOLR1 cell surface expression, or high FOLR1 cell surface expression. In some embodiments, the IHC distinguishes staining intensity and uniformity in cancerous samples or biological samples expressing FOLR1 compared to reference samples. In some embodiments, the IHC is performed manually. In some embodiments, the IHC is performed using an automated system. In some embodiments, the FOLR1 score is determined from the IHC. In some embodiments, IHC using the antibodies or antigen-binding fragments described herein produces a series of staining for cells with increased FOLR1 expression, particularly those cells with a staining level equal to or greater than 2.

[0094] In some embodiments, a score of at least 2 indicates that the patient will benefit from administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, a score of at least 2 homogeneous (>75% uniformity) or at least 2 heterogeneous (25%-75% uniformity) indicates that the patient will benefit from administration of an active agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer or endometrial cancer.

[0095] In some embodiments, a score of at least 3 indicates that the patient will benefit from administration of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof. In some embodiments, a score of at least 3 homogeneous (>75% uniformity) or at least 3 heterogeneous (25%-75% uniformity) indicates that the patient will benefit from administration of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0096] In some embodiments, a score of at least 2 indicates that the patient will benefit from administration of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof. In some embodiments, a score of at least 2 homogeneous (>75% uniformity) or at least 2 heterogeneous (25%-75% uniformity) indicates that the patient will benefit from administration of an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0097] In some embodiments, a score of at least 2 indicates that a reduced dose of the active agent should be administered. In some embodiments, a score of at least 2 homogeneous (>75% uniformity) or at least 2 heterogeneous (25%-75% uniformity) indicates that a reduced dose of the active agent should be administered. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0098] In some embodiments, a score of at least 2 identifies the cancer as likely to respond to low-dose anti-FOLR1 therapy. In some embodiments, a score of at least 2 homogeneous (>75% uniformity) or 2 heterogeneous (25%-75% uniformity) identifies the cancer as likely to respond to low-dose anti-FOLR1 therapy. In some embodiments, the cancer is lung cancer or endometrial cancer.

[0099] In some embodiments, a score of at least 3 identifies the cancer as likely to respond to low-dose anti-FOLR1 therapy. In some embodiments, a score of at least 3 homogeneous (>75% uniformity) or at least 3 heterogeneous (25%-75% uniformity) identifies the cancer as likely to respond to low-dose anti-FOLR1 therapy. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0100] In some embodiments, a score of at least 2 identifies the cancer as likely to respond to low-dose anti-FOLR1 therapy. In some embodiments, a score of at least 2 homogeneous (>75% uniformity) or at least 2 heterogeneous (25%-75% uniformity) identifies the cancer as likely to respond to low-dose anti-FOLR1 therapy. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0101] In some embodiments, a score of at least 2 identifies the cancer as sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, a score of at least 2 homogeneous (>75% uniformity) or at least 2 heterogeneous (25%-75% uniformity) identifies the cancer as sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer or endometrial cancer.

[0102] In some embodiments, a score of at least 3 identifies the cancer as being sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, a score of at least 3 homogeneous (>75% uniformity) or at least 3 heterogeneous (25%-75% uniformity) identifies the cancer as being sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0103] In some embodiments, a score of at least 2 identifies the cancer as sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, a score of at least 2 homogeneous (>75% uniformity) or at least 2 heterogeneous (25%-75% uniformity) identifies the cancer as sensitive to treatment with an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer is lung cancer, endometrial cancer, or ovarian cancer.

[0104] In some embodiments, the reference sample is a positive reference sample or a negative reference sample. In some embodiments, the reference sample comprises a cell, a cell mass, or a tissue.

[0105] In some embodiments of the active agent or anti-FOLR1 antibody or antigen-binding fragment thereof for use provided herein, the antibody, antigen-binding fragment or polypeptide provided herein further comprises a detection agent selected from the group consisting of an enzyme, a fluorophore, a radiolabel and a luminophore. In some embodiments, the detection agent is selected from the group consisting of biotin, digoxigenin, fluorescein, tritium and rhodamine.

[0106] In some embodiments of the active agent or anti-FOLR1 antibody or antigen-binding fragment thereof for use provided herein, the cancer is a FOLR1-positive cancer. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, brain cancer, breast cancer, uterine cancer, endometrial cancer, pancreatic cancer, kidney cancer, and lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer or bronchioloalveolar carcinoma. In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, the cancer is platinum-resistant, recurrent, or refractory.

[0107] In some embodiments of the active agent or anti-FOLR1 antibody or antigen-binding fragment thereof for the purposes provided herein, FOLR1 expression is detected using at least one additional anti-FOLR1 antibody or antigen-binding fragment thereof. In some embodiments, FOLR1 expression is measured using two anti-FOLR1 antibodies or antigen-binding fragments thereof. In some embodiments, at least one antibody or antigen-binding fragment thereof is bound to a solid support. In some embodiments, at least one antibody or antigen-binding fragment thereof is bound to a microtiter plate. In some embodiments, at least one antibody or antigen-binding fragment thereof comprises a detection agent. In some embodiments, the detection agent is a chromogenic detection agent, a fluorescent detection agent, an enzyme detection agent, or an electrochemiluminescent detection agent. In some embodiments, the detection agent is horseradish peroxidase (HRP). In some embodiments, the ELISA is a sandwich ELISA.

[0108] In some embodiments of the active agent or anti-FOLR1 antibody or antigen-binding fragment thereof for use provided herein, the active agent comprises or is the FOLR1 antibody huMov19. In some embodiments, the active agent is administered as an antibody maytansinoid conjugate further comprising maytansinoid DM4 and a cleavable sulfo-SPDB linker (IMGN853).

[0109] In some embodiments, the antibodies, antigen-binding fragments, polypeptides, or compositions provided herein are used as diagnostic agents.

[0110] In some embodiments, the antibodies, antigen-binding fragments, polypeptides or compositions provided herein are for use in a method of diagnosing cancer in a patient suffering from cancer. In some embodiments, the cancer is associated with elevated FOLR1 levels.

[0111] In some embodiments, the binding affinity of the antibody, antigen-binding fragment, or polypeptide is the binding affinity obtained in Example 3 and / or shown in Figures 4, 5, and / or 6. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] Figure 1 Provides images of IHC staining of NSCLC and ovarian endometrioid adenocarcinoma specimens using 353.2-1 and 353.9-20 antibodies.

[0113] Figure 2 Provided are images of IHC staining of normal salivary gland and pancreas specimens using 353.2-1 and 353.9-20 antibodies.

[0114] Figure 3 Images of Western blots of cell lysates using 353.9-21, 353.2-1, 353.3-8, and 353.5-7 antibodies are provided.

[0115] FIG. 4 shows the binding of 353.2-1, 353.3-1, 353.5-7, and 353.9-21 antibodies to denatured KB cells (A) and non-denatured T47D cells (B) as determined using fluorescence activated cell sorter (FACS).

[0116] Figure 5 Binding of 353.2-1, 353.3-1, 353.5-7 and 353.9-21 antibodies to recombinant human FOLR1 as determined using ELISA is shown.

[0117] 6 shows the binding of anti-FOLR2 antibodies and 353.2-1, 353.3-1, 353.5-7 and 353.9-21 antibodies to FOLR2 (A) and the binding of anti-FOLR3 antibodies and 353.2-1, 353.3-1, 353.5-7 and 353.9-21 antibodies to FOLR3 (B) as determined by ELISA.

[0118] Figure 7 Shown is the binding of anti-FOLR1 antibodies 2.1 and huMov19 to deglycosylated and untreated recombinant human FOLR1 as determined by ELISA.

[0119] Figure 8 Shown is the binding of anti-FOLR1 antibodies 2.1, huMov19 and BN3.2 to deglycosylated and untreated KB and Igrov-1 cell lysates as determined by Western blot analysis.

[0120] Fig. 9 The relevant amino acids used for resurfacing of the anti-FOLR1 FRIHC2-1 antibody and the kabat position corresponding to each residue are shown.

[0121] Fig.10 An alignment of the mouse and humanized FRIHC2-1 antibody sequences used for surface remodeling is shown. The mouse heavy and light chain sequences correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively. The surface remodeled humanized heavy chain sequence corresponds to SEQ ID NO: 62, and the surface remodeled human light chain version 1.0 and version 1.1 sequences correspond to SEQ ID NO: 63 and SEQ ID NO: 64, respectively. The leader sequence "S" (framework position -1) in the light chain sequence was not considered for humanization and was not used in the humanized antibody sequence, so it is not shown in the figure.

[0122] Fig.11 The relevant amino acids used for CDR grafting of the anti-FOLR1 FRIHC2-1 antibody and the kabat position corresponding to each residue are shown.

[0123] Fig.12 An alignment of the murine and humanized FRIHC2-1 sequences used for CDR grafting is shown. The murine heavy and light chain sequences correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively. The grafted humanized heavy chain sequence corresponds to SEQ ID NO: 65, and the grafted human light chain version 1.0 and version 1.1 sequences correspond to SEQ ID NO: 66 and SEQ ID NO: 67, respectively. The leader sequence "S" (framework position -1) in the light chain sequence was not considered for humanization and was not used in the humanized antibody sequence, so it is not shown in the figure.

[0124] Fig.13 Provided are images of IHC staining of lung adenocarcinoma tissue using FOLR1-2.1 (353-2.1) antibody at different dilutions.

[0125] Figure 14 provides images of IHC staining of positive normal tissue (fallopian tube) (A) and cells (FOLR1 transfected cells (B)) and negative cells (untransfected cells (C)) using FOLR1-2.1 (353-2.1) antibody.

[0126] FIG. 15 provides images of IHC staining of ovarian cancer tissue (A) and lung adenocarcinoma tissue samples (B) using FOLR1-2.1 (353-2.1) antibody.

[0127] Fig.16 Shown is membrane staining of tumor cells in an endometrial cancer specimen using the FOLR1-2.1 assay. Stromal cells were not stained.

[0128] Fig.17 Shown is a comparison of staining and scoring differences between (A) FOLR1-2.1 assay and (B) BN3.2 assay. DETAILED DESCRIPTION OF THE INVENTION

[0129] The present disclosure provides methods for detecting human folate receptor 1 (FOLR1) on circulating tumor cells (including membrane FOLR1, shed FOLR1, and FOLR1) and methods for improving the efficacy of treatment of cancers characterized by overexpression of FOLR1 or the likelihood of responding to the treatment. The detection methods can detect a clinically relevant dynamic range of FOLR1 and can therefore be used for patient stratification, for monitoring or determining the efficacy of treatment or the likelihood of responding to treatment of cancers characterized by overexpression of FOLR1. Also disclosed are novel FOLR1 binding polypeptides, such as antibodies, suitable for FOLR1 detection methods (e.g., IHC for membrane-bound and cell-associated FOLR1). Also provided are related polypeptides and polynucleotides, compositions comprising the FOLR1 binders, and methods for preparing FOLR1 binders.

[0130] I. Definitions

[0131] To facilitate the understanding of the present invention, certain terms and phrases are defined below.

[0132] Unless otherwise indicated, the terms "human folate receptor 1", "FOLR1" or "folate receptor alpha (FR-α)" as used herein refer to any native human FOLR1. Therefore, all of these terms may refer to a protein or nucleic acid sequence as indicated herein. The term "FOLR1" encompasses "full-length," unprocessed FOLR1 as well as any form of FOLR1 produced by processing within a cell. The term also encompasses naturally occurring FOLR1 protein or nucleic acid variants, such as splice variants, allelic variants, and isoforms. The FOLR1 polypeptides and polynucleotides described herein can be isolated from a variety of sources, such as a human tissue type or 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.

[0133] The terms "shed antigen" and "shed FOLR1" are used interchangeably herein. These terms refer to a FOLR1 protein that is soluble and not cell-associated. In some embodiments, it includes an extracellular domain (ECD) and a glycosylphosphatidylinositol (GPI) linker. In one embodiment, shed FOLR1 includes only the ECD. The FOLR1 protein includes a signal peptide (amino acids 1-24), a FOLR1 protein chain (amino acids 25-233 or 234), and a cleavable propeptide (amino acids 235 to 257). The mature FOLR1 protein lacks a signal peptide. Shedding FOLR1 may include amino acids 1 to 257, 1 to 233, 1 to 234, 25 to 233, 25 to 234, or any other fragment thereof. In some embodiments, the signal sequence is cleaved. In other embodiments, the ECD and GPI moieties may be embedded in a membrane (e.g., a soluble lipid raft). In one embodiment, shed FOLR1 may include amino acids 1-233 or a fragment thereof.

[0134] The term "antibody" means 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 foregoing) through at least one antigen recognition site in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies (such as bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antigenic determinants of antibodies, and any other modified immunoglobulin molecules comprising antigen recognition sites, as long as the antibody exhibits the desired biological activity. The antibody can be any of the following five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM (referred to as α, δ, ε, γ, and μ, respectively, based on the identity of their heavy chain constant regions), or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0135] In some embodiments, the antibody is a non-naturally occurring antibody. In some embodiments, the antibody is purified from natural components. In some embodiments, the antibody is recombinantly produced. In some embodiments, the antibody is produced by a hybridoma.

[0136] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds, such as FOLR1. In certain embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. Ideally, the biological activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95% or even 100%.

[0137] The term "anti-FOLR1 antibody" or "antibody that binds to FOLR1" refers to an antibody that is capable of binding to FOLR1 with sufficient affinity to make the antibody useful as a diagnostic and / or therapeutic agent in targeting FOLR1. Unless otherwise specified, the extent of binding of the anti-FOLR1 antibody to unrelated, non-FOLR1 proteins is less than about 10% of the binding of the antibody to FOLR1, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to FOLR1 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In one embodiment, the anti-FOLR1 antibody does not bind to FOLR2, FOLR3, FOLR4, or folate. Examples of FOLR1 antibodies are known in the art and are disclosed in U.S. Published Application Nos. 2012 / 0009181 and 2012 / 0282175 and U.S. Provisional Application Nos. 61 / 695,791 and 61 / 756,254 and PCT Publication No. WO2011 / 106528, each of which is incorporated herein by reference. The sequences of anti-FOLR1 antibodies and antigen-binding fragments thereof are provided in Tables 1-8.

[0138] The term "antibody fragment" refers to a portion of an intact antibody and refers to 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, single-chain antibodies, and multispecific antibodies formed from antibody fragments. The term "antigen-binding fragment" of an antibody includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by specific fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains (e.g., an antibody digested with papain produces three fragments: two antigen-binding Fab fragments and one Fc fragment that does not bind antigen); (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region (e.g., an antibody digested with pepsin produces two fragments: a bivalent antigen-binding F(ab')2 fragment and a pFc' fragment that does not bind antigen) and its associated F(ab') monovalent unit; (iii) a Fd fragment consisting of the VH and CH1 domains (i.e., the portion of the heavy chain included in Fab); (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, and the associated disulfide-linked Fv; (v) a dAb (domain antibody) or sdAb (single domain antibody) fragment consisting of the VH domain (Ward et al., Nature 2003). 341:544-546, 1989); and (vi) isolated complementarity determining regions (CDRs).

[0139] "Monoclonal antibody" refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies against different antigenic determinants. The term "monoclonal antibody" encompasses complete and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising an antigen recognition site. In addition, "monoclonal antibody" refers to such antibodies formed in any number of ways, including but not limited to by hybridomas, phage selection, recombinant expression, and transgenic animals.

[0140] The term "humanized antibody" refers to a non-human (e.g., mouse) antibody form of a specific immunoglobulin chain, chimeric immunoglobulin or its fragment containing minimal non-human (e.g., mouse) sequences. Generally, humanized antibodies are human immunoglobulins (Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-327; Verhoeyen et al., 1988, Science, 239: 1534-1536) in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) with the desired specificity, affinity and ability. In some cases, the Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding residues with the desired specificity, affinity and ability in antibodies from non-human species. Humanized antibodies can be further modified by replacement of other residues in the Fv framework region and / or the replaced non-human residues to improve and optimize antibody specificity, affinity and / or ability. In general, humanized antibodies will substantially comprise at least one and usually two or three variable domains, the variable domains containing all or substantially all CDR regions corresponding to non-human immunoglobulins, and all or substantially all FR regions being those of human immunoglobulin consensus sequences. Humanized antibodies may also comprise at least a portion (Fc) of an immunoglobulin constant region or domain, typically a portion of a human immunoglobulin. Examples of methods for producing humanized antibodies are described in U.S. Patents 5,225,539 and 5,639,641, Roguska et al., Proc.Natl.Acad.Sci., USA, 91 (3): 969-973 (1994) and Roguska et al., Protein Eng.9 (10): 895-904 (1996). In some embodiments, a "humanized antibody" is a resurfaced antibody. In some embodiments, a "humanized antibody" is a CDR-grafted antibody.

[0141] The "variable region" of an antibody refers to the variable region of the antibody light chain alone or in combination or the variable region of the antibody heavy chain. The variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementary determining regions (CDRs) (also referred to as hypervariable regions). The CDRs in each chain are closely held together by the FRs and contribute to the formation of the antigen binding site of the antibody together with the CDRs from the other chain. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variation (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda Md.)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273: 927-948)). In addition, a combination of these two methods is sometimes used in the art to determine CDRs.

[0142] When referring to residues in the variable domain, the Kabat numbering system (approximately residues 1-107 for the light chain and residues 1-113 for the heavy chain) is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0143] The amino acid position numbering as in Kabat refers to the numbering system of the heavy chain variable domain or light chain variable domain used for the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may include a shortening of the FR or CDR corresponding to the variable domain or several or additional amino acids inserted therein. For example, the heavy chain variable domain may include a single amino acid insertion after the residue 52 of H2 (according to the residue 52a of Kabat) and multiple insertion residues after the heavy chain FR residue 82 (for example, according to the residue 82a, 82b and 82c of Kabat, etc.). For a given antibody, the Kabat numbering of the residue can be determined by comparing the antibody sequence with the sequence of the "standard" Kabat numbering at the region with homology. Chothia refers to the position of the structural loop (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). When numbering using the Kabat numbering convention, the end of the Chothia CDR-H1 loop 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; 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 by Oxford Molecular's AbM antibody modeling software.

[0144]

[0145] The term "human antibody" means an antibody produced by a human or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to an antibody produced by a human. This definition of a human antibody includes complete or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide, such as, for example, antibodies comprising a mouse light chain polypeptide and a human heavy chain polypeptide.

[0146] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid causing an immune response in the species.

[0147] The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to the portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, an epitope may be formed by contiguous amino acids and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by contiguous amino acids are generally retained upon protein denaturation, whereas epitopes formed by tertiary folding are generally lost upon protein denaturation. An epitope generally comprises at least 3 and more generally at least 5 or 8 to 10 amino acids in a unique spatial conformation.

[0148] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity of a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its partner Y can be generally represented by a dissociation constant (Kd) or a half-maximal effective concentration (EC50). Affinity can be measured by conventional methods known in the art (including those described herein). Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described herein.

[0149] "Or better" as used herein when referring to binding affinity refers to a stronger binding between a molecule and its binding partner. As used herein, "or better" refers to a stronger binding represented by a smaller numerical Kd value. For example, an antibody having an affinity of "0.6 nM or better" for an antigen has an affinity for the antigen that 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 an antibody as measured by Kd will be between about 10 -3 to about 10 -12 M, between about 10 -6 to about 10 -11 M, between about 10 -6 to about 10 -10M, between about 10 -6 to about 10 -9 M, between about 10 -6 to about 10 -8 M or between about 10 -6 to about 10 -7 Between M.

[0150] As used herein, the phrase "substantially similar" or "substantially the same" means a sufficiently high degree of similarity between two values ​​(usually one value associated with an antibody of the invention and the other associated with a reference / comparison antibody) so that one skilled in the art will consider the difference between the two values ​​to be almost or not biologically and / or statistically significant in the context of the biological property measured by the value (e.g., Kd value). The difference between the two values ​​varies with the value of the reference / comparison antibody by less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%.

[0151] 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 following general formula: ALC, wherein C = cytotoxin, L = linker, and A = cell-binding agent or anti-FOLR1 antibody or antibody fragment. An immunoconjugate may also be defined by the following general formula in reverse order: CLA.

[0152] A "linker" is any chemical moiety that is capable of linking a compound (usually a drug, such as maytansinoids) to a cell binding agent, such as an anti-FOLR1 antibody or fragment thereof, in a stable, covalent manner. The linker may be susceptible to or substantially resist acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions where the compound or antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups, and esterase-labile groups. Linkers also include charged linkers and hydrophilic forms thereof as described herein and known in the art.

[0153] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is one that is 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, the isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0154] As used herein, "substantially pure" refers to a material that is at least 50% pure (ie, free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0155] The terms "elevated" FOLR1, "increased expression" of FOLR1, and "overexpression" of FOLR1 refer to a sample containing elevated expression levels of FOLR1. FOLR1 may be elevated, increased, or overexpressed compared to a control value (e.g., expression levels in a biological sample, tissue, or cell from a subject without cancer, a sample or cancer known to express no or low FOLR1, a normal sample, or a cancer that does not have an elevated FOLR1 value). For example, a sample with increased expression may contain an increase of at least 2-fold, at least 3-fold, or at least 5-fold relative to a control value.

[0156] FOLR1 expression can be measured by immunohistochemistry and given a staining intensity score or a staining uniformity score by comparison with a calibrated control showing a determined score (e.g., if the intensity is comparable to a 3-level calibrated control, an intensity score of 3 is given to the test sample, or if the intensity is comparable to a 2-level calibrated control, an intensity of 2 is given to the test sample). For example, a score of 1, 2, or 3 (3+), preferably a score of 2 or 3 (3+), as measured by immunohistochemistry, indicates increased FOLR1 expression. Heterogeneous or homogeneous staining uniformity also indicates FOLR1 expression. Staining intensity and staining uniformity scores can be used alone or in combination (e.g., 2 homogeneous, 2 heterogeneous, 3 homogeneous, 3 heterogeneous, etc.). See Table 11. In another example, an increase in FOLR1 expression can be determined by detecting an increase of at least 2-fold, at least 3-fold, or at least 5-fold relative to a control value (e.g., expression levels in tissues or cells from subjects without cancer or with cancer whose FOLR1 values ​​are not elevated).

[0157] A "reference sample" can be used to correlate and compare the results obtained from a test sample in the method of the present invention. The reference sample can be a cell (e.g., a cell line, a cell mass) or a tissue. The level of FOLR1 in the "reference sample" can be an absolute or relative amount of FOLR1, a range of amounts, a minimum and / or maximum amount, an average amount and / or a median amount. The "reference sample" can also serve as a baseline for FOLR1 expression with which the test sample is compared. The "reference sample" may include a previous sample or baseline sample from the same patient, a normal reference with a known FOLR1 expression level, or a reference from a related patient population with a known FOLR1 expression level. The FOLR1 level can also be expressed as a value in 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 comprising purified FOLR1 or FOLR1-Fc. The diagnostic method of the present invention may involve a comparison between the FOLR1 expression level in the test sample and a "reference value". In some embodiments, the reference value is the expression level of FOLR1 in the reference sample. Reference value can be a predetermined value and can also be determined from a reference sample (e.g., a control biological sample or a reference sample) tested simultaneously with the test sample. Reference value can be a single cutoff value, such as a median or mean value or a range of values, such as a confidence interval. Reference values ​​can be established for different subgroups of individuals, such as individuals susceptible to cancer, individuals with early or advanced 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 and are also described in Example 1 and Example 8-10 of WO2012 / 135675, which are incorporated herein by reference.

[0158] In some embodiments, the reference sample is a sample from a healthy tissue, specifically a corresponding tissue that is not affected by cancer or a corresponding tissue that is not affected by cancer that overexpresses FOLR1 or a corresponding healthy tissue that is known not to express a detectable level of FOLR. These types of reference samples are referred to as negative control samples or "normal" control samples. In other embodiments, the reference sample is a sample from a tumor or healthy tissue that expresses detectable FOLR1. These types of reference samples are referred to as positive controls or positive reference samples. Positive control samples can also be used as a comparison indicator of the type (heterogeneous to homogeneous) and / or degree (0, 1, 2, 3) of staining intensity associated with FOLR1 expression levels. Positive control comparison samples are also referred to as calibrated reference samples. Low or non-FOLR1 expression references are described in the examples herein and also include all structures of the esophagus, pancreatic acinar cells / islets, lung interalveolar connective tissue, and salivary gland acinar cells. For cell lines, exemplary non-expressors include BxPC3, Panc-1, and ASPC1. Positive FOLR1 references are described herein (e.g., in the Examples) and also include the ducts of the pancreas, the respiratory epithelium of the normal lung, and the intercalated ducts of the salivary glands. In some embodiments, positive FOLR1 references include the ducts of the pancreas and the intercalated ducts of the salivary glands. For cell lines, exemplary high FOLR1 expressers are described herein (e.g., in the Examples) and also include KB, HeLa, 300.19 cells transfected with FOLR1, Igrov-1, and Wish, and exemplary low FOLR1 expressers include Ovcar-3, Caov-3, SW620, T47D, and Skov-3. Another positive high FOLR1 reference is a cell line stably or transiently transfected with FOLR1. Additional positive and negative samples of FOLR1 are described in Table 13. Appropriate positive and negative reference FOLR1 levels for a particular cancer can be determined by measuring the level of FOLR1 in one or more appropriate subjects, and such reference levels can be tailored for a particular population of subjects (e.g., reference levels can be age-matched so that comparisons can be made between FOLR1 levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group). Such reference levels can also be tailored for the specific technique used to measure the level of FOLR1 in a biological sample (e.g., immunoassay, etc.).

[0159] As used herein, "immunohistochemistry" refers to histochemical and immunological methods used to analyze, for example, cells or tissues. Therefore, the terms "immunohistochemistry", "immunocytochemistry" and "immunochemistry" can be used interchangeably.

[0160] The term "primary antibody" herein refers to an antibody that specifically binds to a target protein antigen in a sample. The primary antibody is typically the first antibody used in an ELISA assay or an IHC procedure. In one embodiment, the primary antibody is the only antibody used in an IHC procedure. The term "secondary antibody" herein refers to an antibody that specifically binds to the primary antibody, thereby forming a bridge or connection between the primary antibody and subsequent reagents (if any). The secondary antibody is typically the second antibody used in an immunohistochemical procedure.

[0161] In specific embodiments, a "sample" or "biological sample" of the present invention is derived from an organism, such as from a eukaryotic organism. In some embodiments, the sample is a human sample, but animal samples may also be used. Non-limiting examples of sources for samples used in the present invention include solid tissue, biopsy aspirates, ascites, fluid extracts, blood, plasma, serum, cerebrospinal fluid, lymph fluid, external sections of skin, respiratory tract, intestinal and urogenital tracts, tears, saliva, milk, tumors, organs, cell cultures and / or cell culture components. A "cancerous sample" is a sample that contains cancerous cells. The methods can be used to examine aspects of the expression of FOLR1 or the state of a sample, including but not limited to: comparing different types of cells or tissues, comparing different developmental stages, and detecting or determining the presence and / or type of a disease or abnormality.

[0162] For purposes herein, " section " of tissue sample refers to a single part or a slice of tissue sample, such as a thin slice of tissue or a cell cut from a tissue sample. It should be appreciated that multiple sections of tissue sample can be obtained and analyzed according to the present invention. In some cases, selected tissue portion or section comprises a homologous cell colony. In other cases, the selected portion comprises a region of tissue, such as the lumen as a non-limiting example. For example, the selected portion can be such as small as a cell or two cells, or can represent tens of thousands of cells. In most cases, the collection of cells is important, and although the present invention has been described for use in the detection of cellular components, the method can also be used for detecting the non-cellular components of an organism (such as soluble components in blood as a non-limiting example).

[0163] As used herein, the term "capture reagent" refers to a reagent that is capable of binding and capturing a target molecule in a sample so that the capture reagent-target molecule complex can be separated from the rest of the sample under suitable conditions. In one embodiment, the capture reagent is immobilized. In one embodiment, the capture reagent in a sandwich immunoassay is an antibody or a mixture of different antibodies against a target antigen.

[0164] As used herein, the term "detectable antibody" refers to an antibody that can be detected directly by a label amplified by a detection device or indirectly by another antibody, such as a label. For direct labels, the antibody is typically coupled to a portion that can be detected by some device. In one embodiment, the detectable antibody is a biotinylated antibody.

[0165] As used herein, the term "detection means" refers to a moiety or technique for detecting the presence of a detectable antibody and includes detection reagents that amplify fixed labels such as labels captured on a microtiter plate. In one embodiment, the detection means is a fluorescent detection agent such as avidin or streptavidin.

[0166] Typically, a "sandwich ELISA" employs the following steps: (1) a microtiter plate is coated with a capture antibody; (2) the sample is added, and any antigen present binds to the capture antibody; (3) the detection antibody is added and it binds to the antigen; (4) an enzyme-linked secondary antibody is added and it binds to the detection antibody; and (5) a substrate is added and converted to a detectable form by an enzyme.

[0167] The term "label" as used herein refers to a detectable compound or composition that is coupled directly or indirectly to an antibody to produce a "labeled" antibody. The label is itself detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, catalyzes chemical alteration of a detectable substrate compound or composition.

[0168] By "correlate" or "correlating" is meant comparing the performance and / or results of a first analysis with the performance and / or results of a second analysis in any manner. For example, the results of a first analysis can be used to perform a second analysis, and / or the results of a first analysis can be used to determine whether a second analysis should be performed, and / or the results of a first analysis can be compared with the results of a second analysis. In one embodiment, increased FOLR1 expression is associated with an increased likelihood of effectiveness of a FOLR1 targeted anti-cancer therapy.

[0169] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include cancers of endothelial, mesenchymal or epithelial origin, such as lung cancer (e.g., squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, mesothelioma and squamous lung carcinoma), peritoneal cancer (e.g., primary peritoneal cancer), hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer (serous or endometrioid), liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer (e.g., endometrioid adenocarcinoma) or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, brain cancer (e.g., glioblastoma, tumors of the choroid plexus) and various types of head and neck cancer, as well as tumors of blood vessels and fallopian tubes. Cancers also encompass cancers comprising elevated expression levels of FOLR1. Such cancers with elevated FOLR1 include, but are not limited to, ovarian cancer, non-small cell lung cancer (adenocarcinoma), uterine cancer, endometrial cancer, pancreatic cancer, renal cancer, lung cancer, and breast cancer.

[0170] "Tumor" and "neoplasm" refer to any mass of tissue resulting from excessive cell growth or proliferation, whether benign (noncancerous) or malignant (cancerous), including precancerous lesions.

[0171] The terms "cancer cells", "tumor cells" and grammatical equivalents refer to the total cell population derived from a tumor or precancerous lesion, including both non-tumorigenic cells (including tumor cell masses) and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cell" when referring only to those tumor cells that lack renewal and differentiation capabilities will be modified by the term "non-tumorigenic" to distinguish those tumor cells from cancer stem cells.

[0172] The term "subject" refers to any animal (e.g., mammal) that is to be the recipient of a particular treatment, including but not limited to humans, non-human primates, rodents, etc. In general, the terms "subject" and "patient" are used interchangeably herein when referring to a human subject.

[0173] The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and contains no additional components that are unacceptably toxic to a subject to which the formulation is administered. Such preparations may be sterile.

[0174] An "effective amount" of an antibody or immunoconjugate as disclosed herein is an amount sufficient to perform the explicitly stated purpose. An "effective amount" for the stated purpose can be determined empirically and in a routine manner.

[0175] The term "therapeutically effective amount" refers to an amount of an antibody or other drug that is effective to "treat" a disease or condition in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down to some extent and in a certain embodiment stop) cancer cell infiltration of peripheral organs; inhibit (i.e., slow down to some extent and in a certain embodiment stop) tumor metastasis; inhibit tumor growth to some extent; alleviate one or more symptoms associated with cancer to some extent; and / or produce a favorable response such as increased progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR) or in some cases, stable disease (SD), reduction in progressive disease (PD), reduction in time to progression (TTP), reduction in CA125 in the case of ovarian cancer, or any combination thereof. . See the definition of "treatment" herein. To the extent that a drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. In certain embodiments, identification of increased FOLR1 levels allows for administration of reduced amounts of FOLR1 targeted therapeutics to achieve the same therapeutic effect as seen with higher doses. A "prophylactically effective amount" refers to an amount effective, at dosages and durations necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects before or at an early stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0176] The term "good response" generally refers to causing a favorable state in a subject. With respect to cancer treatment, the term refers to providing a therapeutic effect on a subject. The positive therapeutic effect in cancer can be measured in many ways (see, WA Weber, J. Nucl. Med. 50: 1S-10S (2009)). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to assess the therapeutic efficacy of anticancer therapeutics. With respect to tumor growth inhibition, according to NCI standards, T / C ≤ 42% is the lowest level of antitumor activity. T / C < 10% is considered to be a high level of antitumor activity, where T / C (%) = median tumor volume of treated object / median tumor volume of control object × 100. A favorable response can be assessed, for example, by increased progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), a complete response (CR), a partial response (PR) or in some cases, stable disease (SD), a reduction in progressive disease (PD), a reduction in time to progression (TTP), a reduction in CA125 in the case of ovarian cancer, or any combination thereof.

[0177] PFS, DFS and OS can be measured by the standards set by the National Cancer Institute and the US Food and Drug Administration for the approval of new drugs. See Johnson et al., (2003) J. Clin. Oncol. 21(7): 1404-1411.

[0178] "Progression-free survival" (PFS) refers to the time from enrollment to disease progression or death. PFS is usually measured using the Kaplan-Meier method and solid tumor response evaluation criteria (RECIST) 1.1 criteria. Usually, progression-free survival refers to a situation in which a patient remains alive without cancer worsening.

[0179] "Time to tumor progression" (TTP) is defined as the time from enrollment to disease progression. TTP is usually measured using the RECIST 1.1 criteria.

[0180] A "complete response" or "complete remission" or "CR" indicates the disappearance of all signs of a tumor or cancer in response to treatment. It does not always mean that the cancer has been cured.

[0181] "Partial response" or "PR" refers to a decrease in the size or volume of one or more tumors or lesions, or the extent of cancer in the body in response to treatment.

[0182] "Stable disease" refers to disease that has not progressed or recurred. In stable disease, there is neither enough tumor shrinkage to qualify as a partial response nor enough tumor increase to qualify as progressive disease.

[0183] "Progressive disease" refers to the appearance and / or unequivocal progression of a new lesion or tumor that is not an existing target lesion. Progressive disease may also refer to tumor growth of more than 20% since the start of treatment due to an increase in the mass or spread of the tumor.

[0184] "Disease-free survival" (DFS) refers to the length of time a patient remains disease-free during or after treatment.

[0185] "Overall survival" (OS) refers to the time from patient enrollment to death or censoring at the last known date of survival. OS includes an extension of life expectancy compared to an original or untreated individual or patient. Overall survival refers to a situation in which a patient remains alive for a defined period of time, such as (e.g., from the time of diagnosis or treatment) one year, five years, etc.

[0186] "Reduction of CA125 levels" can be assessed according to the Gynecologic Cancer Collaborative Group (GCIG) guidelines. For example, CA125 levels can be measured before treatment to establish baseline CA125 levels. CA125 levels can be measured once or multiple times during or after treatment, and a reduction in CA125 levels over time compared to baseline levels is considered a reduction in CA125 levels.

[0187] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow down, alleviate the symptoms of, and / or prevent the progression of a diagnosed pathological condition or disorder. Thus, those in need of treatment include those already diagnosed with or suspected of having the disorder. In certain embodiments, a subject is successfully "treated" for cancer according to the methods of the invention if the patient exhibits one or more of the following: a decrease in the number of cancer cells or the complete absence of cancer cells; a decrease in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs, including, for example, cancer spread to soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; alleviation of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; improved quality of life; a decrease in tumorigenicity, the frequency of tumorigenicity, or the tumorigenic capacity of a tumor; a decrease in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells into a non-tumorigenic state; increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a decrease in time to progression (TTP), a decrease in CA125 in the case of ovarian cancer, or any combination thereof.

[0188] Prophylactic or preventative measures refer to therapeutic measures that prevent and / or slow the development of a targeted pathological condition or disorder. Thus, those in need of prophylactic or preventative measures include those susceptible to the disorder and those in which the disorder is to be prevented.

[0189] As used herein, the term "health care provider" refers to an individual or institution that directly interacts with and administers to a living subject (e.g., a human patient). Non-limiting examples of health care providers include physicians, nurses, technicians, therapists, pharmacists, consultants, alternative medical practitioners, medical institutions, physician's offices, hospitals, emergency rooms, clinics, urgent care centers, alternative medical clinics / institutions, and any other entity that provides general and / or specialized treatment, assessment, maintenance, therapy, medication, and / or advice concerning all or any part of a patient's health status, including, but not limited to, general medical, specialized medical, surgical, and / or any other type of treatment, assessment, maintenance, therapy, medication, and / or advice.

[0190] In some aspects, a healthcare provider may administer or instruct another healthcare provider to administer a therapy to treat cancer. "Administration" of a therapy as described herein includes prescribing a therapy to a subject as well as delivering, applying, or administering the therapy to a subject. A healthcare provider may perform or instruct another healthcare provider or a patient to perform the following actions: obtain a sample; process a sample; submit a sample; receive a sample; transfer a sample; analyze or measure a sample; quantify a sample; provide a result obtained after analyzing / measuring / quantifying a sample; receive a result obtained after analyzing / measuring / quantifying a sample; compare / score a result obtained after analyzing / measuring / quantifying one or more samples; provide a comparison / score from one or more samples; obtain a comparison / score from one or more samples; administer a therapy or therapeutic agent (e.g., a FOLR1 binding agent); begin administering a therapy; terminate administering a therapy; continue administering a therapy; temporarily interrupt administering a therapy; increase the amount of the administered therapeutic agent; decrease the amount of the administered therapeutic agent; continue administering an amount of the therapeutic agent; increase the frequency of administering a therapeutic agent; decrease the frequency of administering a therapeutic agent; maintain the same dosing frequency of a therapeutic agent; replace a therapy or therapeutic agent with at least another therapy or therapeutic agent; combine a therapy or therapeutic agent with at least another therapy or additional therapeutic agent. These actions may be performed automatically by a healthcare provider using a computer-implemented method (e.g., via a network service or a stand-alone computer system).

[0191] "Polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any matrix that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by coupling with a labeling component. Other types of modifications include, for example, "capping", substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of one or more polynucleotides. In addition, any hydroxyl group that is usually present in sugars can be replaced by (for example) phosphonate group, phosphate group, protected by standard protecting groups or activated to provide other linkages with other nucleotides, or can be coupled with solid support.5' and 3' terminal OH can be replaced by phosphorylation or by amine or organic end-capping group part with 1 to 20 carbon atoms.Other hydroxyls can also be derivatized into standard protecting groups.Polynucleotides can also contain ribose or deoxyribose of similar form commonly known in the art, including for example 2'-O-methyl-, 2'-O-allyl, 2'-fluorine or 2'-azido-ribose, carbocyclic sugar analogs, α-end group isomerized sugars, epimerized sugars (such as arabinose, xylose or lyxose), pyranose, furanose, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl nucleosides.One or more phosphodiester linkages can be replaced by optional linking groups.These optional linkers include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl optionally containing an ether (--O--) linkage. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0192] The term "vector" means a construct capable of delivering and expressing one or more target genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (such as producer cells).

[0193] 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 have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as coupling with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that because the polypeptides of the present invention are based on antibodies, in certain embodiments, the polypeptides may exist as single chains or associated chains. In some embodiments, polypeptides, peptides or proteins are non-naturally occurring. In some embodiments, polypeptides, peptides or proteins are purified from other naturally occurring components. In some embodiments, polypeptides, peptides or proteins are recombinantly produced.

[0194] 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 identical or have the same specified percentage of nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps if necessary), 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. Different algorithms and software that can be used to obtain alignments 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, 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 may 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 other publicly available software programs that can be used for aligning 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 GAP program in the GCG software package incorporating the Needleman and Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the Myers and Miller algorithm (CABIOS, 4: 11-17 (1989)) is used to determine the percent identity between nucleotide or amino acid sequences.For example, the ALIGN program (version 2.0) and PAM120 with a residue table, a gap length penalty of 12, and a gap penalty of 4 can be used to determine the identity percentage. Appropriate parameters for achieving maximum alignment by specific alignment software can be determined by those skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the identity percentage "X" of a first amino acid sequence to a second amino acid sequence is calculated as 100 × (Y / Z), wherein Y is the number of amino acid residues scored as identical matches in the comparison of the first sequence and the second sequence (such as compared by visual inspection or 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 the second sequence, the identity percentage of the first sequence to the second sequence will be longer than the identity percentage of the second sequence to the first sequence.

[0195] As a non-limiting example, in certain embodiments, the Bestfit program (Wisconsin Sequence Analysis Package, Unix Version 8, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) can be used to determine whether any particular polynucleotide has a certain percentage of sequence identity (e.g., at least 80% identity, at least 85% identity, at least 90% identity and in some embodiments at least 95%, 96%, 97%, 98% or 99% identity) with a reference sequence. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482 489 (1981)) to find the best fragment with homology between two sequences. When Bestfit or any other sequence alignment program is used according to the present invention to determine whether a particular sequence has, for example, 95% identity with a reference sequence, parameters are set so that the identity percentage is calculated over the full length of the reference nucleotide sequence and spaces in the homology are allowed to be up to 5% of the total number of nucleotides in the reference sequence.

[0196] In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning that when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection, 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. In certain embodiments, the identity is present in a region of the sequence of at least about 10, about 20, about 40-60 residues or any integer value therebetween, or in a region longer than 60-80 residues, e.g., at least about 90-100 residues, or the sequence is substantially identical over the full length of the compared sequence (e.g., such as the coding region of a nucleotide sequence).

[0197] A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include 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). For example, a substitution of tyrosine with phenylalanine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not eliminate binding of the polypeptide or antibody containing the amino acid sequence to the antigen (i.e., 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, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0198] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0199] It should be understood that wherever herein an embodiment is described with the language "comprising," other similar embodiments described with the phrases "consisting of" and / or "consisting essentially of" are also provided.

[0200] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A," and "B." Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover 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).

[0201] II. FOLR1 Binders

[0202] The present invention provides agents that specifically bind to human FOLR1. These agents are referred to herein as "FOLR1 binding agents". In certain embodiments, the FOLR1 binding agent is an antibody, immunoconjugate or polypeptide. The amino acid and nucleotide sequences of human FOLR1 are known in the art and are also provided herein as represented by SEQ ID NO: 1 and SEQ ID NO: 2.

[0203] Human folate receptor 1:

[0204]

[0205] Human folate receptor 1 nucleic acid sequence:

[0206]

[0207] Thus, in some embodiments, a FOLR1 binding agent can bind to an epitope of SEQ ID NO:1.

[0208] In some embodiments, the anti-FOLR1 antibody can specifically bind to an epitope of FOLR1 (SEQ ID NO: 1), wherein the epitope comprises an N-glycosylated amino acid. Such antibodies will therefore bind to FOLR1 when they are glycosylated and will not bind to FOLR1 when they are not glycosylated. In other words, the binding of these antibodies is glycol-dependent. These antibodies are advantageous in that they can be used to distinguish between glycosylated and non-glycosylated forms of FOLR1. Given that glycosylation may be required for membrane localization, the antibodies can be advantageously used for membrane-specific staining.

[0209] In some embodiments, the anti-FOLR1 antibody can specifically bind to an epitope of FOLR1 comprising N-glycosylated amino acid 69 of FOLR1. In some embodiments, the anti-FOLR1 antibody can specifically bind to an epitope of FOLR1 comprising N-glycosylated amino acid 161 of FOLR1. In some embodiments, the anti-FOLR1 antibody can specifically bind to an epitope of FOLR1 comprising N-glycosylated amino acid 201 of FOLR1.

[0210] In certain embodiments, the anti-FOLR1 antibody is an antibody produced by a murine hybridoma deposited with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, VA 20110, under the terms of the Budapest Treaty on April 16, 2013, and having ATCC Accession No. PTA-120196 ("FOLR1-9.20", also referred to as "IMGN 353.9-20", "353.9-20", or "9.20"). In certain embodiments, the anti-FOLR1 antibody is an antibody produced by a murine hybridoma deposited with the ATCC on April 16, 2013, and having ATCC Accession No. PTA-120197 ("FOLR1-2.1", also referred to as "IMGN 353.2-1", "353.2-1", "2.1", or "muFRIHC2-1").

[0211] FOLR1 binders include FOLR1 binders comprising heavy and light chain CDR sequences of: (i) muFRIHC2-1, which is also referred to as "FOLR1-2.1", "IMGN 353.2-1", "353.2-1" or "2.1"; (ii) muFRIHC5-7, which is also referred to as "IMGN 353.5-7", "353.5-7" or "5.7"; (iii) "muFRIHC9-20", which is also referred to as "FOLR1-9.20", "IMGN 353.9-20", "353.9-20" or "9.20"; (iv) resurfaced huFRIHC2-1 version 1.0 or 1.01; or (v) CDR-grafted huFRIHC2-1 version 1.0 or 1.01, which are provided in Tables 1 and 2 below. FOLR1 binders also include FOLR1 binders comprising the heavy and light chain CDR sequences of the composite CDRs provided in Tables 1 and 2 below.

[0212] Table 1: Variable heavy chain CDR amino acid sequences

[0213]

[0214] Table 2: Variable light chain CDR amino acid sequences

[0215]

[0216] The FOLR1 binding molecule may be an antibody or antigen binding fragment that specifically binds to FOLR1, the antibody or antigen binding fragment comprising the CDRs of antibody 2.1 (i.e., SEQ ID NOs: 3-8), 5.7 (i.e., SEQ ID NOs: 9-14) or 9.20 (i.e., SEQ ID NOs: 15-20), wherein each CDR has up to four (i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutions. The FOLR1 binding molecule may be an antibody or antigen binding fragment that specifically binds to FOLR1, the antibody or antigen binding fragment comprising the CDRs of the composite sequence shown above (i.e., SEQ ID NOs: 21-26), wherein each CDR has up to four (i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutions.

[0217] The FOLR1 binding molecule may be an antibody or antigen-binding fragment that specifically binds to FOLR1, the antibody or antigen-binding fragment comprising the CDRs of the antibody produced by the murine hybridoma with ATCC accession number PTA-120196 or PTA-120197.

[0218] The polypeptide may comprise one of the individual variable light chains or variable heavy chains described herein. Antibodies and polypeptides may also comprise variable light chains and variable heavy chains. The variable light and variable heavy chain sequences of murine antibodies 2.1, 5.7 and 9.20 and humanized 2.1 are provided in Tables 3 and 4 below.

[0219] Table 3: Variable heavy chain amino acid sequences

[0220]

[0221] Table 4: Variable light chain amino acid sequences

[0222]

[0223] Also provided are polypeptides comprising: (a) a polypeptide having at least about 90% sequence identity to SEQ ID NO: 27, 29, 31, 62, or 65; and / or (b) a polypeptide having at least about 90% sequence identity to SEQ ID NO: 28, 30, 32, 63, 64, 66, or 67. 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 NO: 27-32 or 62-67. Thus, in certain embodiments, the polypeptide comprises: (a) a polypeptide having at least about 95% sequence identity to SEQ ID NO: 27, 29, 31, 62, or 65; and / or (b) a polypeptide having at least about 95% sequence identity to SEQ ID NO: 28, 30, 32, 63, 64, 66, or 67. In certain embodiments, the polypeptide comprises (a) a polypeptide having an amino acid sequence of SEQ ID NO:27, 29, 31, 62 or 65; and / or (b) a polypeptide having an amino acid sequence of SEQ ID NO:28, 30, 32, 63, 64, 66 or 67. 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, a polypeptide having a certain percentage of sequence identity to SEQ ID NO:27-32 or 62-67 differs from SEQ ID NO:27-32 or 62-67 only by conservative amino acid substitutions.

[0224] Also provided are polypeptides comprising a variable light chain that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical or identical to the variable light chain sequence of an antibody produced by a murine hybridoma having ATCC Accession No. PTA-120196 or PTA-120197.

[0225] Also provided are polypeptides comprising a variable heavy chain that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical or identical to the variable heavy chain sequence of an antibody produced by a murine hybridoma having ATCC Accession No. PTA-120196 or PTA-120197.

[0226] Also provided are antibodies and antigen-binding fragments thereof comprising variable heavy chain and variable light chain sequences that are at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical or identical to the variable heavy chain and variable light chain sequences of an antibody produced by a murine hybridoma having ATCC Accession No. PTA-120196 or PTA-120197.

[0227] In certain embodiments, the antibody or antigen-binding fragment is an antibody or antigen-binding fragment thereof produced by a murine hybridoma having ATCC Accession No. PTA-120196.

[0228] In certain embodiments, the antibody or antigen-binding fragment is an antibody or antigen-binding fragment thereof produced by a murine hybridoma having ATCC Accession No. PTA-120197.

[0229] The polypeptide may comprise one of the individual light chains or heavy chains described herein. Antibodies and polypeptides may also comprise both light and heavy chains. The light and heavy chain sequences of antibodies 2.1, 5.7 and 9.20 are provided in Tables 5 and 6 below.

[0230] Table 5: Full length heavy chain amino acid sequence

[0231]

[0232]

[0233] Table 6: Full length light chain amino acid sequence

[0234]

[0235]

[0236] Also provided are polypeptides comprising: (a) a polypeptide having at least about 90% sequence identity to SEQ ID NO:33, 35 or 37; and / or (b) a polypeptide having at least about 90% sequence identity to SEQ ID NO:34, 36 or 38. 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 NO:33-38. Thus, in certain embodiments, the polypeptide comprises (a) a polypeptide having at least about 95% sequence identity to SEQ ID NO:33, 35 or 37, and / or (b) a polypeptide having at least about 95% sequence identity to SEQ ID NO:34, 36 or 38. In certain embodiments, the polypeptide comprises (a) a polypeptide having the amino acid sequence of SEQ ID NO:33, 35 or 37; and / or (b) a polypeptide having the amino acid sequence of SEQ ID NO:34, 36 or 38. 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, a polypeptide having a certain percentage of sequence identity to SEQ ID NOs: 33-38 differs from SEQ ID NOs: 33-38 only by conservative amino acid substitutions.

[0237] Also provided are polypeptides comprising a light chain that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical or identical to the light chain sequence of an antibody produced by a murine hybridoma having ATCC Accession No. PTA-120196 or PTA-120197.

[0238] Also provided are polypeptides comprising a heavy chain that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical or identical to the heavy chain sequence of an antibody produced by a murine hybridoma having ATCC Accession No. PTA-120196 or PTA-120197.

[0239] Also provided are antibodies and antigen-binding fragments thereof comprising heavy and light chain sequences that are at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical or identical to the heavy and light chain sequences of an antibody produced by a murine hybridoma having ATCC Accession No. PTA-120196 or PTA-120197.

[0240] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) or kinetics (e.g., BIACORE TM Analysis). Direct binding assays as well as competitive binding assays 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 the methods described herein. The measured affinity of a particular antibody-antigen interaction may vary if measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other antigen binding parameters (e.g., KD or Kd, Kd) can be measured using standardized solutions of antibody and antigen and standardized buffers (such as those known in the art and as described herein). on , K off ) for measurement.

[0241] In one aspect, binding assays can be performed on cells expressing the FOLR1 antigen on the surface using flow cytometry. For example, FOLR1 positive cells such as SKOV3 can be assayed using 1×10 5Cells / samples were incubated with different concentrations of anti-FOLR1 antibodies. Subsequently, the cells were pelleted, washed and incubated with 100 μL of FITC-coupled goat anti-mouse or goat anti-human IgG antibodies (such as can be obtained, for example, from Jackson Laboratory, 6 μg / mL in FACS buffer) for 1 hour. The cells were then pelleted again, washed with FACS buffer and resuspended in 200 μL of PBS containing 1% formaldehyde. Samples can be obtained, for example, using a FACSCalibur flow cytometer with an HTS multi-well sampler and analyzed using CellQuest Pro (all from BD Biosciences, San Diego, US). For each sample, the mean fluorescence intensity (MFI) of FL1 can be output and plotted against antibody concentration in a semi-logarithmic curve to generate a binding curve. The sigmoidal dose response curve was fitted to the binding curve and the EC50 value was calculated using a program such as GraphPad Prism v4 with default parameters (GraphPad Software, San Diego, CA). The EC50 value can be used as a measure of the apparent dissociation constant "Kd" or "KD" for each antibody.

[0242] Monoclonal antibodies can be prepared using hybridoma methods (such as those described by Kohler and Milstein (1975) Nature 256: 495). Using the hybridoma method, mice, hamsters or other appropriate host animals are immunized to induce lymphocytes to produce antibodies that will specifically bind to the immunizing antigen. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes are separated and fused with suitable myeloma cell lines using (for example) polyethylene glycol to form hybridoma cells, which can then be selected from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies specifically for the selected antigen (such as determined by immunoprecipitation, immunoblotting or by in vitro binding assays (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA))) can then be propagated in vitro or in vivo in animals as ascites tumors using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986). The monoclonal antibodies can then be purified from the culture medium or ascites fluid as described for the polyclonal antibodies.

[0243] Alternatively, monoclonal antibodies can also be prepared using recombinant DNA methods as described in U.S. Pat. No. 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the heavy and light chains of the antibody; and their sequences are determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, which, when transfected into a host cell that does not otherwise produce immunoglobulins (such as E. coli cells, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells), produces monoclonal antibodies by the host cell. In addition, 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).

[0244] Recombinant DNA technology can be used to further modify the polynucleotide encoding the monoclonal antibody in many different ways to produce a surrogate antibody. In some embodiments, the constant domains of the light and heavy chains of (e.g.) mouse monoclonal antibodies can be replaced with: 1) (e.g.) those of human antibodies to produce chimeric antibodies or 2) non-immunoglobulin polypeptides to produce fusion antibodies. In some embodiments, the constant region is truncated or removed to produce the desired antibody fragment of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0245] In some embodiments, the monoclonal antibodies against human FOLR1 are humanized antibodies. In certain embodiments, such antibodies are used therapeutically to reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to human subjects.

[0246] The method for engineering transformation, humanization or surface reconstruction of non-human or human antibodies can also be used and is well known in the art. Humanization, surface reconstruction or similar engineered antibodies can have one or more amino acid residues from non-human sources, such as but not limited to mouse, rat, rabbit, non-human primate or other mammals. These non-human amino acid residues are often referred to as "introducing" residue residue replacement, and the "introducing" residue is usually taken from the "introducing" variable, constant or other domains of known human sequences.

[0247] Such introduced sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life or any other suitable characteristic, as known in the art. Typically, the CDR residues are directly or substantially involved in influencing FOLR1 binding. Thus, some or all of the non-human or human CDR sequences are maintained, while the non-human sequences of the variable and constant regions may be replaced with human or other amino acids.

[0248] The antibody may also be optionally humanized, resurfaced, engineered, or engineered while retaining high affinity for the antigen FOLR1 and other favorable biological properties. To achieve this goal, three-dimensional models of the parental sequences, engineered sequences, and humanized sequences may be used to optionally prepare humanized (or human) or engineered anti-FOLR1 antibodies and resurfaced antibodies by analytical methods of the parental sequences and various conceptually humanized and engineered products. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Inspection of these displays allows analysis of the possible role of residues in the function of candidate immunoglobulin sequences, i.e., analysis of residues that affect the ability of candidate immunoglobulins to bind to their antigens, such as FOLR1. In this way, framework (FR) residues may be selected and combined with consensus sequences and introduced sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen.

[0249] Humanization, resurfacing or engineering of the antibodies of the invention can be performed using any known method, such as, but not limited to, those described in Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 333:334 (1989); 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); U.S. Patent Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763 ,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567; PCT / : US98 / 1 US96 / 18978 229246; 7,557,189; 7,538,195; and 7,342,110, each of which is incorporated herein by reference in its entirety, including the references cited herein.

[0250] In certain alternative embodiments, the antibody to FOLR1 is a human antibody. Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes isolated from immune individuals that produce antibodies against a target antigen or immunized in vitro can be generated (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol., 147 (1): 86-95; and U.S. Pat. No. 5,750,373). In addition, the human antibodies can be selected from a phage library, wherein the phage library expresses human antibodies as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314; Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; and Marks et al., 1991, J. Mol. Biol., 222:581. Techniques for the production and use of antibody phage libraries are also described in U.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and in Rothe et al., 2007, J. Mol. Bio., doi: 10.1016 / j.jmb.2007.12.018, each of which is incorporated herein by reference in its entirety. Affinity maturation strategies and chain shuffling strategies (Marks et al., 1992, Bio / Technology 10:779-783, entirely incorporated by reference) are known in the art and can be used to generate high affinity human antibodies.

[0251] Humanized antibodies can also be prepared in transgenic mice containing human immunoglobulin loci that are capable, upon immunization, of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016

[0252] In certain embodiments, antibody fragments are provided for, for example, increasing tumor penetration. Various techniques for producing antibody fragments are known. Traditionally, these fragments are produced via 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 can all be expressed in E. coli or other host cells and secreted therefrom, thereby allowing the production of large quantities of these fragments. Such antibody fragments can also be isolated from antibody phage libraries. The antibody fragments can also be linear antibodies as described in, for example, U.S. Patent No. 5,641,870, and can be monospecific or bispecific. Other techniques for producing antibody fragments will be apparent to skilled practitioners.

[0253] For the purposes of the present invention, it should be recognized that the modified antibodies may comprise any type of variable region that provides for the association of antibodies with polypeptides of human FOLR1. In this regard, the variable region may comprise or be derived from any type of mammal that can be induced to produce a humoral response and produce an immunoglobulin against the desired tumor-associated antigen. Thus, the variable region of the modified antibody may be, for example, human, mouse, non-human primate (e.g., cynomolgus monkey, macaque, etc.) or wolf origin. In some embodiments, the variable region and constant region of the modified immunoglobulin are both human. In other embodiments, the variable region of a compatible antibody (usually derived from a non-human source) may be engineered or specifically modified to improve binding properties or reduce the immunogenicity of the molecule. In this regard, the variable region useful in the present invention may be humanized or otherwise altered by including an introduced amino acid sequence.

[0254] In certain embodiments, the variable domains in both the heavy chain and the light chain are changed by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence changes. Although CDRs may be derived from antibodies of the same class or even subclass as the framework region derived from the antibody therein, it should be envisioned that CDRs will be derived from antibodies of different classes and, in certain embodiments, from antibodies from different species. It may not be necessary to replace all CDRs with complete CDRs from donor variable regions to transfer the antigen binding capacity of one variable domain to the antigen binding capacity of another variable domain. On the contrary, it may only be necessary to transfer those residues necessary for the activity of maintaining the antigen binding site. In view of the explanations proposed in U.S. Patent Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within the capabilities of those skilled in the art to obtain functional antibodies with reduced immunogenicity by performing routine experiments or by trial and error testing.

[0255] Although the variable region is changed, it will be understood by those skilled in the art that the modified antibodies of the present invention will include antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) wherein at least a portion of one or more constant region domains is deleted or otherwise altered to provide desired biochemical properties, such as increased tumor localization or reduced serum half-life when compared to antibodies having approximately the same immunogenicity comprising a natural or unaltered constant region. In some embodiments, the constant region of the modified antibody will include a human constant region. The modification of the constant region consistent with the present invention includes the addition, deletion or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may include changes or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and / or to the light chain constant domain (CL). In some embodiments, it is contemplated that one or more domains are partially or completely deleted in a modified constant region. In some embodiments, the modified antibody will include a domain-deleted construct or variant in which the entire CH2 domain has been removed (ΔCH2 construct). In some embodiments, the omitted constant region domain will be replaced by a short amino acid spacer (eg, 10 residues) that provides some of the molecular flexibility normally conferred by the absent constant region.

[0256] It should be noted that in certain embodiments, the modified antibodies may be engineered to fuse the CH3 domain directly to the hinge region of the corresponding modified antibody. In other constructs, it may be necessary to provide a peptide spacer between the hinge region and the modified CH2 and / or CH3 domains. For example, a compatible construct may be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is connected to the hinge region by a 5 to 20 amino acid spacer. Such a spacer may be added in order to, for example, ensure that the regulatory elements of the constant domain remain free and accessible or to keep the hinge region flexible. However, it should be noted that in some cases, the amino acid spacer may prove to be immunogenic and induce an undesired immune response to the construct. Therefore, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic, or even completely omitted, in order to maintain the desired biochemical properties of the modified antibody.

[0257] In addition to the deletion of the entire constant region domain, it will be recognized that the antibodies of the present invention can be provided by partial deletion or substitution of a few or even single amino acids. For example, a mutation of a single amino acid in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding and thus increase tumor localization. Similarly, it may be necessary to only delete that part of one or more constant region domains that control the effector function to be regulated (e.g., complement C1Q binding). This type of partial deletion of the constant region can improve the selected characteristics of the antibody (serum half-life) while keeping other desired functions associated with the constant region domain intact. In addition, as mentioned above, the constant region of the disclosed antibody can be modified by mutation or substitution of one or more amino acids that enhance the properties of the resulting construct. In this regard, it may be possible to destroy the activity provided by the conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic properties of the modified antibody. Certain embodiments may include adding one or more amino acids to the constant region to enhance the desired characteristics, such as reducing or increasing effector functions or providing more cytotoxins or carbohydrate attachments. In such embodiments, it may be desirable to insert or replicate specific sequences derived from selected constant region domains.

[0258] The present invention further includes variants and equivalents substantially homologous to chimeric, humanized and human antibodies, or antibody fragments thereof proposed herein. These may include, for example, conservative substitution mutations, i.e., one or more amino acids are replaced by similar amino acids. For example, conservative substitutions refer to amino acids being replaced by another amino acid in the same general classification, such as, for example, an acidic amino acid is replaced by another acidic amino acid, a basic amino acid is replaced by another basic amino acid, or a neutral amino acid is replaced by another neutral amino acid. The purpose of conservative amino acid substitutions is well known in the art.

[0259] The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, including antibodies or fragments thereof directed against human FOLR1. It will be recognized in the art that some amino acid sequences of the present invention may be altered without significantly affecting the structure or function of the protein. Therefore, the present invention further includes variants of polypeptides that exhibit substantial activity or comprise regions of antibodies or fragments thereof directed against human folate receptor protein. Such mutants include deletions, insertions, inversions, duplications and type substitutions.

[0260] Polypeptides and analogs may be further modified to include additional chemical moieties that are not normally part of the protein. These derived moieties may improve the solubility, biological half-life, or absorption of the protein. The moieties may also reduce or eliminate any desired side effects of the protein, etc. A review of those moieties may be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th edition, Mack Publishing Co., Easton, PA (2000).

[0261] The isolated polypeptide described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences encoding isolated polypeptide sequences and expressing those sequences in suitable transformed hosts. In some embodiments, recombinant technology is used to construct DNA sequences by separating or synthesizing DNA sequences encoding target wild-type proteins. Optionally, sequences can be mutated by site-specific mutagenesis to provide functional analogs thereof. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81: 5662-5066 (1984) and U.S. Patent No. 4,588,585.

[0262] In some embodiments, the DNA sequence encoding the target polypeptide will be constructed by chemical synthesis using an oligonucleotide synthesizer. This type of oligonucleotide can be designed based on the amino acid sequence of the desired polypeptide and selected to be advantageous in the host cell, and the target recombinant polypeptide will be produced in the host cell. Standard methods can be applied to the isolated polynucleotide sequence of the polypeptide separated by the synthetic coding target. For example, a complete amino acid sequence can be used to construct a gene for reverse translation. In addition, a DNA oligomer containing a nucleotide sequence encoding a specific isolated polypeptide can be synthesized. For example, some small oligonucleotides of the part of the desired polypeptide can be synthesized and then connected. Each oligonucleotide usually comprises a 5' or 3' protrusion for complementary assembly.

[0263] Once assembled (by synthesis, site-directed mutagenesis or other methods), the polynucleotide sequence encoding the specific target isolated polypeptide will be inserted into an expression vector and operably linked to an expression control sequence suitable for the expression of the protein in the desired host. Appropriate assembly can be confirmed by nucleotide sequencing, restriction enzyme cutting patterns, and expression of the biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of the transfected gene in the host, the gene must be operably linked to transcription and translation expression control sequences that are functional in the selected expression host.

[0264] In certain embodiments, a recombinant expression vector is used to amplify and express DNA encoding an antibody or fragment thereof against human FOLR1. A recombinant expression vector is a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain of an anti-FOLR1 antibody or fragment thereof, or a fragment thereof operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcription unit generally comprises an assembly of: (1) one or more genetic elements that have a regulatory role in gene expression, e.g., a transcriptional promoter and / or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational initiation and termination sequences. Such regulatory elements may include an operator sequence to control transcription. The ability to replicate in a host, which is usually conferred by an origin of replication, and a selection gene that aids in the recognition of transformants may be additionally incorporated. DNA regions are operably linked when they are functionally related to each other. For example, if the DNA of a signal peptide (secretion leader sequence) is expressed as a precursor that participates in the secretion of a polypeptide, it is operably linked to the DNA of the polypeptide; if a promoter controls the transcription of a coding sequence, it is operably linked to the coding sequence; or if a ribosome binding site is positioned to allow translation, it is operably linked to the coding sequence. The structural elements intended to be used in a yeast expression system include a leader sequence that enables the host cell to secrete translated proteins extracellularly. Alternatively, when a recombinant protein is expressed without a leader sequence or a transport sequence, it may include an N-terminal methionine residue. The residue may optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0265] The selection of expression control sequences and expression vectors will depend on the selection of the host. A wide range of expression host / vector combinations can be adopted. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli (including pCR 1, pBR322, pMB9 and derivatives thereof), wide host range plasmids (such as M13), and filamentous single-stranded DNA phages.

[0266] Suitable host cells for expressing FOLR1 binding polypeptides or antibodies (or FOLR1 proteins for use as antigens) include prokaryotes, yeast, insects, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985), the relevant disclosure of which is hereby incorporated by reference. Additional information on methods of protein production (including antibody production) can be found in, for example, U.S. Patent Publication No. 2008 / 0187954, U.S. Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO 04009823, each of which is hereby incorporated by reference in its entirety.

[0267] Different mammals or insect cell culture systems are also advantageously used to express recombinant proteins. Expression of recombinant proteins in mammalian cells can be carried out because such proteins are usually correctly folded, appropriately modified and fully functional. Examples of suitable mammalian host cell lines include HEK-293 and HEK-293T, the COS-7 system of monkey kidney cells described by Gluzman (Cell 23:175, 1981) and other cell lines, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors can include non-transcriptional elements such as replication origins, promoters and enhancers suitable for being connected to the gene to be expressed, and other 5' or 3' side non-transcribed sequences and 5' or 3' non-translated sequences, such as essential ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites and transcription termination sequences. The baculovirus system for producing heterologous proteins in insect cells is reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0268] The protein produced by the transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be attached to the protein to allow easy purification by appropriate affinity columns. The separated protein can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance and x-ray crystallography.

[0269] For example, the supernatant from a system that secretes the recombinant protein into the culture medium can first be concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be used, for example, a matrix or substrate with diethylaminoethyl (DEAE) side groups. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reversed phase high performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium (e.g., silica gel with methyl or other aliphatic side groups) can be used to further purify the FOLR1 binder. Some or all of the aforementioned purification steps in various combinations can also be used to provide a homogeneous recombinant protein.

[0270] Recombinant proteins produced in bacterial culture can be isolated by, for example, initial extraction from the cell mass, followed by one or more concentration, salting out, aqueous ion exchange, or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be used for the final purification step. Microbial cells employed in the expression of recombinant proteins can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lytic agents.

[0271] Methods known in the art for purifying antibodies and other proteins also include, for example, those described in U.S. Patent Publication Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is hereby incorporated by reference in its entirety.

[0272] III. Polynucleotides

[0273] In certain embodiments, the present invention encompasses polynucleotides including polynucleotides encoding polypeptides that specifically bind to human FOLR1 receptors or fragments of such polypeptides. For example, the present invention provides polynucleotides comprising a nucleic acid sequence encoding an antibody to human FOLR1 or a fragment encoding such an antibody. The polynucleotides of the present invention may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotide is a cDNA or DNA lacking one or more endogenous introns.

[0274] In some embodiments, the polynucleotide is a non-naturally occurring polynucleotide.In some embodiments, the polynucleotide is recombinantly produced.

[0275] In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure. In some embodiments, the polynucleotide is purified from natural components.

[0276] The present invention provides polynucleotides including polynucleotides encoding polypeptides, the polypeptides comprising a sequence selected from the group consisting of SEQ ID NOs: 3-38 and 59-67. Also provided are polynucleotides encoding polypeptides 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: 3-38 and 59-67.

[0277] The present invention also provides a polynucleotide comprising a sequence selected from those shown in Tables 7 and 8 below.

[0278] Table 7: Variable heavy chain polynucleotide sequences

[0279]

[0280]

[0281] Table 8: Variable light chain polynucleotide sequences

[0282]

[0283]

[0284] Also provided are polynucleotides 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 any one of SEQ ID NOs: 39-44.

[0285] Also provided are polynucleotides encoding a variable light chain that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical or identical to the variable light chain sequence of an antibody produced by a murine hybridoma having ATCC Accession No. PTA-120196 or PTA-120197.

[0286] Also provided are polynucleotides comprising a variable light chain coding sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical or identical to a variable light chain coding sequence encoding the variable light chain of an antibody produced by a murine hybridoma having ATCC Accession No. PTA-120196 or PTA-120197.

[0287] Also provided are polynucleotides encoding a variable heavy chain that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical or identical to the variable heavy chain sequence of an antibody produced by a murine hybridoma having ATCC Accession No. PTA-120196 or PTA-120197.

[0288] Also provided are polynucleotides comprising a variable heavy chain coding sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical or identical to a variable heavy chain coding sequence encoding a variable heavy chain of an antibody produced by a murine hybridoma having ATCC Accession No. PTA-120196 or PTA-120197.

[0289] In certain embodiments, the polynucleotide comprises a coding sequence for a mature polypeptide fused to a polynucleotide that helps, for example, the expression and secretion of a polypeptide from a host cell in the same reading frame (e.g., a leader sequence that serves as a secretory sequence for controlling the transport of a polypeptide from the cell). A polypeptide with a leader sequence is a preprotein and may have a leader sequence that is cleaved by a host cell to form a mature form of the polypeptide. A polynucleotide may also encode a proprotein that is a mature protein plus an additional 5' amino acid residue. A mature protein with a prosequence is a proprotein and is an inactive form of a protein. Once the prosequence is cleaved, the active mature protein is retained.

[0290] In certain embodiments, the polynucleotide comprises a coding sequence for a mature polypeptide fused in the same reading frame to a marker sequence that allows, for example, purification of the encoded polypeptide. For example, in the case of a bacterial host, the marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide purification of a mature polypeptide fused to the marker, or when a mammalian host (e.g., COS-7 cells) is used, the marker sequence may be a hemagglutinin (HA) tag derived from an influenza hemagglutinin protein.

[0291] The present invention also relates to variants of the polynucleotides encoding, for example, fragments, analogs and derivatives described above.

[0292] Polynucleotide variants may contain changes in coding regions, noncoding regions, or both. In some embodiments, polynucleotide variants include changes that produce silent substitutions, additions, or deletions but do not alter the properties or activity of the encoded polypeptide. In some embodiments, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants may be produced for a variety of reasons, for example, to optimize codon expression for a specific host (changing codons in human mRNA to those preferred by bacterial hosts such as E. coli).

[0293] Also provided are vectors and cells comprising the polynucleotides described herein.

[0294] IV. Biological Samples

[0295] Biological samples are often fixed with a fixative. Aldehyde fixatives such as formalin (formaldehyde) and glutaraldehyde are commonly used. Tissue samples fixed using other fixation techniques such as alcohol immersion (Battifora and Kopinski, J. Histochem. Cytochem. (1986) 34: 1095) are also suitable. The samples used can also be embedded in paraffin. In one embodiment, the sample is both fixed with formalin and embedded in paraffin (FFPE). In another embodiment, the FFPE block is stained with hematoxylin and eosin before selecting one or more parts for analysis to select specific areas for the FFPE core sample. Methods for preparing tissue blocks from these microparticle specimens have been used in previous IHC studies of various prognostic factors and / or are well known to those skilled in the art (see, for example, Abbondanzo et al., Am J Clin Pathol. 1909 May; 93 (5): 698-702; Allred et al., Arch Surg. 1990 Jan; 125 (1): 107-13).

[0296] In short, any intact organ or tissue can be cut into relatively small pieces and incubated in various fixatives (e.g., formalin, alcohol, etc.) for different time periods until the tissue is "fixed". The sample can be virtually any intact tissue surgically removed from the body. The sample can be cut into moderately small pieces suitable for being mounted on equipment routinely used in histopathology laboratories. The size range of the cut pieces is typically from a few millimeters to a few centimeters. The biological sample can also be a fluid extract, blood, plasma, serum, cerebrospinal fluid, lymph fluid, and or spleen preparations.

[0297] V. Correlation between FOLR1 expression and therapeutic efficacy

[0298] Antibody maytansinoid conjugate (AMC) IMGN853 comprises a monoclonal antibody that binds FOLR1, huMov19 (M9346A), conjugated to maytansinoid; the maytansinoid is DM4 (N(2')-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)-maytansine) linked via a cleavable sulfo-SPDB (4-(2-pyridyldithio)2-sulfobutyric acid N-succinimidyl ester) linker. The antibody sequence of IMGN853 (huMov19) is provided below as SEQ ID NOs: 45 and 47, and IMGN853 and huMov19 are described in U.S. Application Publication No. 2012 / 0009181 (notably 8,557,966), which is incorporated herein by reference in its entirety.

[0299] SEQ ID NO: 45—huMov19 vHC

[0300]

[0301] SEQ ID NO: 46-huMov19 vLCv1.00

[0302]

[0303] SEQ ID NO: 47-huMov19 vLCv1.60

[0304]

[0305] SEQ ID NO: 48-huMov19 vLC CDR1

[0306] KASQSVSFAGTSLMH

[0307] SEQ ID NO: 49-huMov19 vLC CDR2

[0308] RASNLEA

[0309] SEQ ID NO: 50-huMov19 vLC CDR3

[0310] QQSREYPYT

[0311] SEQ ID NO: 51-huMov19 vHC CDR1

[0312] GYFMN

[0313] SEQ ID NO:52—huMov19 vHC CDR2—Kabat defined

[0314] RIHPYDGDTFYNQKFQG

[0315] SEQ ID NO: 53—huMov19 vHC CDR2-Abm defined

[0316] RIHPYDGDTF

[0317] SEQ ID NO: 54-huMov19 vHC CDR3

[0318] YDGSRAMDY

[0319] SEQ ID NO: 55—huMov19 HC amino acid sequence

[0320]

[0321] SEQ ID NO: 56-huMov19 LCv1.00

[0322]

[0323] SEQ ID NO: 57-huMov19 LCv1.60

[0324]

[0325] SEQ ID NO:58—muMov19 vHC CDR2—Kabat defined

[0326] RIHPYDGDTFYNQNFKD

[0327] IMGN853 is currently in clinical development for a variety of therapeutic indications, including FOLR1-positive ovarian cancer, non-small cell lung cancer, endometrioid carcinoma, renal cancer, and other epithelial malignancies. Ovarian cancer exhibits the greatest FOLR1 penetrance and is considered the primary indication for treatment with IMGN853 (Antony AC. Ann Rev Nutr 16: 501-21 (1996); Yuan Y et al. Hum Pathol 40 (10): 1453-1460 (2009)). Measuring the level of FOLR1 in patient plasma samples can help identify patient populations that are more likely to respond to AMC treatment.

[0328] In certain embodiments, the present invention provides a method for identifying a subject who may respond favorably to a FOLR1-targeted anti-cancer therapy due to elevated FOLR1 expression levels expressed in the subject, specifically using antibodies and antigen-binding fragments thereof provided herein that can detect a dynamic range of FOLR1 expression levels, e.g., in IHC.

[0329] Evaluation of patient samples and correlation with in vivo efficacy using xenograft models demonstrates the ability of expression analysis to select subjects who are more likely to respond to treatment. IHC provides scores for FOLR1 expression on tumor cells: 0 (no expression) to 3 (or 3+) (very high levels of expression). In vivo data using xenograft models demonstrate that samples with FOLR1 expression scores of 2 or 3 (or 3+) have an increased likelihood of response to FOLR-1 targeted anticancer therapy at clinically relevant doses of FOLR1 immunoconjugates (see, e.g., U.S. Provisional Application Nos. 61 / 823,317 and 61 / 828,586 and International Application No. PCT / US2014 / 037911, all of which are incorporated herein by reference in their entirety). Therefore, identifying individuals with elevated FOLR1 scores will help identify those individuals who may respond to clinically relevant doses. In addition, more uniform levels of expression of FOLR1 provide better correlation with therapeutic benefit. Therefore, a combination of homogeneous staining uniformity or increased staining with heterogeneous staining uniformity may indicate increased FOLR1 expression. For example, a heterogeneity score greater than 2 can be used as a patient selection criterion for treatment with a FOLR1 therapeutic agent (see, e.g., U.S. Published Application No. 2012 / 0282175, which is herein incorporated by reference in its entirety).

[0330] FOLR1 expression analysis also identifies patients in whom reduced levels of FOLR1 targeted anti-cancer therapy ("low-dose therapy") can effectively elicit an anti-tumor response. As is well understood in the art, compounds are generally administered at the minimum dose that achieves the desired therapeutic response. This is particularly important for therapeutic agents that cause clinical side effects. The ability to identify those subjects with elevated FOLR1 expression levels allows the dose of FOLR-1 targeted therapeutic agents to be minimized, thereby reducing possible side effects while maintaining therapeutic efficacy.

[0331] Thus, the antibodies and antigen-binding fragments provided herein are particularly advantageous for use in such methods because they are able to detect a dynamic range of FOLR1 expression levels, for example in IHC.

[0332] VI. Shedding antigen assay

[0333] Measuring the level of circulating antigen (shed antigen) in patient plasma samples can help identify patient populations that are more likely to respond to treatment, such as antibody maytansinoid conjugate (AMC) treatment. High levels of shed antigen have been reported to significantly affect the pharmacokinetics of therapeutic antibodies (Tolcher A. et al. 20th Symposium on Molecular Targets and Cancer Therapeutics; October 21-24, 2008; Geneva, Switzerland: EORTC-NCI-AACR, p163, #514; Baselga J et al. J Clin Oncol 14: 737-744 (1996)). It is possible that the level of shed antigen from patient plasma samples will be variable depending on factors such as antigenic target, disease indication, and course of disease. Currently, the level of shed antigen for the disease indication of the anti-FOLR1 immunoconjugate IMGN853 has not been fully examined, and the correlation with solid tumor expression is limited. Although elevation of FOLR1 has been reported in ovarian adenocarcinoma, data suggest that it is not elevated in other FOLR1+ tumor indications such as small cell lung cancer (Mantovani LT, et al. Eur J Cancer 30A(3):363-9 (1994); Basal E, et al. PLoS ONE 4(7):e6292 (2009)). The methods of the present invention allow for detection of the FOLR1 receptor in the presence of high folate using antibodies and antigen-binding fragments thereof provided herein that are capable of detecting a dynamic range of shed FOLR1. Previous assays used Mov19 in the design of the assay. Because IMGN853 comprises Mov19 and is, in one embodiment, a targeted therapy of the present invention, it is important that the method detects FOLR1 in the presence or absence of Mov19. Previous assays using Mov19 have a competitive effect and will detect significantly less or no FOLR1 in patients treated with IMGN853.

[0334] In one embodiment, the method of the present invention for detecting FOLR1 in a fluid sample of human origin uses a traditional sandwich ELISA format. In one embodiment, the method uses a capture reagent (i.e., an antibody) of FOLR1 attached to a solid support. In one embodiment, the solid support is a microtiter plate. For this, the sample (ascites, plasma, etc.) is added without dilution and detected by different detection agents (different antibodies) that do not interfere with the binding of the first capture agent. The detection agent is then detected by using a second detection agent (biotin / streptavidin, anti-human second monoclonal or polyclonal antibody, etc.), which can bind to the first detection agent more than once, thereby amplifying the detection signal. The second detection agent is then quantified by using some other means (e.g., TMB / peroxidase, scintillation counting, fluorescent probes, etc.). In addition, the assay detects FOLR1 and is not adversely affected by the presence of Mov19, IMGN853, other FOLR1 family members, or folic acid.

[0335] The assays of the present invention include both assays for selecting patients eligible for FOLR1-based therapies and assays for monitoring patient responses. Assays for response prediction are performed before treatment selection, and the level of FOLR1 can affect treatment decisions. For monitoring patient responses, the assay is performed at the beginning of treatment to establish a baseline (or predetermined) level of FOLR1 in the sample. The same sample is then assayed and the level of FOLR1 is compared to the baseline or predetermined level. As used herein, the term "predetermined level" generally refers to an assay cutoff value used to assess a diagnostic result by comparing the assay result with a predetermined level, and wherein the predetermined level has been linked or associated with various clinical parameters (e.g., monitoring whether a subject treated with a drug has achieved an effective blood level of the drug, monitoring the response of a subject receiving cancer treatment with an anticancer drug, monitoring the response of a tumor in a subject receiving treatment of the tumor, etc.). The predetermined level can be an absolute value or a value standardized by subtracting a value obtained from a patient before starting treatment. An example of a predetermined level that can be used is a baseline value obtained from one or more subjects who are optionally suffering from one or more diseases or conditions. The comparison of the level of the determined biomarker to a baseline or predetermined level (or information analysis) can be performed by an automated system, such as a software program or intelligent system that is part of or compatible with the device (e.g., a computer platform) on which the determination is performed. Alternatively, the comparison or information analysis can be performed by a physician. In one embodiment where the level remains the same or decreases, the treatment can be effective and can be continued. In the event of a significant increase relative to the baseline level (or predetermined level), the patient may not respond. In another embodiment, an increase in the level of shed FOLR1 can indicate increased cell death and increased release of shed FOLR1. In this embodiment, an increase in shed FOLR1 indicates therapeutic efficacy.

[0336] The assays of the present invention can be performed by any protein assay method. Protein assays suitable for use in the present invention are well known in the art and include immunoassays involving the binding of specific unlabeled or labeled antibodies or proteins to expressed FOLR1 proteins or fragments. Useful immunoassays include liquid phase assays using any format known in the art, such as but not limited to Biacore, time-resolved fluorescence resonance energy transfer (TR-FRET), ELISA formats (sandwich, forward and reverse competitive inhibition) or fluorescence polarization formats and solid phase assays such as immunohistochemistry. The FOLR1 antibodies and antigen-binding fragments thereof provided herein are particularly suitable for use in these immunoassays because, for example, they are able to detect the dynamic range of FOLR1.

[0337] VII. Circulating Tumor Cell Assay

[0338] The anti-FOLR1 antibodies described herein can also be used to detect FOLR1 in circulating tumor cell assays. Circulating tumor cells (CTCs) are cells that are shed from tumors into blood vessels and circulate in the bloodstream. CTCs are present in the circulation in very low numbers. In general, CTCs are enriched from patient blood or plasma by various techniques known in the art. CTCs can be stained for specific markers using methods known in the art, including but not limited to methods based on flow cytometry and methods based on IHC. CTCs can be stained for protein markers that are unique to tumor cells, which allows identification and distinction between CTCs and normal blood cells. Antibodies provided herein, including but not limited to 2.1, 5.7 and 9.20, can also be used to stain CTCs for FOLR1. CTC analysis can also include quantitative analysis of the number of CTCs and / or the number of FOLR1-positive CTCs. The FOLR1 antibodies described herein can be used to stain CTCs isolated from subjects with cancer to measure FOLR1 present in CTCs. An increase in CTCs expressing FOLR1 can help identify the subject as having a cancer that may respond to FOLR1-based therapy or allow optimization of a treatment regimen using a FOLR1 antibody or immunoconjugate. CTC FOLR1 quantification can provide information about tumor staging, response to treatment, and / or disease progression. It can be used as a prognostic, predictive, or pharmacodynamic biomarker. In addition, staining CTCs for FOLR1 using the antibodies provided herein can be used as a liquid biopsy alone or in combination with additional tumor marker analysis of solid biopsy samples.

[0339] VIII. Detection

[0340] The present invention also provides antibodies to FOLR1, typically of a monoclonal type, which are linked to at least one reagent to form a detection antibody conjugate. In order to increase the efficacy of the antibody molecule as a diagnostic agent, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one reporter molecule. A reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules that have been coupled to the antibody include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and / or ligands, such as biotin.

[0341] Certain examples of antibody conjugates are those in which the antibodies or antigen-binding fragments thereof provided herein are linked to a detectable label. A "detectable label" is a compound and / or element detectable due to their specific functional properties and / or chemical characteristics, the use of which allows the antibody or antigen-binding fragment to be linked thereto to be detected, and / or further quantified if desired.

[0342] Many suitable imaging agents are known in the art, as are methods for their attachment to antibodies (see, e.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each of which is incorporated herein by reference). The imaging moiety used can be, for example, a paramagnetic ion; a radioisotope; a fluorescent dye; an NMR-detectable substance; and / or X-ray imaging.

[0343] Exemplary fluorescent labels contemplated for use as binding agent (e.g., antibody) conjugates include, e.g., Alexa 350, Alexa 430, Alexa 488, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Dylight 488, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phycoerythrin, REG, rhodamine green, rhodamine red, tetramethylrhodamine (TMR) renal contrast agent (Renographin), ROX, TAMRA, TET, tetramethylrhodamine, Texas Red (Texas Red), Red) and derivatives of these labels (ie, halogenated analogs modified with isothiocyanates or other linkers for coupling, etc.). An exemplary radiolabel is tritium.

[0344] Antibody or antigen-binding fragment detection conjugates encompassed in the present invention include those used in vitro, wherein the antibody or fragment is linked to a second binding ligand and / or an enzyme (enzyme tag) that will produce a colored product when contacted with a chromogenic substrate. The FOLR1 antibodies and antigen-binding fragments thereof provided herein are particularly suitable for conjugate methods because, for example, they are able to detect the dynamic range of FOLR1. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) catalase, and / or glucose oxidase. In some embodiments, the second binding ligand is biotin and / or avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.

[0345] Molecules containing azido groups can also be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter & Haley, 1983). Specifically, 2- and 8-azido analogs of purine nucleotides have been used as site-directed photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map the nucleotide-binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et al., 1989) and can be used as antibody binders.

[0346] Several methods for connecting or coupling antibodies to their coupling moieties are known in the art. Some methods of connection involve the use of metal chelate complexes, such as organic chelators such as diethylenetriaminepentaacetic anhydride (DTPA) attached to the binding agent (e.g., antibody); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycoluril-3 (U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Protein-bound (e.g., antibody) conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved, for example, using monoclonal antibodies, and a detectable imaging moiety is conjugated to the antibody using a linker such as methyl-p-hydroxybenzimidate or 3-(4-hydroxyphenyl)propionic acid N-succinimidyl ester.

[0347] In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups within the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody combining site is contemplated. Antibody conjugates produced according to this method are disclosed to show improved longevity, specificity, and sensitivity (U.S. Pat. No. 5,196,066, which is incorporated herein by reference). Site-specific attachment of effector or reporter molecules wherein the reporter or effector molecule is coupled to a carbohydrate residue within the Fc region has also been disclosed in the literature (O'Shannessy et al., 1987).

[0348] In other embodiments of the invention, the immunoglobulin is radiolabeled with a nuclide such as tritium. In other embodiments, nanogold particles (eg, about 0.5 nm to 40 nm in size) and / or quantum dots (Hayward, Calif.) are used.

[0349] When a sandwich assay format is used, the capture antibody will be unlabeled. The detection antibody will be labeled directly, or indirectly by adding (after washing away excess detection antibody) a molar excess of a second labeled antibody directed against the first antibody.

[0350] The label used for the detection antibody is any detectable functional group that does not interfere with the binding of the FOLR1 antibody. Examples of suitable labels are those numerous labels known for use in immunoassays, including directly detectable moieties, such as fluorescent dyes, chemiluminescent labels, and radioactive labels; as well as moieties that must react or derivatize to be detected, such as enzymes. Examples of such labels include radioisotopes, 32 P, 14 C. 125 I. 3 H and 131 I, fluorophores (such as rare earth chelates or fluorescein and its derivatives), rhodamine and its derivatives, dansyl, umbelliferone, luciferase (such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456)), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase) coupled to enzymes that use hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase or microperoxidase), biotin / avidin, biotin / streptavidin, biotin / streptavidin-β-galactosidase with MUG, spin labels, phage labels, stable free radicals, etc. As noted herein, fluorescence detection is an example.

[0351] Conventional methods are available to covalently bind these labels to proteins or polypeptides. For example, coupling agents such as dialdehydes, carbodiimides, dimaleimides, diimidates, bis-azide benzidines, and the like can be used to label antibodies using fluorescent labels, chemiluminescent labels, and enzyme labels as described herein. See, for example, U.S. Patent Nos. 3,940,475 (fluorescence analysis) and 3,645,090 (enzymes); Hunter et al. Nature 144:945 (1962); David et al. Biochemistry 13:1014-1021 (1974); Pain et al. J. Immunol. Methods 40:219-230 (1981); and Nygren J. Histochem. and Cytochem. 30:407-412 (1982). In certain embodiments, the label herein is fluorescent to increase amplification and sensitivity to 8pg / ml, more preferably biotin with streptavidin-β-galactosidase and MUG for amplifying the signal. In certain embodiments, a colorimetric label is used, for example, where the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethylbenzidine.

[0352] The coupling of such labels (including enzymes) to antibodies is a standard operating procedure for those of ordinary skill in the immunoassay technique. See, for example, O'Sullivan et al. "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, JJ Langone and H. Van Vunakis, ed., Vol. 73 (Academic Press, New York, NY, 1981), pp. 147-166.

[0353] After adding the final labeled antibody, the amount of bound antibody is determined by removing excess unbound labeled antibody by washing and then measuring the amount of attached label using a detection method appropriate to the label and correlating the measured amount to the amount of shed FOLR1 in the biological sample. For example, in the case of an enzyme, the amount of color developed and measured would be a direct measure of the amount of shed FOLR1 present. Specifically, if HRP is the label, the color can be detected using the substrate 3,3',5,5'-tetramethylbenzidine at an absorbance of 450 nm.

[0354] IX. Substrates and indicators

[0355] The use of substrates and indicators is contemplated for the detection of FOLR1.

[0356] Horseradish peroxidase (HRP) is an enzyme that first forms a complex with hydrogen peroxide and then causes it to decompose to generate water and atomic oxygen. Like many other enzymes, HRP and some HRP-like activities can be inhibited by excess substrate. The complex formed between HRP and excess hydrogen peroxide is catalytically inactive and is reversibly inhibited in the absence of an electron donor (e.g., a chromogenic substance). The lack of excess hydrogen peroxide and electron donors quenches endogenous HRP activity. When used in an assay system, HRP can also be used to convert a limited substrate into its activated chromophore, thereby causing a color change. The HRP enzyme can be coupled to antibodies, peptides, polymers or other molecules by a variety of methods known in the art. Adding glutaraldehyde to a solution containing a mixture of HRP and antibodies will result in more antibody molecules being coupled to each other than with the enzyme coupling. In a two-step procedure, HRP first reacts with a bifunctional reagent. In the second stage, only activated HRP is mixed with the antibody, thereby causing more effective labeling and no polymerization. A two-step glutaraldehyde procedure is also used to couple HRP to (strept)avidin. This format is used, for example, in procedures where LAB and LSAB are substrates. Coupling with biotin also involves two steps, since biotin must first be derivatized to biotinyl-N-hydroxysuccinimide ester or biotin hydrazide before it can react with the epsilon amino group of the HRP enzyme.

[0357] 3,3'-diaminobenzidine (DAB) is a substrate for enzymes such as HRP that produces a brown end product that is highly insoluble in alcohol and other organic solvents. Oxidation of DAB also causes polymerization, thereby causing the ability to react with osmium tetroxide and thus increasing its staining intensity and electron density. Among several metals and methods for enhancing the optical density of polymerized DAB, gold chloride in combination with silver sulfide appears to be the most successful.

[0358] 3-Amino-9-ethylcarbazole (AEC) is a substrate for enzymes such as HRP and forms an alcohol-soluble rose-red end product upon oxidation. Therefore, specimens treated with AEC must not be immersed in alcohol or alcohol solutions (e.g., Harris hematoxylin). Instead, aqueous counterstains and mounting media should be used.

[0359] 4-Chloro-1-naphthol (CN) is a substrate for enzymes such as HRP and precipitates as a blue end product. Because CN is soluble in alcohol and other organic solvents, the specimen must not be dehydrated, exposed to alcohol counterstains, or covered with a slide using a mounting medium containing organic solvents. Unlike DAB, CN tends to diffuse from the site of precipitation.

[0360] p-phenylenediamine dihydrochloride / catechol (Hanker-Yates reagent) is a substrate for enzymes such as HRP, which gives a blue-black reaction product that is insoluble in alcohol and other organic solvents. Like polymeric DAB, this reaction product can be osmicated. Using Hanker-Yates reagent in immunoperoxidase techniques has achieved mixed results.

[0361] Calf intestinal alkaline phosphatase (AP) (MW 100 kD) removes (by hydrolysis) and transfers the phosphate group from organic esters by breaking the P-0 bond; an intermediate enzyme-substrate bond is temporarily formed. The main metal activators for AP are Mg++, Mn++ and Ca++.

[0362] AP was not widely used in immunohistochemistry until the unlabeled alkaline phosphatase anti-alkaline phosphatase (APAAP) procedure was published. The soluble immune complex utilized in this procedure has a molecular weight of approximately 560 kD. The main advantage of the APAAP procedure over the peroxidase anti-peroxidase (PAP) technique is the absence of interference from endogenous peroxidase activity. Endogenous peroxidase can be blocked using a dilute solution of hydrogen peroxide. Due to the potential interference of endogenous peroxidase activity with PAP staining, the APAAP technique is recommended for blood smears and bone marrow smears. Endogenous alkaline phosphatase activity from bones, kidneys, liver and some white blood cells can be inhibited by adding 1 mM levamisole (although 5 mM has been found to be more effective) to the substrate solution. Intestinal alkaline phosphatase is not adequately inhibited by levamisole.

[0363] In the immunoalkaline phosphatase staining method, the enzyme hydrolyzes naphthyl phosphate (substrate) into a phenolic compound and phosphoric acid. The phenol is coupled to a colorless diazonium salt (chromophore) to produce an insoluble colored azo dye. Several different combinations of substrate and chromophore have been used successfully.

[0364] AS-MX naphthyl phosphate AP substrate can be used in its acid form or as a sodium salt. The chromophore substrates Fast Red TR and Fast Blue BB produce bright red or bright blue final products, respectively. Both are soluble in alcohol and other organic solvents, so aqueous mounting media must be used. Fast Red TR is preferred when staining cell smears.

[0365] Additional exemplary substrates include AS-BI naphthyl phosphate, AS-TR naphthyl phosphate, and 5-bromo-4-chloro-3-indole phosphate (BCIP). Other possible chromophores include, for example, Fast Red LB, Fast Deep Red GBC, Nitro Blue Tetrazolium (NBT), and Iononitro Tetrazolium Violet (INT).

[0366] X. Immunoassay Methods

[0367] In other embodiments, the present invention relates to immunoassays for binding, purifying, removing, quantifying and / or generally otherwise detecting biological components such as ligands as encompassed by the present invention. Antibodies prepared according to the present invention may be used. Some immunoassays include, to name a few, immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluorescent immunoassay, chemiluminescent assay, bioluminescent assay, and Western blot. The steps of various applicable immunoassays have been described in the scientific literature, such as, for example, Doolittle MH and Ben-Zeev O, Methods Mol Biol. 1999; 109: 215-37; Gulbis B and Galand P, Hum Pathol. 1993 Dec; 24 (12): 1271-85; and De Jager R et al., Semin Nucl Med. 1993 Apr; 23 (2): 165-79, each of which is incorporated herein by reference.

[0368] Generally, the immunobinding method comprises obtaining a sample suspected of containing a ligand protein, polypeptide and / or peptide, and contacting the sample with a first ligand binding agent (e.g., an anti-ligand antibody) according to the invention under conditions effective to allow formation of an immune complex, as the case may be.

[0369] In terms of antigen detection, the biological sample analyzed can be any sample in which it is desired to detect FOLR1, such as a fluid extract, blood, plasma, serum, cerebrospinal fluid, lymph fluid, tissue sections or specimens, homogenized tissue extracts, biopsy aspirates, cells, isolated and / or purified forms of compositions containing FOLR1, or any biological fluid. In some embodiments, blood, plasma, or lymph samples or extracts are used.

[0370] Contacting the selected biological sample with the antibody under effective conditions for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time sufficient to allow the antibody to form (i.e., bind to) immune complexes with any ligand protein antigen present. After this period of time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, is usually washed to remove any non-specifically bound antibody species, thereby allowing detection of only those antibodies that are specifically bound within the primary immune complex.

[0371] In general, detection of immune complex formation is well known in the art and can be achieved by applying a variety of methods. These methods are generally based on the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzyme tags. U.S. patents related to the use of these labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference. Of course, additional advantages may be found by using a second binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement as known in the art.

[0372] The anti-ligand antibody used in the detection can itself be connected to a detectable label, wherein this label can then be simply detected, thereby allowing the amount of the primary immune complex in the composition to be determined. Alternatively, the first antibody that becomes bound to the primary immune complex can be detected by a second binding agent with binding affinity to the antibody. In these cases, the second binding agent can be connected to a detectable label. The second binding agent itself is often an antibody, so it can be referred to as a "second" antibody. The primary immune complex is contacted with a labeled second binding agent or antibody under effective conditions and for a period of time sufficient to allow the formation of a second immune complex. The second immune complex is then usually washed to remove any non-specifically bound labeled second antibody or ligand, and the remaining label in the second immune complex is then detected.

[0373] Another method includes detecting primary immune complexes by a two-step method. As described herein, a second binding agent such as an antibody with a binding affinity to the antibody is used to form a second immune complex. After washing, the second immune complex is contacted with a third binding agent or antibody with a binding affinity to the second antibody again under effective conditions for a period of time sufficient to allow immune complexes (third immune complexes) to form. The third ligand or antibody is connected to a detectable label, thereby allowing detection of the third immune complex thus formed. If necessary, this system can provide signal amplification.

[0374] In another embodiment, a biotinylated monoclonal or polyclonal antibody is used to detect the target antigen, and then a second step antibody is used to detect biotin attached to the complex biotin. In the method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, a portion of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubating in a continuous solution of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, wherein each step adds additional biotin sites to the antibody / antigen complex. The amplification step is repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody of anti-biotin. This second step antibody is, for example, labeled with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromophore substrate. In the case of suitable amplification, a conjugate visible to the naked eye can be produced.

[0375] In one embodiment, immunohistochemistry (IHC) is used for immunodetection. Using IHC, detection of FOLR1 in a sample can be achieved by targeting the sample with a probe (e.g., an anti-FOLR1 antibody). The probe can be directly or indirectly linked to a detectable label or can be detected by another probe that is directly or indirectly linked to a detectable label.

[0376] In some embodiments, the IHC can distinguish between different protein expression levels, e.g., calibrated IHC. In some embodiments, the IHC can distinguish between staining intensities of samples with low FOLR1 expression, medium FOLR1 expression, or high FOLR1 expression.

[0377] In one embodiment, immunological detection of FOLR1 (by immunohistochemistry) is scored for both intensity and uniformity (percentage of stained cells - cell membrane only). The correlation of the comparison scale of FOLR1 expression intensity is 0-negative, 0-1-very weak, 1-weak, 1-2-weak to moderate, 2-moderate, 2-3-moderate to strong, 3-strong, 3+-very strong. Quantitatively, a score of 0 represents no membrane staining was observed. A score of 1 represents that faint / barely noticeable membrane staining was detected. For a score of 2, weak to moderate complete membrane staining was observed. Finally, a score of 3 (or 3+) represents that moderate to complete membrane staining was observed. Those samples with a score of 0 or 1 for FOLR1 expression can be characterized as not having elevated FOLR1 expression, while those samples with a score of 2 or 3 can be characterized as overexpressing or having elevated FOLR1. In another embodiment, using the antibodies, antigen-binding fragments thereof, or polypeptides provided herein, those samples with a score of 0 for FOLR1 expression can be characterized as not having elevated FOLR1 expression, those samples with a score of 1 can be characterized as having increased FOLR1 expression, and those samples with a score of 2 or 3 can be characterized as overexpressing or having elevated FOLR1.

[0378] Samples that overexpress FOLR1 can also be assessed by immunohistochemical scores corresponding to the number of copies of FOLR1 molecules expressed per well or the number of antibodies bound per well (ABC), and can be determined biochemically. The comparison scale for FOLR1 uniformity (percent cell membrane staining) is as follows: negative = 0%; focal = <25%; heterogeneity (heterogeneity) = 25%-75% and homogeneity (homogeneity) = >75%.

[0379] In one embodiment, immunodetection of FOLR1 (by immunohistochemistry) is scored using an H score. The H score combines a staining intensity score (e.g., a score from 0 to 3, where 0 represents no staining and 3 represents strong staining) with the percentage of cells that are positive for membrane staining (i.e., uniformity). The H score can be calculated as follows: H score = [0*(percentage of cells stained with intensity 0)] + [1*(percentage of cells stained with intensity 1)] + [2*(percentage of cells stained with intensity 2)] + [3*(percentage of cells stained with intensity 3)]. Thus, the H score can range from 0 (no cell membrane staining) to 300 (all cell membrane staining with intensity 3).

[0380] In one embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is at least 50, the subject is identified as a candidate for treatment with an anti-FOLR1 treatment regimen (e.g., IMGN853). In one embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is at least 75, the subject is identified as a candidate for treatment with an anti-FOLR1 treatment regimen (e.g., IMGN853). In one embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is at least 100, the subject is identified as a candidate for treatment with an anti-FOLR1 treatment regimen (e.g., IMGN853). In one embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is at least 125, the subject is identified as a candidate for treatment with an anti-FOLR1 treatment regimen (e.g., IMGN853). In one embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is at least 150, the subject is identified as a candidate for treatment with an anti-FOLR1 treatment regimen (e.g., IMGN853). In one embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is at least 175, the subject is identified as a candidate for treatment with an anti-FOLR1 treatment regimen (e.g., IMGN853). In one embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is at least 200, the subject is identified as a candidate for treatment with an anti-FOLR1 treatment regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is at least 225, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is at least 250, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is at least 275, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a tumor sample from a subject with cancer is 300, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853).

[0381] In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is 75 to 300, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is at least 75, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is at least 100, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is at least 125, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is at least 150, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is at least 175, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is at least 200, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is at least 225, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is at least 250, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is at least 275, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an ovarian tumor sample from a subject with ovarian cancer is 300, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853).

[0382] In another embodiment, when the H score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is 50 to 300, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is at least 50, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is at least 75, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is at least 100, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is at least 125, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is at least 150, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is at least 175, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is at least 200, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is at least 225, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is at least 250, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in a NSCLC tumor sample from a subject with NSCLC is at least 275, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853).In another embodiment, when the H-score for FOLR expression in a NSCLC tumor sample from a subject with NSCLC is 300, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (eg, IMGN853).

[0383] In another embodiment, when the H score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is 50 to 300, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is at least 50, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is at least 75, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is at least 100, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is at least 125, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is at least 150, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is at least 175, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is at least 200, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is at least 225, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is at least 250, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In another embodiment, when the H-score of FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is at least 275, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853).In another embodiment, when the H-score for FOLR expression in an endometrial tumor sample from a subject with endometrial cancer is 300, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (eg, IMGN853).

[0384] By way of example, the H-score for a subject with ovarian cancer may be as follows:

[0385] H score = (75% intensity 0) + (0% intensity 1) + (0% intensity 2) + (25% intensity 3) = 75; or

[0386] H score=(0% intensity 0)+(75% intensity 1)+(0% intensity 2)+(25% intensity 3)=150.

[0387] In another example, the H-score for a subject with endometrial cancer may be as follows:

[0388] H score = (75% intensity 0) + (0% intensity 1) + (25% intensity 2) + (0% intensity 3) = 50; or

[0389] H score=(0% intensity 0)+(75% intensity 1)+(25% intensity 2)+(0% intensity 3)=125.

[0390] In all four examples above, the subject can be identified as a candidate for treatment with an anti-FOLR1 therapeutic regimen (eg, IMGN853).

[0391] In one embodiment, immunodetection of FOLR1 (by immunohistochemistry) is scored using percentage positivity and intensity across samples. In this embodiment, selection for treatment with an anti-FOLR1 treatment regimen is based on the percentage of cells in the sample that are found to express membrane FOLR1 at a specified level reflecting both staining intensity (e.g., 1, 2, or 3) and uniformity (e.g., heterogeneous or homogeneous (see Table 11)). For example, a sample with at least 25% (i.e., 25%-75% or >75%) FOLR1-positive cell staining at 3 can be characterized as "3 heterogeneous" and "3 homogeneous," or together as "at least 25% positive at 3."

[0392] In one embodiment, when at least 25% of the FOLR1 membrane expression in a tumor sample from a subject with ovarian cancer has an intensity score of 3 as determined by IHC, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In one embodiment, IHC is performed using a FOLR1-2.1 antibody.

[0393] In another embodiment, when at least 25% of the FOLR membranous expression in a tumor sample from a subject with endometrial cancer has an intensity score of at least 2 as determined by IHC, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In one embodiment, IHC is performed using a FOLR1-2.1 antibody.

[0394] In another embodiment, when at least 25% of the FOLR membrane expression in a tumor sample from a subject with NSCLC has an intensity score of at least 2 as determined by IHC, the subject is identified as a candidate for treatment with an anti-FOLR1 regimen (e.g., IMGN853). In one example, IHC is performed using the same FOLR1-2.1 antibody as in IHC.

[0395] IHC can be performed manually or using an automated system (e.g., using an automated stainer). IHC can be performed on cells, cell clumps, tissues, preparations from blood, plasma, serum, or lymph, etc. In some embodiments, the sample is a fixed sample. In some embodiments, the sample is a paraffin-embedded sample. In some embodiments, the sample is a formalin-fixed and paraffin-embedded sample.

[0396] In one embodiment, flow cytometry is used for immunoassay. Thus, for example, flow cytometry can be used to assess the number of antibodies (ABC) bound per cell. A high number of anti-FOLR1 antibodies bound per cell can indicate a high FOLR1 expression level and a high likelihood of susceptibility to treatment with an anti-FOLR1 antibody or its immunoconjugate.

[0397] XI. Compositions and Kits

[0398] The present invention also provides compositions and kits for use in the practice of the present invention as disclosed herein. Such kits may include containers, each container having one or more different reagents (usually in concentrated form) used in the method, including, for example, one or more binding agents (antibodies) that have been linked to a marker, or optionally having reagents for coupling the binding agent to the antibody (and the marker itself); buffers, and / or reagents and instruments for separation (optionally by microdissection) to support the practice of the present invention. A label or indicator describing the kit components in the ligand detection method of the present invention or a set of instructions for their use will generally also be included, wherein the instructions may be associated with the package insert and / or the packaging of the kit or its components.

[0399] In other embodiments, the present invention relates to an immunoassay kit for use with the immunoassay methods described herein. Since antibodies are generally used to detect FOLR1, the antibodies will generally be included in the kit. The immunoassay kit will therefore include a first antibody that binds FOLR1 and / or optionally an immunoassay reagent and / or further optionally a FOLR1 protein or a cell sample containing FOLR1 in a suitable container device.

[0400] The immunodetection reagents of the kit can take any of a variety of forms, including those detectable labels associated with and / or attached to a given antibody. Detectable labels associated with and / or attached to a second binding ligand are also contemplated. Exemplary second ligands are those second antibodies that have binding affinity for the first antibody.

[0401] Other suitable immunodetection reagents for use in the kits of the invention include two-component reagents comprising a second antibody having binding affinity for the first antibody, together with a third antibody having binding affinity for the second antibody, the three antibodies being linked to a detectable label. As described above, a variety of exemplary labels are known in the art and / or all such labels may be suitable for use with the present invention.

[0402] The kit may also contain a therapeutic agent for treating cancer, such as an anti-FOLR1 immunoconjugate.

[0403] The kit may also include a FOLR1 detection reagent for measuring FOLR1 expression in a subject, the kit including a FOLR1 detection reagent and instructions for use. In one embodiment, the FOLR1 detection reagent comprises a FOLR1 binding peptide or an anti-FOLR1 antibody. In another embodiment, the kit further includes a second antibody that binds to the anti-FOLR1 antibody.

[0404] In one embodiment, the following concentration of FOLR1 specific antibody is included: about 0.1 to about 20 μg / mL, about 0.1 to about 15 μg / mL, about 0.1 to about 10 μg / mL, about 0.5 to about 20 μg / mL, about 0.5 to about 15 μg / mL, about 0.5 to about 10 μg / mL, about 1 to about 20 μg / mL, about 1 to about 15 μg / mL, about 1 to about 10 μg / mL, about 2 to about 20 μg / mL, about 2 to about 15 μg / mL or about 2 to about 10 μg / mL. In another embodiment, the following concentration of FOLR1 specific antibody is included: about 1.5 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL or about 10 μg / mL. In another embodiment, the FOLR1 specific antibody is included at a concentration of about 2 μg / mL. In another embodiment, the FOLR1 specific antibody is included at a concentration of about 10 μg / mL.

[0405] In another embodiment, the antibody in concentrated solution is included, with dilution instructions to achieve a final concentration of about 1 to about 20 μg / mL, about 1 to about 15 μg / mL, about 1 to about 10 μg / mL, about 2 to about 20 μg / mL, about 2 to about 15 μg / mL or about 2 to about 10 μg / mL. In another embodiment, the antibody in concentrated solution is included, with dilution instructions to achieve a final concentration of about 1.5 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL or about 10 μg / mL. In another embodiment, the antibody in concentrated solution is included, with dilution instructions to achieve a final concentration of about 2 μg / ml. In another embodiment, the antibody in concentrated solution is included, with dilution instructions to achieve a final concentration of about 10 μg / ml.

[0406] In another embodiment, the kit further comprises a detection agent selected from the group consisting of: an enzyme, a fluorophore, a radiolabel, and a luminophore. In another embodiment, the detection agent is selected from the group consisting of: biotin, digoxigenin, fluorescein, tritium, and rhodamine.

[0407] The kit may also include instructions for detecting and scoring FOLR1 expression. The kit may also include a control or reference sample. Non-limiting examples of control or reference samples include cell agglomerates or tissue culture cell lines derived from normal (normal control) samples or tumor (positive control) samples. Exemplary cell lines include cell lines stably or transiently transfected with expression vectors expressing FOLR1. Additional examples include cell agglomerates and tissue samples described in the Examples.

[0408] In some embodiments, the kit is a packaged combination comprising the following essential elements: (a) a capture reagent comprising a monoclonal antibody against human FOLR1; and (b) a detection reagent, which may also comprise a FOLR1 monoclonal antibody, but may also comprise a detectable (labeled or unlabeled) antibody that binds to FOLR1. These essential elements are defined herein.

[0409] In one embodiment, the kit further comprises a solid support for the capture reagent, which may be provided as a separate element or the capture reagent is already immobilized thereon. Thus, the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such a support included with the kit or provided separately from the kit.

[0410] In one embodiment, the capture reagent is coated on a microtiter plate. The detection reagent can be a labeled antibody for direct detection or an unlabeled antibody detected by a labeled antibody for an unlabeled antibody produced in different species. In the case where the label is an enzyme, the kit will generally include substrates and cofactors required for the enzyme, and in the case where the label is a fluorophore, the kit will generally include a dye precursor providing a detectable chromophore. When the detection reagent is unlabeled, the kit may also include a detection method for detectable antibodies, such as labeled antibodies for unlabeled antibodies, such as in the form of fluorescence detection. In the case where the label is an enzyme, the kit will generally include substrates and cofactors required for the enzyme, in the case where the label is a fluorophore, the kit will generally include a dye precursor providing a detectable chromophore, and in the case where the label is biotin, the kit will generally include avidin, such as avidin, streptavidin, or streptavidin coupled to HRP or β-galactosidase with MUG.

[0411] In one embodiment, the capture reagent is FOLR1 antibody 2.1, 5.7 or 9.20, or an antibody comprising the sequence of antibody 2.1, 5.7 or 9.20. In one embodiment, the detection reagent is FOLR1 antibody 2.1, 5.7 or 9.20, or an antibody comprising the sequence of antibody 2.1, 5.7 or 9.20. In another embodiment, the detection reagent FOLR1 antibody 2.1, 5.7 or 9.20 or an antibody comprising the sequence of antibody 2.1, 5.7 or 9.20 is biotinylated.

[0412] The kit also typically contains instructions for performing the assay, and / or a FOLR1 protein or fragment thereof (e.g., all or part of the FOLR1 extracellular domain or the FOLR1 extracellular domain and the GPI-linked domain) as an antigen standard and other additives, such as stabilizers, washing and incubation buffers, etc. In one embodiment, the FOLR1 antigen standard is FOLR1-Fc immunoadhesin. The kit may also include instructions for detecting and scoring FOLR1 expression.

[0413] The components of the kit may be provided in predetermined ratios, wherein the relative amounts of the various reagents are suitably varied to provide a concentration of solution of the reagents that substantially maximizes the sensitivity of the assay. Specifically, the reagents may be provided as dry powders, typically lyophilized, including excipients, which upon dissolution will provide a reagent solution having an appropriate concentration for combination with a sample to be tested.

[0414] Compositions comprising antibodies or antigen-binding fragments described herein are also provided. In one embodiment, the composition comprises an anti-FOLR1 antibody or antigen-binding fragment described herein and a buffer, such as a buffer that can be used in a detection assay such as FACS, IHC or ELISA. Such buffers are known to those of ordinary skill in the art and include diluents. As an example, certain FACS buffers are provided herein (e.g., in the working examples). FACS buffers may also include, for example, serum or albumin (e.g., bovine serum, goat serum or BSA) and / or sodium azide. FACS buffers may also include PBS, EDTA and / or DNA enzymes or any combination thereof. IHC buffers are also provided herein and are known to those of ordinary skill in the art. IHC buffers may include, for example, casein serum or albumin (e.g., bovine serum, goat serum or BSA), Tween or Triton, PBS and / or sodium azide or any combination thereof. ELISA buffers are also provided herein and are known to those of ordinary skill in the art. ELISA buffers may include, for example, serum or albumin (e.g., bovine serum, goat serum or BSA), skim milk powder, casein and / or gelatin or any combination thereof.

[0415] The embodiments of the present disclosure may be further defined by reference to the following non-limiting examples, which describe in detail the preparation of certain antibodies of the present disclosure and methods for using the antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications to materials and methods may be practiced without departing from the scope of the present disclosure. Example

[0416] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes thereto will be suggested to those skilled in the art and are included within the spirit and scope of the present application.

[0417] Example 1: Generation of FOLR1 Hybridomas

[0418] Hybridomas producing anti-human FOLR1 monoclonal antibodies suitable for immunohistochemistry (IHC) staining (antibodies of the invention) were selected from more than 16,000 hybridomas. The hybridomas were produced by immunizing wild-type Balb / c mice with different antigens, including: formalin-fixed 300-19 cells that had been transfected with human FOLR1, human FOLR1-mouse IgG2a Fc recombinant protein, and human FOLR1 recombinant protein. Immunization with fixed 300-19 cells was performed by subcutaneous injection of transfected 300-19 cells in PBS in the absence of any adjuvant (5E6 cells / mouse / injection). Immunization with FOLR1 recombinant protein was performed by subcutaneous injection of protein emulsified in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (Sigma) or Magic mouse adjuvant (Creative Diagnostics) for boosting. Typically, mice were immunized five times (two weeks apart) before receiving a final boost, which was performed by intraperitoneal injection of the immunogen three days before fusion.

[0419] A total of 16 independent fusions (including fusions 352, 353 and 354) were performed using spleen cells and mouse myeloma P3X63Ag8.653 cells (P3 cells) derived from immunized wild-type Balb / c mice. Cell fusions were performed according to standard protocols using an ECM200 electrofusion machine (BTXHarvard Apparatus). More than 1,000 hybridomas were produced per fusion. The antibodies produced by these hybridomas were screened and confirmed using denatured FOLR1-positive cells and FOLR1-negative cells by a FACS-based method. Among the more than 16,000 hybridomas screened, 14 hybridomas that were positive by FACS screening were found. All positive hybridomas were derived from mice immunized with human FOLR1-mouse IgG2a Fc recombinant protein.

[0420] Of the 14 hybridomas that were initially positive by FACS screening, only ten showed sufficient IgG concentrations for further analysis.

[0421] Example 2. Immunohistochemical evaluation of hybridoma supernatants

[0422] Ten of the initial 14 hybridomas were analyzed by IHC. Analysis was performed using a Leica Bond RX autostainer and the reagents and conditions listed in Table 9.

[0423] Table 9. IHC reagents and assay conditions

[0424]

[0425] Slides containing formalin-fixed paraffin-embedded (FFPE) cells, normal tissue, patient lung tumor biopsies, and patient ovarian tumor biopsies were baked at 60°C and dewaxed using Bond dewaxing solution and 100% ethanol. Heat-induced epitope repair was performed using Bond epitope repair 2 (a solution based on ethylenediaminetetraacetic acid (pH 9.0)) for 20 minutes and endogenous peroxidase was blocked with peroxide for 5 minutes. The slides were incubated for 15 minutes with antibodies produced by ImmunoGen, Inc. at different concentrations or Leica / Novocastra muIgG1 control antibodies. The bound antibodies were detected by incubation with the Leica BondRefine detection system. After applying the antibodies, the slides were incubated with Post Primary Reagent (rabbit anti-mouse IgG) for 8 minutes, with polymer (goat anti-rabbit polymer) for 8 minutes and with DAB (3,3-diaminobenzidine tetrahydrochloride) for 10 minutes, which produced a brown color signal. Slides were counterstained with hematoxylin for 5 minutes.

[0426] As outlined below, FFPE tissue samples are derived from human tissue blocks obtained from Proteogenex and Cooperative Human Tissue Network (CHTN). FFPE cell samples are derived from KB cell lines supplied by the American Tissue Culture Collection. Slides containing sample sections are prepared from FFPE blocks using a microtome set at 5 μm and fixed on positively charged slides. Before staining, these slides are air dried overnight.

[0427] Table 10. FFPE test samples

[0428] Human Organization Type Commercial Sources Normal lung CHTN Normal pancreas CHTN Normal salivary glands CHTN Ovarian papillary serous adenocarcinoma Proteogenex Lung adenocarcinoma CHTN

[0429] FOLR1 staining intensity and distribution pattern were scored relative to control IgG staining (nonspecific). Intensity was scored on a scale of 0 to 3, where 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The uniformity of staining was scored as negative (no cells exhibiting positive staining), focal (<25% of cells staining), heterogeneous (25%-75% of cells staining), and homogeneous (>75% of cells staining). Staining intensity and scoring scales are described below. All staining was evaluated by a board-certified pathologist.

[0430] Table 11. Staining intensity and uniformity

[0431]

[0432] IHC selection method for characterization of hybridomas for FFPE FOLR1 IHC

[0433] Primary clones (ten clones in total) that were positive by FACS on FOLR1-positive degenerated cells were evaluated by IHC. Two clones were obtained from fusion 352 (clones 352.1 and 352.2). Six clones were obtained from fusion 353 (clones 353.1, 353.2, 353.3, 353.5, 353.9, 353.15), and two clones were obtained from fusion 354 (clones 354.1 and 354.2). Hybridoma supernatants were collected from cultured hybridoma cells and used for analysis. Anti-mouse L-chain-specific polyclonal antibodies for capturing mouse monoclonal antibodies from the supernatant and anti-mouse Fc-specific polyclonal antibodies for detecting captured antibodies were used to determine the antibody concentration in the hybridoma supernatant by ELISA; a mouse monoclonal IgG1 sample with a known concentration was used as a standard to calculate the IgG concentration. Cell culture medium (undiluted) showed no interference with the IHC staining method (no background / nonspecific staining was noted when culture medium was used instead of the primary antibody). Ten supernatants (IMGN 352.1, 352.2, 353.1, 353.2, 353.3, 353.5, 353.9, 353.15, 354.1 and 354.2) diluted in Leica Antibody Diluent at different concentrations up to 10 μg / mL were stained using FOLR1 known positive control samples (human normal lung cells, patient-derived ovarian serous papillary adenocarcinoma cells and KB cells) and evaluated to identify positive candidate clones (clones depicting acceptable membrane staining and specificity in FOLR1 positive samples). Five of the ten clones demonstrated acceptable membrane staining and good specificity in FOLR1 positive samples. Suitable staining concentrations were experimentally determined for each of the five candidate clones as follows: 353.1 (0.7 μg / mL), 353.2 (2.3 μg / mL), 353.3 (2.3 μg / mL), 353.5 (2 μg / mL and 10 μg / mL), and 353.9 (2 μg / mL and 10 μg / mL). Of the remaining 5 clones, clone 353.15 (stained at 2 and 10 μg / mL) showed acceptable membrane staining in KB cells and normal lung tissue; however, cytoplasmic staining was only observed in the ovarian tumor tissue of the patient tested. Clones 352.1, 352.2, and 354.1 did not show visible staining in any sample and clone 354.2 showed only obvious nonspecific cytoplasmic staining, all of which were considered unacceptable.

[0434] Five candidate clones were further subcloned. Subclones of four clones (353.2, 353.3, 353.5 and 353.9) were successfully identified, and no subclones of clone 353.1 were generated. A total of eight subclones were purified. Two subclones were obtained from clone 353.5 (353.5-7 and 353.5-10). Two subclones were obtained from clone 353.9 (353.9-20 and 353.9-21). Two subclones were obtained from clone 353.3 (353.3-8 and 353.3-9), and two subclones were obtained from clone 353.2 (353.2-1 and 353.2-12). (Note that these subclones are also referred to as 5.7, 5.10, 9.20, 9.21, 3.8, 3.9, 2.1, and 2.12, respectively.) These subclones were characterized by IHC using the methods described above at antibody concentrations of 2 and 10 μg / mL (Table 9: IHC reagents and assay conditions) Eight subclones were also sequenced as described below in Example 3. Candidate subclones were further evaluated to identify the best membrane staining and specificity and were ranked as follows: [353.2-1, 353.2-12], [353.9-20, 353.9-21], [353.5-7, 353.5-10], and [353.3-8, 353.3-9] and selected for further characterization (subclones were bracketed together based on sequence identity, as described in Example 3).

[0435] Two subclones were selected for further IHC assay optimization: 353.2-1 and 353.9-20. Both antibodies were used to stain human normal lung, human normal salivary gland, human normal pancreas, patient ovarian cancer biopsy, patient non-small cell lung cancer (NSCLC) biopsy, and patient clear cell renal cell carcinoma biopsy. Under optimal conditions (see Tables 12 and 12 below), the expression of 353.2-1 and 353.9-20 was detected in 1% 4% 5% 2 ... Fig.13 ), both subclones showed specific and reasonably sensitive staining in human normal tissue and patient tumor tissue. The ducts of the pancreas, the respiratory epithelium of the normal lung, and the intercalated ducts showed positive membrane-associated staining. The acinar cells / islets of the pancreas, the interalveolar connective tissue of the lung, and the acinar cells of the salivary glands, which were expected to be negative, did not show positive staining with either subclone. Tumor cells from ovarian cancer, NSCLC, and clear cell renal cell carcinoma samples, which were expected to be positive, showed positive membrane-associated staining localized to the tumor cells. Tumor substructures (stroma, blood vessels, and lymphocytes) that were expected to be negative did not show positive staining with 353.2-1 or 353.9-20. Additional staining of normal tissues with 353-2.1 (FOLR1-2.1) is summarized in Table 13 below and shown in Figure 14. In summary, the IHC characterization data indicate that 353.2-1 and 353.9-20 are specific for FOLR1 in FFPE tissues (see Figure 1 and Figure 2 ).

[0436] Table 12. Optimized assay conditions

[0437]

[0438] Table 13. Optimized assay conditions

[0439]

[0440]

[0441] Example 3. Characterization of selected anti-FOLR1 antibodies

[0442] As described above in Example 2, in the fourteen hybridoma clones selected based on primary and confirmation FACS screening, ten primary clones were analyzed by immunohistochemistry (IHC) analysis. In ten primary clones (i.e., 352.1, 352.2, 353.1, 353.2, 353.3, 353.5, 353.9, 353.15, 354.1 and 354.2), five were positive by IHC (i.e., 353.1, 353.2 and 353.3, 353.5 and 353.9), and all five were derived from the same fusion (fusion 353). Four of the five were successfully subcloned. A subclone of primary clone 353.2 was selected and named 353.2-1 ("2.1"). A subclone of primary clone 353.3 was selected and named 353.3-8 ("3.8"). One subclone of primary clone 353.5 was selected and named 353.5-7 ("5.7"), and two subclones of primary clone 353.9 were selected and named 353.9-20 ("9.20") and 353.9-21 ("9.21"). Subclones 9.20 and 9.21 were sequenced, and as expected, both clones had identical sequences. In addition, two of the clones, 2.1 and 9.20, were deposited with the ATCC on April 16, 2013 as PTA-120197 and PTA-120196, respectively.

[0443] Specificity of anti-FOLR1 antibodies determined by western blotting

[0444] The specificity of the generated antibodies was analyzed by Western blotting with a panel of cell lysates prepared from FOLR1 positive (Igrov-1, Ovcar-3, Caov-3, Wish and Skov-3) and FOLR1 negative (BxPC3, Panc-1 and ASPC1) cell lines. For the assay, the lysates were run in SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by standard procedures. The membranes were incubated with anti-FOLR1 antibodies of the invention, and the antigen-antibody complexes formed were detected with a secondary anti-mouse antibody coupled to horseradish peroxidase (hrp). Figure 3 ). All tested anti-FOLR1 antibodies recognized FOLR1 in cell lines with high FOLR1 expression levels (i.e., Igrov-1 and Wish). FOLR1 in low expressing cell lines Ovcar-3, Caov-3, and Skov-3 was detected only by anti-FOLR1 clones 2.1 and 9.21; clones 3.8 and 5.7 did not stain these cell lysates, probably due to insufficient antibody sensitivity. No additional nonspecific bands were detected in FOLR1 positive cell lines by the clones; no staining of FOLR1 negative cell lines was observed.

[0445] Binding of anti-FOLR1 antibodies to denatured and non-denatured cells

[0446] The ability of anti-FOLR1 antibodies to bind to denatured and non-denatured (native conformation) FOLR1 was determined by indirect FACS using FOLR1 positive cells KB and T47D. Cells were harvested by Versine and washed with phosphate buffered saline (PBS). Denatured cells were prepared by incubating cells overnight at 4°C in PBS containing 10% formaldehyde, followed by washing with PBS and incubation at 95°C for 30 minutes. Denatured and non-denatured cells were then incubated on ice for 2 hours with anti-FOLR1 antibodies diluted in FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum). The cells were centrifuged, washed with PBS and incubated with FITC-conjugated goat anti-mouse IgG antibodies for 40 minutes. The cells were centrifuged again, washed with PBS and resuspended with 0.2 ml of PBS containing 1% formaldehyde. Cell-associated fluorescence was measured using a FACSCalibur flow cytometer with HTS multiwell and analyzed using CellQuest Pro (BD Biosciences, San Diego, US). As shown on FIG. 4 , all anti-FOLR1 antibodies bound to both denatured and non-denatured cells.

[0447] Affinity of anti-FOLR1 antibodies determined by ELISA

[0448] The binding affinity of anti-FOLR1 antibodies was examined by ELISA, in which recombinant human FOLR1-murine Fc2a protein was used as antigen. The recombinant protein was immobilized on a microtiter plate, and the antibody was added to the plate at a series of concentrations. The plate was incubated for two hours at room temperature, washed with PBS supplemented with 0.05% Tween-20, and incubated with hrp-labeled goat anti-mouse secondary antibody for one hour at room temperature. The plate was washed again with PBS / Tween-20, and the bound hrp-conjugated antibody was detected by adding the hrp-substrate TMB (Bio-FX). Representative results are shown in Figure 5 The anti-FOLR1 antibodies had similar affinities for human FOLR1 at half-maximal effective concentrations (EC50) ranging from 0.5 to 0.9 nM.

[0449] No cross-reactivity of anti-FOLR1 antibodies with FOLR2 and FOLR3

[0450] FOLR1 is a member of the folate receptor family. The cross-reactivity of anti-FOLR1 antibodies with other members of the family FOLR2 and FOLR3 was determined by ELISA. Recombinant proteins FOLR2-His or FOLR3-His (R&D Systems) were fixed to Ni-NTA plates (QIAGEN) and anti-FOLR1 antibodies were added to the plates and incubated for 2 hours at room temperature. As positive controls for FOLR2 and FOLR3, ELISA polyclonal anti-FOLR2 and FOLR3 antibodies (R&D systems) were used, respectively. The antibody-antigen complex formed was detected with a HRP-labeled goat anti-mouse secondary antibody. As shown in Figure 6, the anti-FOLR1 antibodies of the present invention did not bind to FOLR2 or FOLR3; only the control antibodies detected the corresponding antigens.

[0451] Example 4. Epitope Characterization

[0452] Human FOLR1 has three potential sites for N-glycosylation at positions 69, 161 and 201 (UniProt), and all three sites are glycosylated as reported in the literature. To characterize the nature of the epitope recognized by the anti-FOLR1 antibodies described herein, binding experiments were performed with deglycosylated and untreated receptors. Of the anti-FOLR1 clones generated, only clone 2.1 was used in the study because, based on sequencing data, the clones were related and likely to bind to the same epitope. In addition to clone 2.1, two other anti-FOLR1 antibodies were included: huMov19 (WO 2011 / 106528) and clone BN3.2 (Leica). To deglycosylate FOLR1, lysates of recombinant human FOLR1 or FOLR1-positive KB or Igrov-1 cells were treated with a mixture of deglycosylation enzymes according to the manufacturer's protocol (Enzymatic DeGlycoMX kit, QA-bio). Samples of treated and untreated FOLR1 were then used in ELISA and Western blot analysis. For ELISA, deglycosylated and untreated FOLR1 were fixed to ELISA plates (Immulon), and anti-FOLR1 antibody FRIHC2-1 ("2.1") or huMov19 was added. After a 2-hour incubation, antibody-antigen complexes were detected with HRP-labeled goat anti-human (for huMov19) or anti-mouse (for 2.1) secondary antibodies ( Figure 7 For Western blot analysis, samples of deglycosylated and untreated lysates or huFOLR1 recombinant protein were separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by standard procedures. The membranes were incubated with anti-FOLR1 antibodies 2.1, huMov19, or BN3.2, and the antigen-antibody complexes were detected with appropriate secondary anti-mouse or anti-human antibodies conjugated to horseradish peroxidase ( Figure 8 ).like Figure 7 and 8 As shown, the binding of antibody 2.1 to deglycosylated FOLR1 was significantly reduced relative to untreated FOLR1, indicating that the antibody binds to a glycodependent epitope. In contrast, the other two anti-FOLR1 antibodies, huMov19 and BN3.2, bound similarly to deglycosylated and untreated receptors, indicating that (i) the FOLR1 protein was not damaged during the deglycosylation procedure, and (ii) huMov19 and BN3.2 recognized a protein epitope of FOLR1.

[0453] Example 5. Cloning and sequencing of the VL and VH regions of anti-human FOLR1 antibodies

[0454] 5 × 10 of the FOLR1 hybridoma described in Example 1 were isolated using the RNeasy kit (QIAgen) according to the manufacturer's protocol. 6 Total cellular RNA was prepared from 10 cells. The cDNA of eight subclones (2.1, 2.12, 3.8, 3.9, 5.7, 5.10, 9.20 and 9.21) was then synthesized from the total RNA using the SuperScript II cDNA Synthesis Kit (Invitrogen).

[0455] The PCR procedure for amplifying antibody variable region cDNA derived from hybridoma cells is based on the methods described in Wang et al. ((2000) J Immunol Methods. 233: 167-77) and Co et al. ((1992) J Immunol. 148: 1149-54). The variable light chain (VL) and variable heavy chain (VH) sequences were amplified by degenerate primers on the 5' end and by mouse κ or IgG1 constant region specific primers on the 3' end, respectively. The PCR reaction was then run on a 1% low melting point agarose gel, followed by excision of the 300 to 400 bp amplicon band, which was subsequently purified using a Zymo DNA mini column. The purified amplicon was sent to Beckman Coulter Genomics for sequencing using the same 5' and 3' primers of the PCR reaction to generate variable region cDNA sequences from both directions.

[0456] Because the degenerate primers used to clone VL and VH cDNA sequences change the 5' end, additional sequencing efforts are needed to verify the complete cDNA sequence. The preliminary sequence is entered into the search query of NCBI IgBlast website (www.ncbi.nlm.nih.gov / igblast / ) to identify the mouse germline sequence from which the antibody sequence is derived. Then design PCR primers to anneal with the leader sequence connected to the germline of the mouse antibody so that this new PCR reaction will produce the complete variable region cDNA sequence that is not changed by the PCR primers. The PCR reaction, band purification and sequencing are performed as described above.

[0457] Quality measurement for sequence confirmation

[0458] The variable region cDNA sequences obtained for each of the anti-FOLR1 antibodies were combined with the germline constant region sequences to obtain the full-length antibody cDNA sequences. The molecular weights of the heavy and light chains were then calculated from the translation of the cDNA sequences and compared to the molecular weights obtained by LC / MS analysis of the purified mouse anti-FOLR1 antibodies. LC / MS was performed by deglycosylation and reduction of the antibodies to separate the full-chain light and heavy chain peptides. The observed molecular weights of each of the heavy chains matched the expected molecular weights, but each of the light chains differed by approximately 85 Da. Subsequent peptide fragmentation analysis by LC / MS of the light chain fragments indicated that the final serine of the light chain leader peptide was in fact retained on the mature light chain, thereby adding approximately 87 Da to the expected MW, thereby confirming the cDNA sequences of each of the FOLR1 antibodies.

[0459] Composite CDR sequences of anti-FOLR1 antibodies

[0460] Comparison of the antibody sequences of the 8 subclones revealed that 3 of the 4 original hybridomas produced closely related but unique antibodies. As expected, each of the 4 sister subclone pairs was identical. In addition, the two groups of subclones were also identical, resulting in 3 unique antibody sequences (SEQ ID NOs: 27-32) (2.1, 5.7, and 9.20). The light chain variable framework sequences and heavy chain variable framework sequences of these 3 unique antibodies were closely related, but each antibody contained unique CDRs, which may be due to somatic amino acid substitutions (see Table 14 below). Because these CDR variants of the mouse anti-FOLR1 antibody were found to be functionally identical, they provide some structural insights into the sequence flexibility of the CDRs of the anti-FOLR1 antibodies of the present invention. Light chain CDRs 2 and 3 are identical in each of the antibodies, indicating that these strictly conserved CDRs can provide a consistent structural basis for FOLR1 binding. On the other hand, amino acid substitutions in the remaining CDRs (especially those in heavy chain CDRs 2 and 3) indicate that these positions are critical for the improvement of the affinity and specificity of these antibodies. Specific residue substitutions in these CDR positions also provide examples of residues that can be incorporated into engineered versions of these antibodies. Table 14 provides a list of composite CDR sequences compiled from anti-FOLR1 antibodies of the invention. The composite CDRs identified herein can be used to design recombinant antibodies that would be expected to retain the functional attributes of the anti-FOLR1 antibodies of the invention.

[0461] Table 14 Composite CDR

[0462]

[0463] Antibody humanization

[0464] The FRIHC2-1 antibody was humanized according to a previously described surface remodeling method, such as described in Roguska et al., Proc. Natl. Acad. Sci., USA, 91 (3): 969-973 (1994) and Roguska et al., Protein Eng. 9 (10): 895-904 (1996), which are incorporated herein by reference in their entirety. The surface remodeling involves identifying variable region framework surface residues in both the light chain and the heavy chain and replacing the residues with human equivalents. The mouse CDRs are retained in the surface remodeled antibody. Exemplary CDRs of the FRIHC2-1 antibody are defined as indicated in Table 14. In order to minimize concerns about the effects of coupled lysines falling into the CDRs, lysine 24 and lysine 27 in the light chain CDR1 of the mouse FRIHC2-1 antibody were replaced with arginine to obtain humanized version 1.0 (shown in italics), thus giving two versions of LC CDR1. In addition to the AbM heavy chain CDR2 definition used for resurfacing, the table provides exemplary Kabat defined heavy chain CDR2s for the murine and human versions of the FRIHC2-1 antibody. Underlined sequences mark portions of the Kabat heavy chain CDR2 that were not considered to be part of the CDR used for resurfacing.

[0465] The surface residue position was defined as any position with a relative accessibility of 30% or greater (Pedersen J.T. et al., J. Mol. Biol. 1994; 235: 959-973). The calculated surface residues were then aligned with human germline surface sequences to identify the most homologous human surface sequence. The human germline sequence used as the replacement surface for the VL domain of the FRIHC2-1 antibody was IGKV2-30*01, while IHV1-69*10 was used as the replacement surface for the VH domain of the FRIHC2-1 antibody. The specific framework surface residue changes for the FRIHC2-1 antibody were Fig. 9 Because the resurfaced light chain included a preferred form of CDR1 lysine substitutions, a resurfaced version (v1.01) was also generated in which the murine lysine was retained in CDR-L1. Fig.10 Shown is an alignment of the resurfaced sequences of the FRIHC2-1 variable domains of both the light and heavy chains with their murine counterparts.

[0466] In addition to humanization by resurfacing of the variable domains, the FRIHC2-1 antibody was humanized according to the complementarity determining region (CDR) grafting technique (Jones et al., Nature 321: 604-608 (1986) and Verhoeyen et al., Science 239: 1534-1536 (1988)). The CDR grafting method involves grafting the CDRs from naturally evolved murine antibodies onto the Fv framework region (FR) of a human antibody. The Kabat numbering scheme and Kabat CDR definition were used for CDR grafting of the FRIHC2-1 antibody. Exemplary CDRs of FRIHC2-1 used for CDR grafting are given in Table 14. The human immunoglobulin germline sequences with the highest homology to the murine FRIHC2-1 antibody were obtained from the International ImMunoGeneTics information (identified by V-QUEST, an interactive tool of IMGT (http: / / imgt.cines.fr / ), as described in Lefranc, Nucleic Acids Res. 29:207-209 (2001). The human germline sequences used as acceptor frameworks for the VL and VH domains of the FRIHC2-1 antibody were IGKV2D-29*02 and IGHV1-2*02, respectively. To minimize concerns about the impact of coupled lysines falling into the CDRs, lysine 24 and lysine 27 in the light chain CDR1 of the murine FRIHC2-1 antibody were replaced with arginine in the CDR-grafted constructs (Table 15). Specific framework residue changes and substitutions in CDR-L1 in the CDR grafting of the FRIHC2-1 antibody were Fig.11 The alignment of the CDR-grafted sequences of the FRIHC2-1 antibody variable domains with their murine counterparts is given in Fig.12 Shown in.

[0467] Table 15

[0468]

[0469] Example 6. IHC evaluation of 353-2.1 (FOLR1-2.1) antibody using human tumor samples

[0470] Human tumor samples representing ovarian cancer (n=63), lung adenocarcinoma (n=104), and endometrioid adenocarcinoma (n=58) were evaluated for FOLR1 expression by IHC using the 353-2.1 antibody. The intensity and score distribution of FOLR1 staining are summarized in Table 16 below. Figure 15 shows an example of staining ovarian cancer and lung adenocarcinoma tissues with the 353-2.1 antibody. These results demonstrate the utility of 353-2.1 as an IHC assay to identify patients as potential candidates for therapy using FOLR1 targeting agents (e.g., IMGN853).

[0471] Table 16: Score distribution (positive %)

[0472]

[0473]

[0474] The unique antigen specificity and high binding affinity of the FOLR1-2.1 (FOLR1 353-2.1) antibody were further demonstrated using an additional IHC assay. The IHC assay utilized the OptiView DAB detection kit on a Ventana BenchMark XT automated slide stainer for semi-quantitative determination of FOLR1 protein expression in formalin-fixed paraffin-embedded tissue specimens. The assay has been optimized and validated for specificity, sensitivity, and accuracy using normal and tumor tissue controls. Under the optimized conditions, significant membrane staining was clearly observed in tumor cells, while normal stromal tissue was completely negative. ( Fig.16 In addition, the assay also achieves a wider dynamic range, allowing the discrimination of moderate staining intensities (grade 2, moderate brown staining, Fig.17 ) and the strongest staining intensity (grade 3, dark brown staining, Fig.17 ). The enhanced dynamic range improves the ability to rank FOLR1 positive samples based on staining intensity and enables further identification of subpopulations of patients with the highest FOLR1 expression levels.

[0475] Ovarian cancer tissue microarray (TMA) was used to compare BN3.2 (Leica) antibody and FOLR1-2.1 (FOLR1353-2.1) antibody. In IHC assay (BN3.2 assay), using BN3.2 (Leica) antibody, close to 50% of the samples (16 of 35) were scored in the highest category (3 levels of staining intensity on at least 25% tumor cells). In contrast, in the above-described IHC assay (FOLR1-2.1 assay) utilizing OptiView DAB detection kit on Ventana BenchMark XT automatic slide stainer, using FOLR1-2.1 (FOLR1 353-2.1) antibody allowed these 16 samples to be further divided into 2 different categories: 6 in the highest category (3 levels of staining intensity on at least 25% tumor cells, Table 17), and the other 10 in the second highest category (2 levels of staining intensity on at least 25% tumor cells, Table 17). Thus, the more discreet staining obtained with the FOLR1-2.1 antibody in the FOLR1-2.1 assay allowed the use of the BN3.2 antibody in the BN3.2 assay to distinguish among samples that were all grouped together as having level 3 expression.

[0476] Table 17: Comparison of FOLR1 prevalence in ovarian cancer TMA (n=35)

[0477]

[0478]

[0479] All publications, patents, patent applications, internet websites, 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 website, or accession number / database sequence was specifically and individually indicated to be so incorporated by reference. Sequence Listing <110> IMUNOKIN <120> Antibodies and assays for detecting folate receptor 1 <130> 2921.044PC03 / EKS / CLD <150> 61 / 940,184 <151> 2014-02-14 <150> 61 / 875,475 <151> 2013-09-09 <150> 61 / 872,407 <151> 2013-08-30 <160> 75 <170> PatentIn version 3.5 <210> 1 <211> 257 <212> PRT <213> Homo sapiens <400> 1 Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val 1 5 10 15 Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu 20 25 30 Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly 35 40 45 Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala 50 55 60 Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr 65 70 75 80 Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys 85 90 95 Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn 100 105 110 Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg 115 120 125 Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu 130 135 140 Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp 145 150 155 160 Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln 165 170 175 Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile 180 185 190 Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg 195 200 205 Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu 210 215 220 Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala 225 230 235 240 Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu 245 250 255 Ser <210> 2 <211> 771 <212> DNA <213> Homo sapiens <400> 2 atggctcagc ggatgacaac acagctgctg ctccttctag tgtgggtggc tgtagtaggg 60 gaggctcaga caaggattgc atgggccagg actgagcttc tcaatgtctg catgaacgcc 120 aagcaccaca aggaaaagcc aggccccgag gacaagttgc atgagcagtg tcgaccctgg 180 aggaagaatg cctgctgttc taccaacacc agccaggaag cccataagga tgtttcctac 240 300 atccaggaca cctgcctcta cgagtgctcc cccaacttgg ggccctggat ccagcaggtg 360 gatcagagct ggcgcaaaga gcgggtactg aacgtgcccc tgtgcaaaga ggactgtgag 420 caatggtggg aagattgtcg cacctcctac acctgcaaga gcaactggca caagggctgg 480 aactggactt cagggtttaa caagtgcgca gtgggagctg cctgccaacc tttccatttc 540 tacttcccca caccactgt tctgtgcaat gaatctgga ctcactccta caaggtcagc 600 aactacagcc gagggagtgg ccgctgcatc cagatgtggt tcgacccagc ccagggcaac 660 cccaatgagg aggtggcgag gttctatgct gcagccatga gtggggctgg gccctgggca 720 gcctggcctt tcctgcttag cctggcccta atgctgctgt ggctgctcag c 771 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> muFRIHC2-1 VH-CDR1 <400> 3 Asn Ser Tyr Ile His 1 5 <210> 4 <211> 17 <212> PRT <213> Artificial sequence <220> <223> muFRIHC2-1 VH-CDR2 <400> 4 Trp Ile Tyr Pro Glu Ser Leu Asn Thr Gln Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ala <210> 5 <211> 13 <212> PRT <213> Artificial sequence <220> <223> muFRIHC2-1 VH-CDR3 <400> 5 Arg Gly Ile Tyr Tyr Tyr Ser Pro Tyr Ala Leu Asp His 1 5 10 <210> 6 <211> 16 <212> PRT <213> Artificial sequence <220> <223> muFRIHC2-1 VL-CDR1 <400> 6 Lys Ser Ser Lys Ser Leu Leu Asn Ser Asp Gly Phe Thr Tyr Leu Asp 1 5 10 15 <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> muFRIHC2-1 VL-CDR2 <400> 7 Leu Val Ser Asn His Phe Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> muFRIHC2-1 VL-CDR3 <400> 8 Phe Gln Ser Asn Tyr Leu Pro Leu Thr 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial sequence <220> <223> muFRIHC5-7 VH-CDR1 <400> 9 Asn Tyr Tyr Ile His 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial sequence <220> <223> muFRIHC5-7 VH-CDR2 <400> 10 Trp Ile Tyr Pro Gly Ser Phe Asn Val Glu Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ala <210> 11 <211> 13 <212> PRT <213> Artificial sequence <220> <223> muFRIHC5-7 VH-CDR3 <400> 11 Arg Gly Ile Tyr Phe Tyr Ser Pro Tyr Ala Leu Asp Tyr 1 5 10 <210> 12 <211> 16 <212> PRT <213> Artificial sequence <220> <223> muFRIHC5-7 VL-CDR1 <400> 12 Lys Ser Thr Glu Ser Leu Leu Asn Ser Asp Gly Phe Thr Tyr Leu Asp 1 5 10 15 <210> 13 <211> 7 <212> PRT <213> Artificial sequence <220> <223> muFRIHC5-7 VL-CDR2 <400> 13 Leu Val Ser Asn His Phe Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> muFRIHC5-7 VL-CDR3 <400> 14 Phe Gln Ser Asn Tyr Leu Pro Leu Thr 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> muFRIHC9-20 VH-CDR1 <400> 15 Asn Tyr Tyr Ile His 1 5 <210> 16 <211> 17 <212> PRT <213> Artificial sequence <220> <223> muFRIHC9-20 VH-CDR2 <400> 16 Trp Ile Tyr Pro Glu Asn Val Asn Val Arg Tyr Asn Asp Lys Phe Lys 1 5 10 15 Ala <210> 17 <211> 13 <212> PRT <213> Artificial sequence <220> <223> muFRIHC9-20 VH-CDR3 <400> 17 Arg Gly Ile Tyr Tyr Tyr Ser Pro Tyr Ala Met Asp Tyr 1 5 10 <210> 18 <211> 16 <212> PRT <213> Artificial sequence <220> <223> muFRIHC9-20 VL-CDR1 <400> 18 Lys Ser Thr Lys Ser Leu Leu Asn Ser Asp Gly Phe Thr Tyr Leu Asp 1 5 10 15 <210> 19 <211> 7 <212> PRT <213> Artificial sequence <220> <223> muFRIHC9-20 VL-CDR2 <400> 19 Leu Val Ser Asn His Phe Ser 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial sequence <220> <223> muFRIHC9-20 VL-CDR3 <400> 20 Phe Gln Ser Asn Tyr Leu Pro Leu Thr 1 5 <210> twenty one <211> 5 <212> PRT <213> Artificial sequence <220> <223> Composite VH-CDR1 <220> <221> misc_feature <222> (2) <223> Xaa can be either tyrosine or serine <400> twenty one Asn Xaa Tyr Ile His 1 5 <210> twenty two <211> 17 <212> PRT <213> Artificial sequence <220> <223> Composite VH-CDR2 <220> <221> misc_feature <222> (5) <223> Xaa can be glycine or glutamic acid <220> <221> misc_feature <222> (6) <223> Xaa can be serine or asparagine <220> <221> misc_feature <222> (7) <223> Xaa can be phenylalanine or valine and leucine <220> <221> misc_feature <222> (9)..(9) <223> Xaa can be valine or threonine <220> <221> misc_feature <222> (10)..(10) <223> Xaa can be glutamic acid or arginine or glutamine <220> <221> misc_feature <222> (13) <223> Xaa can be either glutamic acid or aspartic acid <400> twenty two Trp Ile Tyr Pro Xaa Xaa Xaa Asn Xaa Xaa Tyr Asn Xaa Lys Phe Lys 1 5 10 15 Ala <210> twenty three <211> 13 <212> PRT <213> Artificial sequence <220> <223> Composite VH-CDR3 <220> <221> misc_feature <222> (5) <223> Xaa can be phenylalanine or tyrosine <220> <221> misc_feature <222> (11)..(11) <223> Xaa can be leucine or methionine <220> <221> misc_feature <222> (13) <223> Xaa can be tyrosine or histidine <400> twenty three Arg Gly Ile Tyr Xaa Tyr Ser Pro Tyr Ala Xaa Asp Xaa 1 5 10 <210> twenty four <211> 16 <212> PRT <213> Artificial sequence <220> <223> Composite VL-CDR1 <220> <221> misc_feature <222> (3) <223> Xaa can be threonine or serine <220> <221> misc_feature <222> (4) <223> Xaa can be lysine or glutamic acid <400> twenty four Lys Ser Xaa Xaa Ser Leu Leu Asn Ser Asp Gly Phe Thr Tyr Leu Asp 1 5 10 15 <210> 25 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Composite VL-CDR2 <400> 25 Leu Val Ser Asn His Phe Ser 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Composite VL-CDR3 <400> 26 Phe Gln Ser Asn Tyr Leu Pro Leu Thr 1 5 <210> 27 <211> 122 <212> PRT <213> Artificial sequence <220> <223> muFRIHC2-1 VH chain <400> 27 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Ser 20 25 30 Tyr Ile His Trp Val Lys Lys Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Glu Ser Leu Asn Thr Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ser Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Ile Tyr Tyr Tyr Ser Pro Tyr Ala Leu Asp His Trp 100 105 110 Gly Gln Gly Ala Ser Val Thr Val Ser Ser 115 120 <210> 28 <211> 114 <212> PRT <213> Artificial sequence <220> <223> muFRIHC2-1 VL chain <400> 28 Ser Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Ile 1 5 10 15 Gly Asp Gln Ala Ser Ile Ser Cys Lys Ser Ser Lys Ser Leu Leu Asn 20 25 30 Ser Asp Gly Phe Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn His Phe Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln 85 90 95 Ser Asn Tyr Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg <210> 29 <211> 122 <212> PRT <213> Artificial sequence <220> <223> muFRIHC5-7 VH chain <400> 29 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Ser Phe Asn Val Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Ile Tyr Phe Tyr Ser Pro Tyr Ala Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Ala Ser Val Thr Val Ser Ser 115 120 <210> 30 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> muFRIHC5-7 VL chain <400> 30 Ser Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Ile 1 5 10 15 Gly Asp Gln Ala Ser Ile Ser Cys Lys Ser Thr Glu Ser Leu Leu Asn 20 25 30 Ser Asp Gly Phe Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn His Phe Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln 85 90 95 Ser Asn Tyr Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Val 100 105 110 Lys Arg <210> 31 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> muFRIHC9-20 VH chain <400> 31 Gln Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Glu Asn Val Asn Val Arg Tyr Asn Asp Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Ile Tyr Tyr Tyr Ser Pro Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Ala Ser Val Thr Val Ser Ser 115 120 <210> 32 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> muFRIHC9-20 VL chain <400> 32 Ser Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Leu 1 5 10 15 Gly Asp Gln Ala Ser Ile Ser Cys Lys Ser Thr Lys Ser Leu Leu Asn 20 25 30 Ser Asp Gly Phe Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn His Phe Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln 85 90 95 Ser Asn Tyr Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg <210> 33 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> muFRIHC2-1 full-length heavy chain <400> 33 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Ser 20 25 30 Tyr Ile His Trp Val Lys Lys Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Glu Ser Leu Asn Thr Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ser Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Ile Tyr Tyr Tyr Ser Pro Tyr Ala Leu Asp His Trp 100 105 110 Gly Gln Gly Ala Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Glu Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185 190 Pro Ser Ser Met Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 210 215 220 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe 225 230 235 240 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 245 250 255 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 260 265 270 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 290 295 300 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 305 310 315 320 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 340 345 350 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 355 360 365 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 370 375 380 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn 385 390 395 400 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 405 410 415 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 420 425 430 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445 <210> 34 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> muFRIHC2-1 full-length light chain <400> 34 Ser Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Ile 1 5 10 15 Gly Asp Gln Ala Ser Ile Ser Cys Lys Ser Ser Lys Ser Leu Leu Asn 20 25 30 Ser Asp Gly Phe Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn His Phe Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln 85 90 95 Ser Asn Tyr Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser 115 120 125 Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn 130 135 140 Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu 145 150 155 160 Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr 180 185 190 Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr 195 200 205 Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 220 <210> 35 <211> 446 <212> PRT <213> Artificial sequence <220> <223> muFRIHC5-7 full-length heavy chain <400> 35 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Ser Phe Asn Val Glu Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Ile Tyr Phe Tyr Ser Pro Tyr Ala Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Ala Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Glu Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185 190 Pro Ser Ser Met Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 210 215 220 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe 225 230 235 240 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 245 250 255 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 260 265 270 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 290 295 300 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 305 310 315 320 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 340 345 350 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 355 360 365 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 370 375 380 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn 385 390 395 400 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 405 410 415 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 420 425 430 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445 <210> 36 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> muFRIHC5-7 full-length light chain <400> 36 Ser Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Ile 1 5 10 15 Gly Asp Gln Ala Ser Ile Ser Cys Lys Ser Thr Glu Ser Leu Leu Asn 20 25 30 Ser Asp Gly Phe Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn His Phe Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln 85 90 95 Ser Asn Tyr Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Val 100 105 110 Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser 115 120 125 Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn 130 135 140 Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu 145 150 155 160 Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr 180 185 190 Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr 195 200 205 Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 220 <210> 37 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> muFRIHC9-20 full-length heavy chain <400> 37 Gln Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Glu Asn Val Asn Val Arg Tyr Asn Asp Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Gly Ile Tyr Tyr Tyr Ser Pro Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Ala Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Glu Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185 190 Pro Ser Ser Met Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 210 215 220 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe 225 230 235 240 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 245 250 255 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 260 265 270 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 290 295 300 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 305 310 315 320 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 340 345 350 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 355 360 365 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 370 375 380 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn 385 390 395 400 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 405 410 415 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 420 425 430 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445 <210> 38 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> muFRIHC9-20 full-length light chain <400> 38 Ser Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Leu 1 5 10 15 Gly Asp Gln Ala Ser Ile Ser Cys Lys Ser Thr Lys Ser Leu Leu Asn 20 25 30 Ser Asp Gly Phe Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn His Phe Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln 85 90 95 Ser Asn Tyr Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser 115 120 125 Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn 130 135 140 Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu 145 150 155 160 Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr 180 185 190 Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr 195 200 205 Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 220 <210> 39 <211> 366 <212> DNA <213> Artificial sequence <220> <223> muFRIHC2-1 VH chain <400> 39 caggtccaac tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaggata 60 tcctgcaagg cttctggcta caccttcaca aactcctata ttcactgggt gaaaaagagg 120 cctggacagg gacttgagtg gattggatgg atttatcctg aaagtcttaa tactcaatac 180 aatgagaagt tcaaggccaa ggccacactg actgctgaca agtcctccag cacatcctac 240 atgcagctca gcagtctgac ctctgaggac tctgcggtct atttctgtgc aagaaggggt 300 atttattact actctcccta tgctctggac cactggggtc aaggagcctc agtcaccgtc 360 tcctca 366 <210> 40 <211> 342 <212> DNA <213> Artificial sequence <220> <223> muFRIHC2-1 VL chain <400> 40 agtgatgttg ttctgaccca aactccactc tctctgcctg tcaatattgg agatcaagcc 60 tctatctctt gcaagtcttc taagagtctt ctgaatagtg atggattcac ttatttggac 120 tggtacctgc agaagccagg ccagtctcca cagctcctaa tatatttggt ttctaatcat 180 ttttctggag ttccagacag gttcagtggc agtgggtcag gaacagattt cacactcaag 240 atcagcagag tggaggctga ggatttggga gtttattatt gcttccagag taactatctt 300 cctctcacgt tcggaggggg gaccaagctg gaaataaaac gg 342 <210> 41 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> muFRIHC5-7 VH chain <400> 41 caggtccaac tgcagcagtc tggacctgag gtggtgaagc ctggggcttc agtgaggata 60 tcctgcaagg cttctggcta caccttcaca aactactata tacactgggt gaagcagagg 120 cctggacagg gacttgagtg gattggatgg atttatcctg gaagttttaa tgttgagtac 180 aatgagaagt tcaaggccaa ggccacactg actgcagaca aatcctccag cacagtctac 240 atgcaactca gcagcctgac ctctgaggac tctgcggtct atttctgtgc aagaaggggt 300 atttatttct actctcccta tgctttggac tactggggtc aaggagcctc agtcaccgtc 360 tcctca 366 <210> 42 <211> 342 <212> DNA <213> Artificial sequence <220> <223> muFRIHC5-7 VL chain <400> 42 agtgatgttg ttctgaccca aactccactc tctctgcctg tcaatattgg agatcaagcc 60 tctatctctt gcaagtctac tgagagtctt ctgaatagtg atggattcac ttatttggac 120 tggtacctgc agaagccagg ccagtctcca cagctcctaa tatatttggt ttctaatcat 180 ttttctggag ttccagacag gttcagtggc agtgggtcag gaacagattt cacactcaag 240 atcagcagag tggaggctga ggatttggga gtttattatt gcttccagag taactatctt 300 cctctcacgt tcggaggggg gaccaagctg gaagtaaaac gg 342 <210> 43 <211> 366 <212> DNA <213> Artificial sequence <220> <223> muFRIHC9-20 VH chain <400> 43 caggtccaac tgcagcagtc tggacctgac ctggtgaagc ctggggcttc agtgaggata 60 tcctgcaagg cttctggctt caccttcaca aactactata tacactgggt gaagcagagg 120 cctggacagg gacttgagtg gattggatgg atttatcctg aaaatgttaa tgttaggtac 180 aatgacaagt tcaaggccaa ggccacactg actgcagaca aatcctccag cacagcctac 240 atgcagctca gcagcctgac ctctgaggac tctgcggtct atttctgtgc aagaaggggt 300 atttattact actctcccta tgctatggac tactggggtc aaggagcctc agtcaccgtc 360 tcctca 366 <210> 44 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> muFRIHC9-20 VL chain <400> 44 agtgatgttg ttctgaccca aactccactc tctctgcctg tcaatcttgg agatcaagcc 60 tctatctctt gcaagtctac taagagtctt ctgaatagtg atggattcac ttatttggac 120 tggtacctgc agaagccagg ccagtctcca cagctcctaa tatatttggt ttctaatcat 180 ttttctggag ttccagacag gttcagtggc agtgggtcag gaacagattt caccctcaag 240 atcagcagag tggaggctga ggatttggga gtttattatt gcttccagag taactatctt 300 cctctcacgt tcggaggggg gaccaagctg gaaataaaac gg 342 <210> 45 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> huMov19 vHC <400> 45 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala His 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Phe Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Asp Gly Ser Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 46 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> huMov19 vLCv1.00 <400> 46 Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Pro Ala Ile Ile Ser Cys Lys Ala Ser Gln Ser Val Ser Phe Ala 20 25 30 Gly Thr Ser Leu Met His Trp Tyr His Gln Lys Pro Gly Gln Gln Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ala Gly Val Pro...

Claims

1. A kit comprising: An antibody or antigen-binding fragment thereof that specifically binds to FOLR1 for use in diagnosis, wherein the antibody or antigen-binding fragment thereof comprises a VH CDR1-3 and a VL CDR1-3 polypeptide sequence selected from the group consisting of: (a) are SEQ ID NOs: 3-8, respectively; (b) SEQ ID NO:9-14, respectively; (c) SEQ ID NO: 15-20, respectively; (d) SEQ ID NOs: 3-5 and SEQ ID NOs: 59, 7 and 8, respectively; (e) SEQ ID NOs: 3, 60 and 5 and SEQ ID NOs: 6-8, respectively; (f) SEQ ID NOs: 3, 61 and 5 and SEQ ID NOs: 6-8, respectively; (g) SEQ ID NOs: 3, 60 and 5 and SEQ ID NOs: 59, 7 and 8, respectively; and (h) are SEQ ID NOs: 3, 61 and 5 and SEQ ID NOs: 59, 7 and 8, respectively.

2. The kit of claim 1, wherein the antibody or antigen-binding fragment thereof for diagnosis comprises VH and VL polypeptide sequences selected from the group consisting of: (a) SEQ ID NO:27 and SEQ ID NO:28, respectively; (b) SEQ ID NO:29 and SEQ ID NO:30, respectively; (c) SEQ ID NO:31 and SEQ ID NO:32, respectively; (d) SEQ ID NO:62 and SEQ ID NO:63 or SEQ ID NO:64, respectively; and (e) SEQ ID NO:65 and SEQ ID NO:66 or SEQ ID NO:67, respectively.

3. The kit of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof for diagnosis is recombinantly produced.

4. The kit of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof for diagnosis is murine, humanized, chimeric, resurfaced or human.

5. The kit according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof for diagnosis binds to human FOLR1 but does not bind to FOLR2 or FOLR3.

6. The kit of claim 1 or 2, wherein the antibody used for diagnosis is a full-length antibody.

7. The kit of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof for diagnosis is an antigen-binding fragment.

8. The kit of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof for diagnosis binds to human folate receptor 1 with a Kd of 0.5 to 10 nM.

9. The kit according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof for diagnosis binds to human folate receptor 1 with a Kd of 1.0 nM or higher.

10. The kit of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof for diagnosis is detectably labeled.

11. The kit according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof for diagnosis detects FOLR1 expression by IHC.

12. The kit according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof for diagnosis detects FOLR1 expression by ELISA.

13. The kit of claim 1 or 2, further comprising an anti-FOLR1 immunoconjugate for treatment, the immunoconjugate comprising an anti-FOLR1 monoclonal antibody conjugated to DM4 (N(2′)-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine) connected by a cleavable sulfo-SPDB (N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutyrate) linker, wherein the anti-FOLR1 antibody comprises VH and VL comprising the amino acid sequences of SEQ ID NOs: 45 and 47, respectively.

14. A polypeptide that specifically binds to FOLR1, wherein the polypeptide comprises an anti-FOLR-1 antigen binding fragment, the fragment comprising VH and VL, the VH and VL comprising VH CDR1-3 and VL CDR1-3 sequences, respectively, which are selected from the group consisting of: (a) are SEQ ID NO:3-8 respectively; (b) are SEQ ID NO:9-14, respectively; (c) are SEQ ID NO: 15-20, respectively; (d) SEQ ID NOs: 3-5 and SEQ ID NOs: 59, 7 and 8, respectively; (e) SEQ ID NOs: 3, 60 and 5 and SEQ ID NOs: 6-8, respectively; (f) SEQ ID NOs: 3, 61 and 5 and SEQ ID NOs: 6-8, respectively; (g) SEQ ID NOs: 3, 60 and 5 and SEQ ID NOs: 59, 7 and 8, respectively; and (h) are SEQ ID NOs: 3, 61 and 5 and SEQ ID NOs: 59, 7 and 8, respectively.

15. The polypeptide of claim 14, wherein the polypeptide is recombinantly produced.

16. The polypeptide of claim 14, wherein the polypeptide binds to human FOLR1 but not to FOLR2 or FOLR3.

17. The polypeptide of claim 14, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab')2 and Fv fragments.

18. The polypeptide of claim 14, which binds to human folate receptor 1 with a Kd of 0.5 to 10 nM.

19. The polypeptide of claim 14, which binds to human folate receptor 1 with a Kd of 1.0 nM or higher.

20. The polypeptide of claim 14, wherein the polypeptide is detectably labeled.

21. A cell which produces the polypeptide of any one of claims 14-20.

22. A method for preparing the polypeptide of any one of claims 14-20, the method comprising (a) culturing the cell of claim 21; and (b) isolating the polypeptide from the cultured cell.

23. A composition comprising the polypeptide of any one of claims 14-20 and a buffer selected from the group consisting of: a FACS buffer, an IHC buffer, and an ELISA buffer.

24. Use of the polypeptide according to any one of claims 14 to 20 or the composition according to claim 23 in the preparation of a detection reagent for use in a method for detecting FOLR1 expression in a sample, wherein the method comprises contacting the sample with the polypeptide according to any one of claims 14 to 20 or the composition according to claim 23.

25. The use of claim 24, wherein the polypeptide is detectably labeled.

26. The use according to claim 25, wherein the label is selected from the group consisting of: immunofluorescence label, chemiluminescent label, phosphorescent label, enzyme label, radioactive label, colloidal gold particles, colored particles and magnetic particles.

27. The use according to claim 25, wherein the label is avidin and / or biotin.

28. The use according to any one of claims 24 to 27, wherein FOLR1 expression is determined by radioimmunoassay, Western blot assay, cytometry, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay or immunohistochemical assay.

29. The use according to claim 28, wherein the cell counting is flow cytometry.

30. The use of claim 28, wherein FOLR1 expression is determined by IHC.

31. Use of a polypeptide according to any one of claims 14 to 20 or a composition according to claim 23 in the preparation of a detection reagent for use in a method of increasing the efficacy of cancer therapy using an agent comprising an anti-FOLR1 antibody or an antigen-binding fragment thereof, the method comprising using the detection reagent to detect an increase in FOLR1 expression in a sample from a subject suffering from cancer, and administering the agent to the subject.

32. An antibody or an antigen-binding fragment thereof that specifically binds to FOLR1, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO:

34.

33. An antibody or an antigen-binding fragment thereof that specifically binds to FOLR1, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO:

36.

34. An antibody or an antigen-binding fragment thereof that specifically binds to FOLR1, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and a light chain comprising the amino acid sequence of SEQ ID NO:

38.

35. The antibody or antigen-binding fragment thereof of any one of claims 32-34, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.

36. The antibody or antigen-binding fragment thereof of any one of claims 32-34, wherein the antibody or antigen-binding fragment thereof binds to human FOLR1 but not FOLR2 or FOLR3.

37. The antibody or antigen-binding fragment thereof of any one of claims 32-34, which is a full-length antibody.

38. The antibody or antigen-binding fragment thereof of any one of claims 32-34, which is an antigen-binding fragment.

39. The antibody or antigen-binding fragment thereof of any one of claims 32-34, which binds to human folate receptor 1 with a Kd of 0.5 to 10 nM.

40. The antibody or antigen-binding fragment thereof of any one of claims 32-34, which binds to human folate receptor 1 with a Kd of 1.0 nM or higher.

41. The antibody or antigen-binding fragment thereof of any one of claims 32-34, wherein the antibody or antigen-binding fragment thereof is detectably labeled.

42. A cell that produces the antibody or antigen-binding fragment thereof according to any one of claims 32-40.

43. A method for preparing the antibody or antigen-binding fragment thereof as described in any one of claims 32-41, the method comprising (a) culturing the cells of claim 42; and (b) isolating the antibody or antigen-binding fragment thereof from the cultured cells.

44. A composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 32-41 and a buffer selected from the group consisting of: FACS buffer, IHC buffer and ELISA buffer.

45. Use of the antibody or antigen-binding fragment thereof according to any one of claims 32 to 41 or the composition according to claim 44 in the preparation of a detection reagent for a method for detecting FOLR1 expression in a sample, wherein the method comprises contacting the sample with the antibody, antigen-binding fragment or polypeptide according to any one of claims 32 to 41 or the composition according to claim 44.

46. ​​The use of claim 45, wherein the antibody or antigen-binding fragment thereof is detectably labeled.

47. The use of claim 46, wherein the label is selected from the group consisting of: immunofluorescence label, chemiluminescent label, phosphorescent label, enzyme label, radioactive label, colloidal gold particles, colored particles and magnetic particles.

48. The use according to claim 46, wherein the label is avidin and / or biotin.

49. The use according to any one of claims 45 to 48, wherein FOLR1 expression is determined by radioimmunoassay, Western blot assay, cytometry, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay or immunohistochemical assay.

50. The use of claim 49, wherein the cell counting is flow cytometry.

51. The use of claim 49, wherein FOLR1 expression is determined by IHC.

52. Use of an antibody or antigen-binding fragment thereof as described in any one of claims 32-41 or a composition as described in claim 44 in the preparation of a detection reagent for use in a method of increasing the efficacy of cancer therapy using an active agent comprising an anti-FOLR1 antibody or antigen-binding fragment thereof, the method comprising using the detection reagent to detect an increase in FOLR1 expression in a sample from a subject suffering from cancer, and administering the active agent to the subject.

53. A kit comprising: An antibody, antigen-binding fragment or polypeptide as claimed in any one of claims 14 to 20 and 32 to 41, or a composition as claimed in claim 23 or 44 for use in diagnosis; and An active agent for use in treatment comprising an anti-FOLR1 antibody or antigen-binding fragment thereof.

54. The kit of claim 53, wherein the antibody or antigen-binding fragment thereof or polypeptide for diagnosis detects FOLR1 expression by IHC.

55. The kit of claim 53, wherein the antibody or antigen-binding fragment thereof or polypeptide for diagnosis detects FOLR1 expression by ELISA.

56. The kit of claim 53, further comprising an anti-FOLR1 immunoconjugate for treatment, the immunoconjugate comprising an anti-FOLR1 monoclonal antibody conjugated to DM4 (N(2′)-deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)-maytansine), connected by a cleavable sulfo-SPDB (N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutyrate) linker, wherein the anti-FOLR1 antibody comprises a heavy chain and a light chain, the heavy chain and the light chain comprising the amino acid sequences of SEQ ID NOs: 55 and 57, respectively.

57. The kit of any one of claims 1, 2 and 53, further comprising reagents for immunohistochemistry (IHC) and one or more standardized reference samples.

58. The kit of claim 57, wherein the standardized reference sample comprises a cell, a cell aggregate, or a formalin-fixed paraffin-embedded tissue sample.

59. The kit of claim 57, wherein the one or more normalization reference samples are from cells, cell aggregates or tissues with no FOLR1 expression, low FOLR1 expression or high FOLR1 expression.

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