use of a folate antireceptor immunoconjugate 1 (folr1)

专利摘要:
USE OF FOLATE 1 ANTIREPTECTOR ANTIBODY (FOLR1) IN CANCER THERAPY, METHODS FOR IDENTIFYING CANCER AND TUMOR RESPONSIBLE FOR TREATING WITH THIS ANTIBODY, AS WELL AS A MANUFACTURING ARTICLE AND UNDERSTANDING THE ANTIBODY. The present invention relates to the use of folate antireceptor antibody 1 (FOLR1) or an anti-FOLR1 immunoconjugate in cancer therapy, in which a tumor sample exhibits increased expression of FOLR1 using a detection method that distinguishes between the intensity of staining or uniformity of staining in a cancer sample expressing FOLR1, compared to the intensity of staining or uniformity of staining in one or more reference sample (s), and in which the effectiveness of cancer therapy with an anti-FOLR1 antibody or anti-FOLR1 immunoconjugate is increased. The present invention also relates to methods for identifying cancer and tumor responsive to treatment with said antibody, as well as the article of manufacture and kit comprising said antibody.
公开号:BR112013025415B1
申请号:R112013025415-7
申请日:2012-03-30
公开日:2021-03-16
发明作者:Christina N. Carrigan；Kathleen R. Whiteman；Gillian Payne；Sharron Ladd
申请人:Immunogen, Inc；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED REQUESTS
[0001] This application claims the benefit of Provisional Application US61 / 471,007, filed on April 1, 2011, which is hereby incorporated by reference. BACKGROUND OF THE INVENTION Field of the Invention
[0002] The field of the invention generally refers to increasing the effectiveness of the treatment of cancers characterized by overexpression of the human folate receptor 1 (FOLR1). More specifically, the invention relates to a more effective treatment of individuals susceptible to or diagnosed with cancer, in which tumor cells overexpress FOLR1 as determined by a gene expression assay, with a FOLR1 antagonist, for example, a FOLR1 immunoconjugate. . Background of the Technique
[0003] Cancer is a leading cause of death in the developed world, 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 1 in 3 people will develop some form of cancer during their lifetime. There are more than 200 different types of cancer, four of which - breast, lung, colorectal and prostate - account for more than half of all new cases (Jemal et al., 2003, Cancer J. Clin. 53: 5-26) .
[0004] Folate receptor 1 (FOLR1), also known as Folate-alpha Receptor, 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 various derivatives of reduced folic acid. FOLR1 mediates the release of physiological 5- methyltetrahydrofolate folate into cells.
[0005] FOLR1 is overexpressed in the vast majority of ovarian cancers, as well as in many cancers of the uterus, endometrium, pancreas, kidney, lung and breast, while the expression of FOLR1 in normal tissues is restricted to the apical membrane of epithelial cells in proximal kidney tubules, alveolar pneumocytes of the lung, bladder, testicles, choroid and thyroid plexus (Weitman SD, et al., Cancer Res 52: 3396-3401 (1992); Antony AC, Annu Rev Nutr 16: 501-521 (501-521 ( 1996); Kalli KR, et al. Gynecol Oncol 108: 619-626 (2008)). This pattern of FOLR1 expression makes it a desirable target for cancer therapy targeting FOLR1.
[0006] As ovarian cancer is generally asymptomatic until advanced stages, it is often diagnosed at a late stage and has a poor prognosis when treated with currently available procedures, typically chemotherapy after surgical debulking (von Gruenigen V et al., Cancer 112: 2221-2227 (2008); Ayhan A et al., Am J Obstet Gynecol 196: 81e81-86 (2007); Harry VN et al., ObstetGynecol Surv 64: 548-560 (2009)). Thus, there is an unmet medical clarity for more efficient drugs for ovarian cancers. SUMMARY OF THE INVENTION
[0007] The present invention is based on the discovery of a dynamic range of FOLR1 expression in tumor tissue, and the discovery that tumors with increased levels of FOLR1 expression are more sensitive to treatment with anti-FOLR1 antibodies or anti-immunoconjugates. -FOLR1. The present invention advantageously allows the treatment of individuals who are more likely to respond to treatment by administration of therapeutic agents, that is, anti-FOLR1 antibodies or anti-FOLR1 immunoconjugates, to individuals who are found to have an increased level of expression of FOLR1.
[0008] The present invention provides a method for identifying an individual predisposed to respond favorably to an anti-cancer drug directed at Folate Receptor 1 (FOLR1), the method comprising detecting the expression of FOLR1 in a tissue sample from the individual.
[0009] The present invention also provides a method for increasing the likelihood of the effectiveness of a cancer treatment, the method comprising administering a therapeutically efficient dose of an anti-cancer drug targeting FOLR-1 to an individual, in which expression of FOLR1 in a tissue sample from the individual was found enlarged.
[00010] The present invention also provides a method for predicting the effectiveness of a low-dose cancer treatment, the method comprising administering a therapeutically efficient dose of an anti-cancer drug targeting FOLR1 to an individual, in which said individual has been found as having increased expression of FOLR1 in a sample.
[00011] In one embodiment, the methods are directed at ovarian carcinoma, non-small cell lung adenocarcinoma (including bronchial alveolar carcinoma), renal carcinomas and endometrial carcinomas.
[00012] In one embodiment, the amplitude and uniformity of FOLR1 expression is detected by immunohistochemistry (IHC), flow cytometry, or nucleic acid hybridization. In another modality, the level of expression of FOLR1 is detected by immunohistochemistry. Non-limiting examples of IHC include IHC methods that distinguish between different levels of FOLR1 and calibrated IHC methods, such as those described herein. FOLR1 expression can be recorded with an appropriate scoring system, including, but not limited to, the grade methods described herein. For example, FOLR1 expression can be scored using a calibrated IHC method that includes a range of 0.1, 2, 3, and 3+ for the staining intensity with 0 being the lowest level of staining intensity and 3+ being the highest level of color intensity. Alternatively or additionally, FOLR1 expression can be scored using a calibrated IHC method that includes a uniformity of staining ranging from focal (<25% stained cells), to heterogeneous (25-75% stained cells), to homogeneous (> 75% of stained cells), where focal staining is the least uniform staining and homogeneous is the most uniform staining.
[00013] In an additional embodiment, the expression of FOLR1 in a sample (for example, a tumor tissue sample) is measured and compared with one or more reference samples and the expression of FOLR1 in the tissue sample of a tumor of the individual, tumor xenograft, or cell line has a specific FOLR1 degree correlating to the extent and uniformity of expression, compared to one or more reference samples. In several examples, a sample of tissue or cell with a level of 1, 2, 3 or 3+ of FOLR1 staining intensity with a homogeneous staining pattern is considered to have increased FOLR1 expression; a tissue or cell sample with level 3 of FOLR1 expression of heterogeneous staining intensity or focal expression staining patterns is considered to have increased FOLR1 expression In another embodiment, the expression of FOLR1 in a sample is measured and compared with one or more reference samples to identify a comparable level of staining. In one embodiment, the reference sample has a pre-assigned IHC grade and / or a predetermined antigen number per cell (or ABC) and the antigen or ABC number for the tissue sample can be determined based on the comparison.
[00014] In one embodiment, the expression of FOLR1 in a sample (for example, a sample of tumor tissue) is measured and compared with one or more control samples and the expression of FOLR1 in the tissue sample of a tumor, xenograft of tumor, or cell line of the individual has a specific FOLR1 degree correlating to the amplitude and uniformity of expression compared to one or more samples control. In one embodiment, the expression of FOLR1 in the sample is compared to a negative control sample that demonstrates low or no detectable FOLR1 expression. In another embodiment, the expression of FOLR1 in the sample is compared to a positive control sample having an increased expression of FOLR1 (level 1, 2, 3 or 3+). In some embodiments, the control samples include, among others, Namalwa, SW2, SW620, T47D, IGROV-1, 300.19 FR1, HeLa or KB cells. In particular embodiments, control samples include cells or cell pellets from cells transfected with the folate receptor (for example, 300.19 FR1).
[00015] In one embodiment, the anti-cancer drug targeting FOLR1 is an FOLR1 immunoconjugate. In one embodiment, the immunoconjugate comprises an anti-FOLR1 antibody, a linker, and a cytotoxin.
[00016] In another embodiment, the anti-FOLR1 antibody is huMOV19. In another embodiment, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a linker based on dicarboxylic acid. In another embodiment, the linker is selected from the group consisting of: N-succinimidyl 4- (2-pyridyldithium) pentanoate (SPP) or N-succinimidyl 4- (2-pyridyldithium) -2-sulfopentanoate (sulfo-SPP), N- succinimidyl 4- (2-pyridyldithio) -butanoate (SPDB) or N-succinimidyl 4 - (2-pyridyldithio) -2-sulfobutanoate (sulfo-SPDB), N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-sulfosuccinimidyl4- (maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC), N-succinimidyl-4- (iodoacetyl) - aminobenzoate (SIAB), and N-succinimidyl ester - [(N-maleimidopropionamido) -tetraethyl-Glycol-Glycol] maleimide). In another embodiment, the linker is N-succinimidyl 4- (2-pyridyldithio) -2-sulfobutanoate (sulfo-SPDB). In another embodiment, the cytotoxic agent is selected from the group consisting of a maytansinoid, maytansinoid analogue, benzodiazepine, taxoid, CC-1065, CC-1065 analogue, duocarmicin, duocarmicin analogue, calicheamicin, dolastatin, dolastatin analogue, auristatin , derived from tomamycin, and derived from leptomycin or a prodrug of the agent. In another embodiment, the cytotoxic agent is a maytansinoid. In another embodiment, the cytotoxic agent is N (2 ') - deacetyl-N- (2') - (3-mercapto-1-oxopropyl) -maitansine or N (2 ') - deacetyl- N2- (4-mercapto- 4-methyl-1-oxopentyl) maytansine. In another embodiment, the cytotoxic agent is N (2 ') - deacetyl-N2- (4-mercapto-4-methyl-1-oxopentyl) -maitansine (DM4). In another embodiment, the immunoconjugate comprises the antibody HUMOV19, sulfo-SPDB and DM4 (IMGN853).
[00017] The invention is also directed to a kit for measuring expression of FOLR1 in an individual comprising a FOLR1 detection reagent, and instructions for use. In one embodiment, the FOLR1 detection reagent comprises a FOLR1-binding peptide, protein or molecular probe (i.e., nucleic acid). In another embodiment, the FOLR1 detection reagent is an anti-FOLR1 antibody. In another embodiment, the kit further comprises a secondary antibody that binds to the anti-FOLR1 antibody. In one embodiment, the antibody is included in a concentration of 0.5 to 7.5 μg / mL, desirably 0.9 to 3.8 +/- 0.5 μg / mL. In several embodiments, the antibody is included at a concentration of 1.0 + / 0.5 μg / mL, 1.5 +/- 0.5 μg / mL, 1.9 +/- 0.5μg / mL, 2 , 5 +/- 0.5 μg / mL, 3.0 +/- 0.5 μg / mL, 3.5 +/- 0.5 μg / mL, 3.8 +/- 0.5 μg / mL , greater than 4.2 μg / mL. In another embodiment, the antibody is included in the concentrated solution, with instructions for dilution until reaching a final concentration of 0.9 to 3.8 +/- 0.5 μg / mL. In another way, the kit also comprises a detection reagent selected from the group consisting of: an enzyme, a fluorophore, a radioactive marker and a luminophore. In another embodiment, the detection reagent is selected from the group consisting of: biotin, digoxigenin, fluorescein, tritium and rhodamine.
[00018] The kit can also include instructions for detecting and scoring FOLR1 expression. The kit can also include control or reference samples. Non-limiting examples of control or reference samples include tissue samples, cell pellets or cells. Control or reference samples can be derived from tissue culture cell samples (normal or tumor), normal tissue (normal control) or tumor tissue (positive control). Examples include cell lines SW620, T47D, IGROV-1, HELA, KB, JEG-3 and cell lines stably or transiently transfected with an expression vector that expresses FOLR1 (for example, 300.19FR1). Examples of tissues that can be used as normal reference tissues in the methods of detecting FOLR1 expression are described herein and include lung, salivary glands, and normal pancreas.
[00019] The invention is also directed to a method to identify a cancer susceptible to respond to an anti-FOLR1 antibody or anti-FOLR1 immunoconjugate comprising: (a) contacting a biological sample comprising cells of said cancer, with an agent that binds to the FOLR1 protein on the cell surface, (b) detecting the binding of said agent that binds to the FOLR1 protein on the cell surface of said biological sample from (a), (c) assigning a degree to said binding of step (b), in that said grade is assigned based on comparison with one or more reference samples, and (d) comparing said grade in step (c) to the grade of a reference tissue or cell, where a grade for said grade of FOLR1 in cancer that is greater than the grade for a normal or low FOLR1 expression level in the reference sample or a grade for a high FOLR1 expression level in the reference sample or a count for said FOLR1 level in cancer that is equal to or greater than the result of one to reference sample expressing FOLR1 identifies the likelihood of said cancer responding to an anti-FOLR1 antibody or anti-FOLR1 immunoconjugate. In certain modalities, cancer is ovarian or lung cancer.
[00020] The invention is also directed to a method for identifying a tumor as sensitive to treatment with an anti-FOLR1 antibody or anti-FOLR1 immunoconjugate, said method comprising: (a) measuring the level of expression of FOLR1 in a sample of tumor tissue obtained from said tumor, wherein said measurement comprises the use of a detection method, which distinguishes between the intensity of staining or uniformity of staining in a cancer sample expressing FOLR1, in comparison with the intensity of staining or uniformity of staining in one or more reference samples, (b) determining a degree of FOLR1 staining intensity for said tumor tissue sample, and (c) comparing the degree of FOLR1 staining intensity determined in step (b) with a relative value of FOLR1 determined by measuring the expression of the protein in at least one reference sample, where said, at least one reference sample is a sample of tissue, cell or cell pellet that is not o is sensitive to treatment with an anti-FOLR1 antibody or anti-FOLR1 immunoconjugate, and in which a degree of intensity of FOLR1 staining for said sample determined in step (b), which is greater than said relative value identifies the said tumor as being sensitive to treatment with an anti-FOLR1 antibody or anti-FOLR1 immunoconjugate. In certain embodiments, the detection method is carried out manually or using an automated system. In one embodiment, the detection method is IHC. In another modality, the IHC is a calibrated IHC that can distinguish different levels of FOLR1 expression.
[00021] The invention is also directed to a method of improving a therapeutic regimen with an anti-FOLR1 antibody or an anti-FOLR1 immunoconjugate for an individual containing ovarian or lung cancer, said method comprising: (a) contacting said sample of said individual with an antibody that specifically binds to cell surface FOLR1, (b) measuring the binding of said antibody in (a) to said cell surface FOLR1 in said sample using a detection method that can distinguish between the intensity of staining or uniformity of staining in a cancer sample expressing FOLR1 compared to the intensity of staining or uniformity of staining in one or more reference samples and assigning a degree of staining to said sample, and (c) administering a high dose of an anti-FOLR1 immunoconjugate when the grade in step (b) is less than or equal to the grade for a reference sample expressing normal or low FOLR1 or administering a dose drop of an anti-FOLR1 immunoconjugate when the grade is greater than the grade for a reference sample expressing normal or low FOLR1.
[00022] The invention is also directed to a method for detecting the expression of FOLR1 on the cell surface in cancer cells in a sample of tumor tissue from an individual, said method comprising: (a) obtaining samples of tumor tissue, in that said cancer sample is fixed in formalin and embedded in paraffin, (b) contacting said sample with an antibody that specifically binds to the cell surface FOLR1, (c) measuring the binding of said antibody, in (b) with said cell surface FOLR1 in said tumor tissue sample using a detection method that can distinguish between staining intensity or staining uniformity in a cancer sample expressing FOLR1, in comparison with staining intensity or staining uniformity in one or more reference samples, and (d) assigning a degree of expression of FOLR1 to said FOLR1 after comparing the level of staining intensity or staining uniformity of cell surface FOLR1 in said sample stra of tumor tissue with one or more reference samples.
[00023] The invention is also directed to a method for identifying an individual containing ovarian or lung cancer, as susceptible to responding to a low-dose anti-FOLR1 antibody or anti-FOLR1 immunoconjugate treatment regimen, said method comprising: (a) contacting a biological sample comprising cells of said ovarian or lung cancer with an agent that binds to the cell surface protein FOLR1, (b) detecting the binding of said agent to said biological sample from (a) , (c) assigning a grade to said link in step (b), wherein said grade is assigned based on comparison with one or more reference samples, and (d) comparing said grade in step (c) with the degree of a reference tissue or cell, in which a degree for the level of FOLR1 in said ovarian or lung cancer that is greater than the degree for a reference sample expressing low or normal FOLR1, or a degree for the level of FOLR1 in said ovarian or lung cancer that is equal to or greater than the degree of a reference sample expressing high FOLR1 identifies the likelihood of said ovarian or lung cancer to respond to a low dose of anti-FOLR1 antibody or anti-FOLR1 immunoconjugate. In certain embodiments, the method further comprises administering a therapeutically effective amount of a humanized anti-FOLR1 antibody or an anti-FOLR1 immunoconjugate to said individual. BRIEF DESCRIPTION OF THE DRAWINGS
[00024] Figure 1. Manual Staining Method: Anti-FOLR1 antibodies detect FOLR1 expression in transfected cells. 300.19 cells were transfected with a polynucleotide encoding human FOLR1. Expression of the FOLR1 protein was detected using the murine antibody BN3.2. Smith, AE et al, Hybridoma (Larchmt). 2007 Oct; 26 (5): 281-8.
[00025] Figure 2. Manual Staining Method: Anti-FOLR1 antibodies can distinguish different levels of FOLR1 expression. The BN3.2 antibody was used to detect FOLR1 expression in several xenograft cells. The detection limit for the BN3.2 antibody was about 4000 cell-bound antibodies (ABC).
[00026] Figure 3. Manual Staining Method: Anti-FOLR1 antibodies can distinguish different levels of FOLR1 expression in tissue samples. BN3.2 was used to detect FOLR1 expression in ovarian tumors (A), as well as non-small cell lung cancer tumors (B).
[00027] Figure 4. Manual Staining Method: Uniform expression of FOLR1 in ovarian and NSCLC tumors. FOLR1 expression was elevated in many of the ovarian carcinomas, as well as lung adenocarcinomas and bronchioles carcinomas tested. Most samples of ovarian carcinoma showed the highest intensity of staining in serous or endometrioid cells. In NSCLC tumors, the highest ABC values were found in bronchial alveolar carcinomas and papillary adenocarcinoma.
[00028] Figure 5. Manual Staining Method: FOLR1 expression is generally confined to the NSCLC cell membrane. High-resolution microscopy revealed that the majority of FOLR1 staining was restricted to the membrane in NSCLC tumors.
[00029] Figure 6. Manual Staining Method: FOLR1 expression is generally confined to the membrane of ovarian cancer cells. High-resolution microscopy revealed that the majority of FOLR1 staining was restricted to the membrane in ovarian tumors.
[00030] Figure 7. In vivo efficacy of huMov19-targeted conjugates in a KB xenograft model. The cleavable conjugate targeting FOLR1huMov19-SPDB-DM4 (B) compared to huC242-SPDB-DM4 not targeting FOLR1 (D), and huMov19-PEG4-Mal-DM4 non-cleavable (C), compared to huC242 Non-targeted PEG4Mal-DM4 (E) were tested using an established KB cell xenograft implanted subcutaneously in SCID mice. The targeting of FOLR1 by huMov19 resulted in a significant reduction in the mean tumor volume.
[00031] Figure 8. Dose-response of antitumor activity of treatment with IMGN853 in human ovarian carcinoma xenografts OVCAR-3. The mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg / kg. A group of control animals received a single intravenous injection of PBS.
[00032] Figure 9. Dose-response of antitumor activity of treatment with IMGN853 in IGROV-1 human ovarian carcinoma xenografts. The mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg / kg. A group of control animals received a single intravenous injection of PBS.
[00033] Figure 10. Dose-response of antitumor activity of treatment with IMGN853 in human ovarian carcinoma xenografts OV-90. The mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg / kg. A group of control animals received a single intravenous injection of PBS.
[00034] Figure 11. Dose-response of antitumor activity of treatment with IMGN853 in three SKOV-3 human ovarian carcinoma xenografts. The mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg / kg. A group of control animals received a single intravenous injection of PBS.
[00035] Figure 12. Dose-response of antitumor activity of treatment with IMGN853 in KB xenografts of human cervical adenocarcinoma. The mice were treated with a single intravenous injection of IMGN853 at 1.0, 2.5 or 5.0 mg / kg. A group of control animals received a single intravenous injection of PBS.
[00036] Figure 13. Automatic Staining Methods: Photographs and Representative Histograms showing FOLR1 Expression in Cell Lines by IHC and Flow Cytometry. SW620, T47D, Igrov-1, 300.19 / FR1, HeLa and KB cells were all scored for intensity and uniformity of FOLR1 staining. SW630 and IGROV-1 cells were scored as 1-3 hetero, T47D was scored as 1-2 hetero, HeLa was scored as 2-3 hetero, while 300.19 / FR1 and KB were scored as 3 homo.
[00037] Figure 14. Automated Staining Methods: Representative Staining of FOLR1 in Serous Ovarian Cancer. Staining patterns demonstrating 3 homo, 2-3 homo, 2 homo, and 2 hetero staining are shown for IHC serous ovarian cancer tissue cuts.
[00038] Figure 15. Automated Staining Methods: Representative Staining of FOLR1 in Endometrioid Ovarian Cancer. Staining patterns demonstrating 3 homo, 2-3 homo, 3 focal, and 1-2 hetero staining are shown for HCI endometrioid cancer tissue cuts.
[00039] Figure 16. Automated Staining Methods: Representative Staining of FOLR1 in NSCLC of the Adenocarcinoma Subtype (excluding alveolar bronchioles). Staining patterns demonstrating 3 homo, 2-3 homo, 2 hetero, 2 homo, and 1-2 hetero staining are shown for sections of non-small cell lung adenocarcinoma tissue, HHC adenocarcinoma subtype.
[00040] Figure 17. Automated Staining Methods: Representative Staining of FOLR1 in Endometrial Adenocarcinoma. Staining patterns demonstrating 3 hetero, 2 hetero, and 1 hetero staining are shown for IHC endometrial adenocarcinoma tissue sections.
[00041] Figure 18. Automated staining methods: Representative staining of FOLR1 in Renal Light Cell Carcinoma. Staining patterns demonstrating 2 homo, 2 hetero, and 1 hetero staining are shown for kidney cell cancer tissue sections by IHC.
[00042] Figure 19. The in vitro cytotoxic activity of IMGN853. Five FOLR1-positive cell lines (KB, IGROV-1, JEG-3 and SKOV-3 OVCAR-3) and two FOLR1 negative cell lines (Namalwa and SW2) were analyzed for their sensitivity to the cytotoxic effects of IMGN853. The cells were exposed to IMGN853 (continuous line), or to IMGN853 plus 0.5 μM huMov19 (M9346A) unconjugated (dashed line), for 5 days and cell survival was determined through an assay based on WST-8. Representative data is shown. The percentage of surviving cells was plotted against the base 10 logarithm of the IMGN853 concentration.
[00043] Figure 20. IMGN853 sensitivity of FOLR1 positive cell lines versus FOLR1 expression level. The potency and specificity of IMGN853 were analyzed against FOLR1 positive cell lines with a wide range of FOLR1 expression. Cell lines were incubated with IMGN853 and KB, Igrov-1, and Jeg-3 were specifically sensitive to IMGN853 while huMov19 (M9346A) unconjugated showed decreased conjugate activity. Skov-3 and Ovcar-3 were not sensitive to IMGN853 and huMov19 (M9346A) unconjugated did not alter the activity of the conjugate.
[00044] Figure 21. Automated Staining Methods: Effectiveness of Ovarian Carcinoma Xenograft models stained for FOLR1. Staining patterns showing 1-3 hetero (Ovcar 3), 1-3 homo (Igrov 1), 1-2 hetero (Ov 90) and negative (SKOV 3) are shown for IHC ovarian cancer xenograft tissue cuts .
[00045] Figure 22. Automated Staining Methods: Mouse Xenograft Models. The staining patterns for FOLR1 in NSCLC Cell Line xenografts (A), Endometrial Carcinoma (B) and Cervical Carcinoma (C) are shown. NSCLC samples demonstrated 2-3 homo or 2 homo staining, endometrial carcinoma demonstrated 2 hetero / 3 focal staining, and cervical carcinoma demonstrated 3 homo staining.
[00046] Figure 23. Guide for automatic staining of control tissues for tests. Staining patterns for negative (esophagus 0) and positive (salivary gland 1-2 hetero, lung 2 homo, pancreas 3 homo) samples are shown as determined by automated IHC.
[00047] Figure 24. Guide for automatic staining of tumor tissues. Representative staining patterns for level 3, level 2 and level 1 staining are shown on control tissue, as determined by automated IHC.
[00048] Figure 25. Guide for automatic staining of tumor tissues. Representative staining patterns for level 3, level 2, and level 1 / negative staining are shown on control tissue, as determined by automated IHC. DETAILED DESCRIPTION OF THE INVENTION
[00049] The present invention provides methods for increasing the effectiveness or likelihood of response to the treatment of cancers characterized by overexpression of FOLR1. The present invention is based on the discovery of a dynamic range of FOLR1 expression in tumor tissue compared to normal tissue and the discovery that tumors with increased levels of FOLR1 expression are more sensitive to treatment with anti-HIV antibodies. FOLR1 or anti-FOLR1 immunoconjugates. We also found differences in sensitivity and detection of dynamic ranges between manual and automated methods. Kits comprising one or more reagents useful for practicing the methods of the invention are further provided. I. Definitions
[00050] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.
[00051] The terms "human folate receptor 1" or "FOLR1", as used herein, refer to any native human FOLR1, unless otherwise indicated. The term "FOLR1" includes unprocessed "full-length" FOLR1, as well as any form of FOLR1 that results from processing within the cell. The term also includes naturally occurring variants of FOLR1, for example, processing variants, allelic variants and isoforms. The FOLR1 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Examples of FOLR1 strings include, but are not limited to, the NCBI reference numbers P15328, NP_001092242.1, AAX29268.1, AAX37119.1, NP_057937.1, and NP_057936.1, and those shown in SEQ ID NOs: 1 and 2.
[00052] The term "increased expression" of FOLR1 refers to a sample that contains high levels of FOLR1 expression. In one example, the expression of FOLR1 is measured by IHC and assigned a degree of staining intensity or a degree of uniformity of staining by comparison with controls (eg, calibrated controls) showing defined degrees (eg, a degree of intensity 3 is given to the test sample, if the intensity is comparable to the calibrated control level 3 or an intensity of 2 is given to the test sample, if the intensity is comparable to the calibrated control level 2). For example, a grade of 1, 2, 3, or 3+ or greater by immunohistochemistry indicates an increase in FOLR1 expression. The uniformity of color, which is heterogeneous or homogeneous, is also indicative of a greater expression of FOLR1. The degrees of staining intensity and staining uniformity can be used alone or in combination (for example, 2 homo, 2 hetero, 3 homo, 3 hetero, etc.) In another example, an increase in FOLR1 expression can be determined by detection of an increase of at least 2-fold, at least 3-fold, or at least 5-fold) over control values (for example, the level of expression in a tissue or cell of an individual without cancer or with a cancer which does not have high values of FOLR1).
[00053] A "reference sample" can be used to correlate and compare the results obtained in the methods of the invention from a test sample. The reference samples can be cells (for example, cell lines, cell pellets) or tissues. The levels of FOLR1 in the "reference sample" can be an absolute or relative value, a value range, a minimum and / or maximum value, an average value, and / or a median value of FOLR1. The diagnostic methods of the invention involve a comparison between the levels of expression of FOLR1 in a test sample and a "reference value". In some embodiments, the reference value is the level of expression of FOLR1 in a reference sample. A reference value can be a predetermined value and can also be determined from reference samples (for example, a biological sample control) tested in parallel with the test samples. A reference value can be a single cut-off value, such as a median or average, or a range of values, such as a confidence interval. Reference values can be established for various subgroups of individuals, such as individuals predisposed to cancer, individuals containing early or late stage cancer, male and / or female individuals, or individuals undergoing cancer therapy. Examples of normal samples or reference values and positive samples or reference values are described here.
[00054] In some embodiments, the reference sample is a sample of healthy tissue, in particular a corresponding tissue, which is not affected by cancer. These types of reference samples are referred to as negative control samples. In other embodiments, the reference sample is a sample of tumor tissue that expresses FOLR1. These types of reference samples are referred to as positive control samples. Positive control samples can also be used as a comparative indicator for uniformity (hetero versus homo) and / or the degree (1, 2, 3, 3+) of the staining intensity, which correlates with the degree of expression of FOLR1. Comparative positive control samples are also referred to as calibrated reference samples that demonstrate a dynamic range of staining intensity or uniformity. As shown in Examples 1-9, reference samples not expressing FOLR1 include human esophageal tissue; low FOLR1 reference includes tissues of the salivary gland (particularly the interim ducts) and lung (particularly the respiratory epithelium); and tissue expressing high FOLR1 includes the pancreas (in particular ductal cells). For cell lines, those that express little include, among others, OVCAR3 and T47D, those that express moderately include, among others, SW620, IGROV-1, JEG3, those that express high include, among others, KB and IGROV1. A particularly desirable positive reference for high FOLR1 is a stable or transiently transfected cell line with Folate Receptor 1 (for example, 300.19 / FR1). Appropriate positive and negative reference levels of FOLR1 for a particular cancer can be determined by measuring the levels of FOLR1 in one or more appropriate individuals, and these reference levels can be customized in specific populations of individuals (for example, a reference level can be compatible with age so that comparisons can be made between FOLR1 levels in samples from individuals of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a given age group). These reference levels can also be customized for specific techniques that are used to measure FOLR1 levels in biological samples (eg, immunological assays, etc.), where FOLR1 levels may differ based on the specific technique that will be used.
[00055] The term "primary antibody" refers here to an antibody that specifically binds to the antigen of target proteins in a tissue sample. A primary antibody is generally the first antibody used in the immunohistochemistry (IHC) process. In one embodiment, the primary antibody is the only antibody used in an IHC process. The term "secondary antibody" used herein refers to an antibody that specifically binds to a primary antibody, thus forming a bridge between the primary antibody and a subsequent reagent, if any. The secondary antibody is generally the second antibody used in the immunohistochemistry process.
[00056] A "sample" or "biological sample" of the present invention is of biological origin, in specific embodiments, such as eukaryotic organisms. In preferred embodiments, the sample is a human sample, but animal samples can also be used in the practice of the invention. Non-limiting sources of a sample for use in the present invention include solid tissue, aspirations of biopsy, ascites, fluid extracts, blood, plasma, serum, spinal fluid, lymphatic fluid, the outer sections of the skin, respiratory, intestinal and genital tracts -urinary, tears, saliva, milk, tumors, organs, cell cultures and / or cell culture constituents, for example. The present invention is particularly useful for cancer samples that generally comprise samples of solid tissues, or other body fluids, such as ascites, where the amount of material available is small. The method can be used to examine an aspect of FOLR1 expression or a sample state, including, but not limited to, comparing different types of cells or tissues, comparing different stages of development, and detecting or determining the presence and / or type of disease or abnormality.
[00057] For purposes here, a "section" of a tissue sample refers to a single piece of a tissue sample, for example, a thin slice of tissue or cut of cells from a tissue sample. It is understood that multiple cuts of tissue samples can be taken and subjected to analysis according to the present invention. In some cases, the selected portion or cut of tissue comprises a homogeneous population of cells. In other cases, the selected portion comprises a region of tissue, for example, the lumen as a non-limiting example. The selected portion can be as small as one cell or two cells, or it could represent many thousands of cells, for example. In most cases, the collection of cells is important, and while the invention has been described for use in the detection of cellular components, the method can also be used for the detection of non-cellular components of an organism (for example, blood-soluble components as a non-limiting example).
[00058] By "correlating" or "correlating" is meant a comparison, in any form, of the performance and / or the results of a first analysis with the performance and / or results of a second analysis. For example, the results of a first analysis can be used to perform the 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 can be compared first analysis with the results of a second analysis. In one embodiment, the increased expression of FOLR1 correlates with the increased likelihood of the effectiveness of an anti-cancer therapy targeting FOLR1.
[00059] 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 combinations of the above by means of at least one recognition site of the antigen within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab ', F (ab') 2, and Fv fragments), single chain Fv mutants (scFv), multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-determining portion of an antibody, and any other modified immunoglobulin molecule comprising a recognition site antigen, while antibodies exhibit the desired biological activity. An antibody can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or subclasses (isotypes) of the same (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on identity of its heavy chain constant domains referred to as alpha, delta, epsilon, gamma and mu, respectively. The different classes of immunoglobulins have different and known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules, such as radioisotopes, toxins, etc.
[00060] A "blocking" antibody or an "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds, such as FOLR1. In a given embodiment, blocking antibodies or antagonistic antibodies substantially or completely inhibit the biological activity of the antigen. Desirably, biological activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95% or even 100%.
[00061] The term "anti-FOLR1 antibody" or "an antibody that binds to FOLR1" refers to an antibody that is capable of binding to FOLR1 with sufficient affinity for the antibody to be useful as a diagnostic agent and / or targeted therapy for FOLR1. The extent of binding of an anti-FOLR1 antibody to an unrelated, non-FOLR1 protein is less than about 10% of the antibody's binding to FOLR1 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to FOLR1 has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. Examples of anti - FOLR1 antibodies are known in the art and are disclosed in US Order Publication 2012/0009181, which is incorporated herein by reference.
[00062] The term "antibody fragment" refers to a portion of an intact antibody and refers to the variable regions of antigenic determination 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.
[00063] A "monoclonal antibody" refers to a homogeneous population of antibodies associated with the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" includes intact, full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab ', F (ab') 2, Fv), single chain mutants (scFv), fusion proteins comprising a portion of the antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site. In addition, "monoclonal antibody" refers to those antibodies produced in any number of ways, including, but not limited to, hybridoma, phage selection, recombinant expression and transgenic animals.
[00064] The terms "epitope" or "antigenic determinant" are used interchangeably here and refer to the portion of an antigen capable of being specifically recognized and bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed from contiguous amino acids and non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically kept under protein denaturation, while epitopes formed by tertiary folds are typically lost in protein denaturation. An epitope typically includes at least 3, and more generally, at least 5 or 8-10 amino acids in a single spatial conformation.
[00065] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site on a molecule (for example, an antibody) and its binding partner (for example, an antigen) . Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (for example, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind to the antigen slowly and tend to dissociate quickly, whereas high-affinity antibodies generally bind to the antigen more quickly and tend to stay on longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific illustrative modalities are described below.
[00066] "Or rather", when used here to refer to binding affinity refers to a stronger bond between the molecule and its binding partner. "Or rather", when used here, refers to a stronger bond, represented by a smaller numerical Kd value. For example, an antibody that has an affinity for an antigen of "0.6 nM or better", the antibody's affinity for antigen is <0.6 nM, that is, 0.59 nM, 0.58 nM, 0 , 57 nM, etc., or any value less than 0.6 nM.
[00067] Aphasis "substantially similar" or "substantially the same", as used herein, denotes a sufficiently high degree of similarity between two numerical values (generally one associated with an antibody of the invention and the other associated with a reference / comparator antibody ) so that a person skilled in the art considers the difference between the two values to be of little or no biological and / or statistical importance within the context of the biological characteristics measured by said values (for example, Kd values). The difference between said two values is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value for the reference antibody / comparator.
[00068] A polypeptide, antibody, polynucleotide, vector, cell, or composition that is "isolated" is a polypeptide, antibody, polynucleotide, vector, cell or composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to a degree that they are no longer in a way that they are found in nature. In some embodiments, an antibody, polynucleotide, vector, cell, or composition that is isolated is substantially pure.
[00069] As used herein, "substantially pure" refers to material that is at least 50% pure (that is, free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
[00070] The term "immunoconjugate" or "conjugate" as used herein refers to a compound or a derivative thereof, which is attached to a cell-binding agent (i.e., an anti-FOLR1 antibody or fragment of the same) and is defined by a generic formula: CLA, where C = cytotoxin, L = ligand, and A = a cell-binding agent or anti-FOLR1 antibody or antibody fragment. Immunoconjugates can also be defined by the generic formula in reverse order: A-L-C.
[00071] A "ligand" is any chemical fraction that is capable of binding a compound, usually a drug, such as maytansinoid, to a cell binding agent, such as an anti FOLR1 antibody or fragment thereof, in a stable covalent form. The ligands may be susceptible to, or substantially resistant to, acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions under which the compound or antibody remains active . Suitable linkers are well known in the art and include, for example, disulfide groups, thio ester groups, acid labile groups, photo labile groups, labile peptidase groups and labile esterase groups. The binders include charged binders, and hydrophilic forms thereof as described herein and known in the art.
[00072] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a cell population is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specific examples of these cancers include squamous cell cancer, small cell lung adenocarcinoma, non-small cell lung adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, peritoneum cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, colon cancer uterine cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, liver carcinoma and various types of head and neck cancers.
[00073] "Tumor" and "neoplasm" refer to a mass of tissue that results from overgrowth or cell proliferation, both benign (non-cancerous) or malignant (cancerous), including precancerous lesions.
[00074] The terms "cancer cells", "tumor cells", and their grammatical equivalents refer to the total population of cells derived from a tumor or a precancerous lesion, including both non-tumorigenic cells, which constitute the most of the tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cell" will be modified by the term "non-tumorigenic" when referring only to those tumor cells that do not have the capacity to renew and differentiate to distinguish tumor cells from tumorigenic stem cells.
[00075] The term "individual" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, that will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably in this document with reference to a human subject.
[00076] Administration "in combination with" one or more therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
[00077] The term "pharmaceutical formulation" refers to a preparation that is such as to allow the biological activity of the active ingredient to be efficient, and that it does not contain any additional components that are unacceptably toxic to the individual to whom the formulation would be administered. This formulation can be sterile.
[00078] An "efficient amount" of an antibody as described herein is an amount sufficient to accomplish a specifically stated purpose. An "efficient amount" can be determined empirically and routinely in relation to the stated objective.
[00079] The term "therapeutically effective amount" refers to an amount of an antibody or other drug effective to "treat" a disease or disorder in an individual or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells, reduce the size of the tumor, inhibit (that is, reduce to some extent and in a certain way, interrupt) the infiltration of cancer cells into peripheral organs; inhibit (that is, reduce to a certain extent and, in a certain way, interrupt) the metastasis of the tumor; inhibit, to some extent, tumor growth, and / or relieve to some extent one or more of the symptoms associated with cancer. See the definition of "treatment" here. Insofar as the drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. In certain embodiments, the identification of increased levels of FOLR1 allows the administration of lower amounts of the FOLR1-targeted therapy to achieve the same therapeutic effect as seen with higher doses. A "prophylactically effective amount" refers to an efficient amount, in dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in individuals before or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
[00080] The term "respond favorably" generally refers to causing a beneficial state in an individual. Regarding the treatment of cancer, the term refers to providing a therapeutic effect to the individual. Positive therapeutic effects on cancer can be measured in several ways (See, W.A. Weber, J. Nucl. Med. 50: 1 S-10S (2009)). For example, inhibition of tumor growth, expression of the molecular marker, expression of the serum marker, and molecular imaging techniques can all be used to assess the therapeutic effectiveness of an anti-cancer drug. With respect to tumor growth inhibition, according to NCI standards, a T / C <42% is the minimum level of antitumor activity. A T / C <10% is considered a high level of anti-tumor activity, with T / C (%) = Median volume of the treated tumor / Median volume of the control tumor x 100.
[00081] The term "labeling" when used herein refers to a compound or composition that is conjugated directly or indirectly with the antibody in order to generate a detectable "labeled" antibody. The label can be detectable by itself (for example, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, it can catalyze a chemical change of compound or composition of a substrate that is detectable.
[00082] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of the mechanism of action. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, poisonous vegetable spindle alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "target therapy" and conventional chemotherapy.
[00083] Terms such as "treating" or "treating" or "treating" or "relieving" or "relieving" refer to 1) therapeutic measures capable of curing, delaying, decreasing the symptoms of, and / or stopping the progression of a pathological condition or disorder diagnosed and 2) prophylactic or preventive measures that prevent and / or delay the development of a target condition or pathological disorder. Thus, those in need of treatment include those already with the disorder; those likely to have the disorder, and those in which the disorder should be prevented. In certain embodiments, an individual is successfully "treated" for cancer according to the methods of the present invention if the patient has one or more of the following: reduced cachexia, increased survival time, prolonged tumor progression time , reduction in tumor mass, reduction in tumor burden and / or a prolongation of tumor metastasis time, tumor recurrence time, tumor response, complete response, partial response, stable disease, progressive disease, progression-free survival ( PFS), global survival (OS), each measured by standards set by the National Cancer Institute and US Food and Drug Administration for the approval of new drugs. See Johnson et al., (2003) J. Clin. Oncol. 21 (7): 1404-1411.
[00084] "Progression-free survival (PFS)", also referred to as "Tumor Progression Time" (YIP), indicates the length of time, during and after treatment that the cancer does not progress. Progression-free survival includes the amount of time that patients experienced a complete or partial response, as well as the amount of time that patients experienced stable disease.
[00085] "Disease-free survival" (DFS) refers to the period of time during and after treatment that the patient remains disease-free.
[00086] "General survival" (OS) refers to an extension of life expectancy compared to naive or untreated individuals or patients.
[00087] As used in the present disclosure and claims, the singular forms "one", "one", "o" and '‘a" include plural forms unless the context clearly dictates otherwise.
[00088] It is understood that, whenever modalities are described here with the language "comprising", other analogous modalities described in terms of "consisting of" and / or "consisting essentially of" are also provided.
[00089] The term "and / or" as used in the sentence as "A and / or B" used herein is intended to include "A and B", "A or B", "A" and "B". Likewise, the term "and / or", as used in the sentence such as "A, B and / or C" is intended to cover each of the following modalities: 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 (only), B (only), and C (only). II.Biological samples
[00090] Biological samples are often fixed with a fixative. Aldehyde fixatives such as formalin (formaldehyde) and glutaraldehyde are typically used. Tissue samples fixed using other fixation techniques, such as immersion in alcohol (Battifora and Kopinski, J. Histochem. Cytochem. (1986) 34: 1095) are also appropriate. The samples can also be embedded in paraffin. In one embodiment, the tissue samples are fixed in formalin and embedded in paraffin (FFPE). In another embodiment, the FFPE block is stained with hematoxylin and eosin before selecting one or more portions for analysis in order to select the specific area for the central FFPE sample. Methods for preparing tissue blocks from these particulate samples have been used in previous IHC studies of several prognostic factors, and / or are well known to those of skill in the art (see, for example, Abbondanzo et al., Am J Clin Pathol May 1990, 93 (5): 698-702; Allred et al., Arch Surg 1990 Jan; .. 125 (1): 107-13).
[00091] In short, any intact organ or tissue can be cut into relatively small pieces and incubated in various fixatives (eg formalin, alcohol, etc.) for several periods of time until the tissue is "fixed". The samples can be virtually any intact tissue surgically removed from the body. The samples can be cut into reasonably small pieces that fit the equipment used routinely in histopathological laboratories. The size of the cut pieces typically ranges from a few millimeters to a few centimeters. III. Detection of Conjugated Antibodies
[00092] The present invention further provides antibodies against FOLR1, in general, of the monoclonal type, which is bound to at least one agent to form a conjugated detection antibody. In order to increase the effectiveness of diagnostic antibody molecules, it is conventional to bind or covalently link or complex at least one desired molecule or fraction. This molecule or fraction can be, among others, at least one reporter molecule. A reporter molecule is defined as any fraction that can be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photo-affinity molecules, colored particles and / or ligands, such as biotin.
[00093] Any cell-binding agent (for example, an antibody or polypeptide) of sufficient selectivity, specificity or affinity can be employed as the basis for detecting the FOLR1 polypeptide. These properties can be evaluated using conventional immunological screening methodologies known to those skilled in the art. Binding sites for molecules with biological activity on the antibody molecule, in addition to the canonical antigen binding sites, include sites that reside in the variable domain that can bind the antigen. In addition, the variable domain is involved in the auto-binding of antibodies (Kang et al., 1988) and contains epitopes (idiotopes) recognized by anti-antibodies (Kohler et al., 1989).
[00094] Certain examples of protein-binding conjugates (for example, antibody) are conjugates in which the protein-binding agent (for example, antibody) is attached to a detectable marker. "Detectable markers" are compounds and / or elements that can be detected, due to their specific functional properties, and / or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and / or further quantified, if wanted.
[00095] Many suitable imaging agents are known in the art, as are the methods for binding them to antibodies (see, for example, US Patent 5,021,236. 4,938,948, and 4,472,509, each incorporated herein by reference. ). The image fractions used can be paramagnetic ions; radioactive isotopes; fluorochromes, substances detectable by NMR, and / or, for example, X-ray image.
[00096] Examples of fluorescent markers intended for use as protein-binding conjugates (eg antibodies) include 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, fluorescent green protein (GFP), HEX, 6 JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phycoerythrin, REG, Rhodamine Green, Rhodamine Red, Tetra Methyl Rhododamine (TMR), Renografin, ROX, TAMRA, TET, Tetramethyl Rhododamine, Texas Red and derivatives of these markers (ie, halogenated analogs, modified with isothiocyanate or other binder to conjugate, etc.), for example. An example of a radioactive marker is tritium.
[00097] Protein binding detection conjugates (for example, antibody) contemplated in the present invention include those for in vitro use, wherein the antibody is bound to a secondary binding ligand and / or an enzyme (an enzyme tag) ) that will generate a colored product in contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and / or glucose oxidase. Preferred secondary linkers are compounds of biotin and / or avidin and streptavidin. The use of such markers is well known to those of skill in the art and are described, for example, in US Patents 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.
[00098] Molecules containing azido groups can also be used to form covalent bonds with proteins, through intermediate nitrene reactives that are generated by low intensity ultraviolet light (Potter & Haley, 1983). In particular, purine nucleotide 2 and 8-azide analogs are used as site probe photos directed to identifying nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985.). Nucleotides 2 - and 8-azido are also for mapping nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia and et al., 1989.) and can be used as agents antibody binding.
[00099] Various methods are known in the art for binding or conjugating an antibody to its conjugate fraction. Some bonding methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such as diethylenetriaminopentacetic acid anhydride (DTPA), ethylene triamine tetra acetic acid, N-chloro-p-toluenesulfonamide, and / or tetra-chloro-3α-6α-diphenylglycouryl-3 linked to the antibody (US Patents 4,472,509 and 4,938,948, each incorporated herein by reference). Monoclonal antibodies can also react with an enzyme in the presence of a binding agent such as glutaraldehyde or periodate. Protein-binding conjugates (e.g., antibody) with fluorescein markers are prepared in the presence of these binding agents or by reaction with an isothiocyanate. In US Patent 4,938,948, imaging of breast tumors, for example, is achieved using monoclonal antibodies, and detectable image fractions are linked to the antibody using ligands, such as methyl-p-hydroxybenzimidate or N-succinimidyl- 3- (4-hydroxyphenyl) -propionate.
[000100] In other modalities, derivatization of immunoglobulins by the selective introduction of sulfhydryl groups in the Fc region of an immunoglobulin using reaction conditions that do not alter the antibody combining site is contemplated. Conjugated antibodies produced according to this methodology are revealed to exhibit improved longevity, specificity and sensitivity (US Patent 5,196,066, incorporated herein by reference). Linking specific site of effector or reporter molecules, in which the effector or reporter molecule is conjugated to a carbohydrate residue in the Fc region, is also revealed in the literature (O'Shannessy et al., 1987).
[000101] In other embodiments of the invention, immunoglobulins are radiolabelled with radionuclides such as tritium. In additional embodiments, gold nano particles (such as sizes from about 0.5 nm - 40 nm) and / or Quantum Dots (Hayward, California) are employed. IV.Enzymes and substrates (Chromogenic)
[000102] The use of substrates and indicators is contemplated for the detection of FOLR1, as examples of the modalities provided below, for example.
[000103] Horseradish peroxidase (HRP) is an enzyme that first forms a complex with hydrogen peroxide and then causes it to decompose, resulting in water and atomic oxygen. Like many other enzymes, HRP and some HRP-like activities can be inhibited by an excess of substrate. The complex formed between HRP and excess hydrogen peroxide is catalytically inactive, and in the absence of an electron donor (eg, chromogenic substance) it is reversibly inhibited. It is the excess of hydrogen peroxide and the absence of an electron donor that cause the suppression of endogenous HRP activities.
[000104] When used in test systems, HRP can also be used to convert a defined substrate to its activated chromogen, thus causing a color change. The HRP enzyme can be conjugated to an antibody, protein, peptide, polymer, or other molecule by a number of methods. Such methods are known in the art. The addition of glutaraldehyde to a solution containing a mixture of HRP and antibody will result in more antibody molecules being conjugated to each other more than to the enzyme. In the two-step process, HRP reacts with the bifunctional reagents first. In the second stage, the activated HRP is only mixed with the antibody, resulting in a much more efficient labeling and without polymerization. HRP is also conjugated to (strept) avidin using the two-step glutaraldehyde procedure. This form is used in procedures where LAB and LSAB are substrates, for example. Conjugation with biotin also involves two steps, since the biotin must first be derivatized with the biotinyl-N-hydroxysuccinimide ester or with the hydrazide biotin before reacting with the epsilonamino groups of the HRP enzyme.
[000105] 3,3'-Diaminobenzidine (DAB) is a substrate for enzymes, such as HRP, which produces a brown final product, which is highly insoluble in alcohol and other organic solvents. The oxidation of DAB also causes polymerization, resulting in the ability to react with osmium tetroxide, thus increasing its color intensity and electronic density. Of the various metals and methods used to enhance the optical density of polymerized DAB, gold chloride in combination with silver sulfide appears to be the most successful.
[000106] 3-Amino-9-ethylcarbazole (AEC) is a substrate for enzymes, such as HRP. After oxidation, it forms a red-pink final product that is soluble in alcohol. Thus, specimens treated with AEC should not be immersed in alcohol or alcoholic solutions (for example, Harris' hematoxylin). Instead, an aqueous counter dye and mounting medium should be used. AEC, unfortunately, is even more susceptible to oxidation and, when exposed to excessive light, will lose intensity. Dark storage is recommended.
[000107] 4-Chlorine-1-naphthol (CN) is a substrate for enzymes, such as HRP and precipitates as a blue final product. Due to the fact that CN is soluble in alcohol and other organic solvents, the sample must not be dehydrated, exposed to alcoholic dyes, or cover slip with assembly medium containing organic solvents. Unlike DAB, CN tends to diffuse from the precipitation site.
[000108] p-Phenylenediamine dihydrochloride / pyrocatechol (Hanker-Yates reagent) is an electron donating substrate for enzymes such as HRP and the reaction product forms a blue-black that is insoluble in alcohol and other organic solvents. As a polymerized DAB, this reaction product can react with osmium tetroxide. Different results were obtained with the Hanker-Yates reagent in immunoperoxidase techniques.
[000109] Calf intestinal alkaline phosphatase (AP) (molecular weight 100 kDa) is an enzyme that removes (by hydrolysis) and transfers the phosphate groups from organic esters, breaking the P-0 bond; an intermediate enzyme-substrate bond is formed quickly. The main metal activators for AP are Mg ++, Mn ++ and Ca ++.
[000110] AP was not used extensively in immunohistochemistry until the publication of the non-labeled anti-alkaline phosphatase procedure (APAAP). The soluble immune complexes used in this process have molecular weights of approximately 560 kD. The main advantage of the APAAP procedure compared to the PAP technique is the lack of interference represented by the endogenous peroxidase activity. Because of the potential distraction of endogenous peroxidase activity in PAP staining, the APAAP technique is recommended for use in blood and bone marrow smears. The endogenous activity of bone alkaline phosphatase, from kidney, liver, and some white blood cells can be inhibited by adding 1 mM levamisole to the substrate solution, although 5 mM is more efficient. Intestinal alkaline phosphatases are not properly inhibited by levamisole.
[000111] In the immuno alkaline phosphatase staining method, the enzyme hydrolyzes naphthol phosphate esters (substrate) for phenolic compounds and phosphates. The phenols couple with colorless diazonium salts (chromogens) to produce insoluble colored azo dyes. Several different combinations of substrates and chromogens are used successfully.
[000112] Naphthol Phosphate AS-MX can be used in its acid form or in the form of sodium salt. The chromogens Fast Red TR and Fast Blue BB produce a final product red or bright blue, respectively. Both are soluble in alcoholic and other organic solvents so aqueous mounting media must be used. FastRed TR is preferred for staining cell smears.
[000113] Examples of additional substrates include naphthol AS-BI phosphate, naphthol AS-TR phosphate and 5-bromo-4-chloro-3-indoxyl phosphate (BCIP). Other possible chromogens include Fast Red LB, Fast Garnet GBC, Nitro Blue Tetrazolium (NBT) iodonitrotetrazolium Violet (INT), and structures derivatives, for example. V. Immunodetection Methods
[000114] In still other embodiments, the present invention relates to immunodetection methods for binding, purifying, removing, quantifying and / or otherwise generally detecting biological components, such as a linker as contemplated by the present invention. Antibodies prepared in accordance with the present invention can be used to detect wild-type and / or mutant binding proteins, polypeptides and / or peptides. As described throughout the present application, the use of specific wild-type and / or mutant binding antibodies is contemplated. Some immunodetection methods include flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to name a few. The steps of various useful immunodetection methods are described in the scientific literature, such as, for example, Doolittle M H and Ben-Zeev O, Methods Mol Biol. 1999; 109: 215-37; Gulbis B and Galand P, Hum Pathol., Dec 1993; 24 (12): 1271-85, and De Jager R et al., Semin Nucl Med. Abr 1993; 23 (2): 165-79, each incorporated herein as reference.
[000115] In general, immunostaining methods include obtaining a sample suspected of comprising a protein, polypeptide and / or linker peptide, and contacting the sample with a first linker binding peptide (e.g., an anti-ligand antibody), in according to the present invention, as the case may be, under efficient conditions to allow the formation of immune complexes.
[000116] In terms of antigen detection, the biological sample analyzed may be a sample that is suspected of comprising a specific antigen of the wild-type or mutant ligand protein, such as a tissue cut or specimen, a homogenized tissue extract, aspiration biopsy, a cell, of any of the separate compositions and / or purified forms containing wild-type or mutant FOLR1, or even any biological fluid that comes in contact with tissue, including blood and / or serum although tissue samples or extracts are preferred.
[000117] Contacting the chosen biological sample with the antibody, under efficient conditions and 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 mixing for a period of time long enough for the antibodies to form immune complexes with, that is, to bind to, any protein binding antigens present. After this time, the sample composition, the antibody, such as a tissue cut, ELISA plates, dot blot or western blot, will generally be washed to remove any kind of antibody not specifically bound, allowing only those antibodies specifically bound in the immune complexes be detected.
[000118] In general, the detection of immune complex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based on the detection of a label or a marker, such as any of the radioactive, fluorescent, biological and enzymatic ones. US patents on the use of these markings include US patents. 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 can be found through the use of a secondary binding ligand as a second binding ligand and / or biotin / avidin array, as is known in the art.
[000119] The anti-ligand antibody used in the detection alone can be linked to a detectable marker, in which one can then simply detect the label, thus allowing the determination of the amount of primary immune complexes in the composition. Alternatively, the first antibody that becomes bound within the primary immune complexes can be detected by means of a second binding agent that has a binding affinity for the antibody. In these cases, the second linker can be linked to a detectable marker. The second binding agent is, in itself, often an antibody, which can therefore be considered a "secondary" antibody, or a polymer detection system. The primary immune complexes are contacted with the labeled secondary binding agent, or antibody / polymer detection system, under efficient conditions, for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then normally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining marking on the secondary immune complexes is then detected.
[000120] Other methods include the detection of primary immune complexes by a two-step approach. A second binding agent, such as an antibody, that has a binding affinity for the antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding agent or antibody that has binding affinity for the second antibody, again under efficient conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). ). The third ligand or antibody is linked to a detectable marker, which allows the detection of the tertiary immune complexes thus formed. This system can provide signal amplification, if that is desirable.
[000121] In another embodiment, a biotinylated monoclonal or polyclonal antibody is used to detect the target antigen, and a second antibody step is then used to detect the biotin bound to the complexed biotin. In this method, the sample to be tested is incubated first in a solution that comprises the first antibody step. If the target antigen is present, some of the antibodies bind to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA solutions at each step by adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until an appropriate level of amplification is achieved, at which point the sample is incubated in a solution that comprises the second biotin antibody step. This second antibody step is marked, as for example, with an enzyme that can be used to detect the presence of the antibody / antigen complex by histology histology using a chromogen substrate. With appropriate amplification, a protein-binding conjugate (for example, an antibody) can be produced, which is macroscopically visible.
[000122] Another well-known method of immunodetection takes advantage of the method of immuno-PCR (Polymerase Chain Reaction). The PCR method uses a DNA / biotinesterptavidin / antibody complex that is washed with a low pH or high salt buffer that releases the antibody. The resulting washing solution is then used to perform a PCR reaction with appropriate primers, with appropriate controls. In specific modalities, the enormous capacity of PCR amplification and specificity can be used to detect a single antigen molecule. This detection can happen in real time. For example, the use of quantitative real-time PCR is contemplated.
[000123] In clinical diagnosis and / or monitoring of patients with various forms of disease, the detection of a FOLR1 mutant, and / or a change in FOLR1 levels, compared to the levels of a corresponding biological sample from a normal individual is indicative of a patient with the disease. However, as is known to those skilled in the art, such a clinical diagnosis would not necessarily have to be made on the basis of this isolation method. Those skilled in the art are familiar with the differentiation between significant differences in types and / or amounts of biomarkers, which represent a positive identification, and / or low level and / or modifications of the background biomarkers. In fact, background expression levels are often used to form a "cut" above, which increased detection will be scored as significant and / or positive.
[000124] In one embodiment, the immunological detection (by immunohistochemistry) of FOLR1 is scored for intensity and uniformity (percentage of stained cells - membrane only). Comparative scales for expression of FOLR1 for intensity correlates as 0 - Negative, 0-1- Very Weak, 1- Weak, 1-2 - Weak amoderate, 2- Moderate, 2-3- Moderate to strong, 3- Strong. Quantitatively, grade 0 represents that no staining of the membrane is observed in tumor cells. A Grade 1 represents the staining of the light / almost imperceptible membrane in the tumor cells. For grade 2, a moderate staining of the membrane is observed in tumor cells. Finally, Grade 3 or 3+ represents moderate to strong membrane staining in tumor cells. Samples with a score of 0 or 1 for expression of FOLR1 can be characterized as not overexpressing FOLR1, while samples with a score of 2 or 3 can be characterized as overexpressing FOLR1. Samples overexpressing FOLR1 can also be evaluated by immunohistochemical grades corresponding to the number of copies of FOLR1 molecules expressed per cell, and have been determined biochemically: 0 = 010,000 copies / cell, 1 = at least about 200,000 copies / cell, 2 = at least about 500,000 copies / cell, and 3 = at least about 2 million copies / cell. Comparative scales for percentage of uniformity of FOLR1 membrane staining per cell correlates as follows: 0 - Negative, Focal - <25%, Heterogeneous (hetero) - 25-75%, and Homogeneous (homo) -> 75%. VI. Nucleic Acid Hybridization
[000125] In situ hybridization is generally performed on cells or tissue sections fixed on slides. In situ hybridization can be performed by several conventional methods (See, for example, Leitch et al. In situ Hybridization: a practical guide, Oxford BIOS Scientific Publishers, Microscopy handbooks v. 27 (1994)). In an in situ procedure, fluorescent dyes (for example, fluorescein isothiocyanate (FITC), which emits green fluorescence when excited by an argon ion laser) are used to identify a nucleic acid sequence probe that is complementary to a sequence of target nucleotide in the cell. Each cell comprising the target nucleotide sequence will bind the labeled probe, producing a fluorescence signal after exposure of the cells to a light source of a wavelength appropriate for the excitation of the specific fluoro chromium used.
[000126] Various degrees of stringency of hybridization can be employed. As hybridization conditions become more stringent, a greater degree of complementarity between the probe and target is required to form and maintain a stable duplex. Strictness is increased by raising the temperature, lowering the salt concentration, or increasing the concentration of formamide. The addition of dextran sulfate or the increase in its concentration can also increase the effective concentration of a labeled probe to increase the hybridization rate and the final signal intensity. After hybridization, the slides are washed in a solution containing reagents generally similar to those found in the hybridization solution with washing time ranging from minutes to hours, depending on the required rigor. Longer or more rigorous washings typically decrease the nonspecific background, but are at risk of decreasing overall sensitivity.
[000127] The probes used in the analysis of nucleic hybridization can be oligonucleotides or RNA or DNA polynucleotides and can contain not only naturally occurring nucleotides, but their analogues, such as digoxigenin dCTP, dcTP biotin 7-azaguanosine, azidothymidine, inosine or uridine, for example. Other useful probes include peptide probes and analogs thereof, branched gene DNA, peptidomethers, peptide nucleic acid (PNA) and / or antibodies, for example.
[000128] The probes must have sufficient complementarity with the target nucleic acid sequence of interest so that stable and specific binding occurs between the target nucleic acid sequence and the probe. The degree of homology required for stable hybridization varies according to the stringency of the hybridization medium and / or washing medium. Preferably, completely homologous probes are employed in the present invention, but those skilled in the art will readily understand that probes exhibiting less but sufficient homology can be used in the present invention (see, for example, Sambrook, J., Fritsch, EF, Maniatis, T., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, (1989)).
[000129] Probes can also be generated and chosen by various means, including, among others, in situ hybridization mapping, somatic cell hybrid panels, or ordered chromosome spot blots; chromosomal binding analysis, or cloned and isolated from chromosomal libraries classified from human cell lines or somatic cell hybrids with human chromosomes, irradiated hybrid somatic cells, microdissection of a region of the chromosome or yeast artificial chromosomes (YACs) identified by PCR primers specific for a single chromosome locus or other appropriate means, such as an adjacent YAC clone. The probes can be genomic DNA, cDNA, or RNA cloned into a plasmid, phage, cosmid, YAC, Bacterial Artificial Chromosomes (BACs), viral vector, or any other appropriate vector. Probes can be cloned or chemically synthesized by conventional methods. When cloned, nucleic acid fragments isolated from the probe are normally inserted into a vector, such as lambda phage, pBR322, M13, or vectors containing the SP6 or T7 promoter and cloned as a library in a bacterial host. [See, for example, Sambrook, J, Fritsch, E.F., Maniatis, T., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, (1989)].
[000130] The probes are preferably labeled, as with a fluorophore, for example. Examples of fluorophores include, but are not limited to, rare earth chelates (europium chelates), Texas red, rhodamine, fluorescein, dansyl, Lissamine, umbeliferone, phycoerythrin, phycocyanin, or commercially available fluorophores, such as SPECTRUM ORANGE ™ and SPECTRUM GREEN ™ and / or derivatives of any one or more of the above. Several probes used in the assay can be labeled with more than one distinguishable fluorescent color or pigment. These color differences provide a means to identify the positions of specific hybridization probes. In addition, probes that are not spatially separated can be identified by a light of a different color or pigment resulting from the mixing of two other colors (for example, light red + green = yellow), pigment (for example, blue + yellow = green ) or using a set of filters that pass only one color at a time.
[000131] The probes can be labeled directly or indirectly with the fluorophore, using conventional methodology known to a person skilled in the art. VII.Composition and Detection Kits
[000132] Kits for use in the practice of the present invention as disclosed herein are also provided by the invention. Such kits may comprise containers, each containing one or more of the various reagents (typically in concentrated form) used in the methods, including, for example, one or more binding agents (antibodies), already attached to a label or optionally with reagents for the coupling of a binding agent to an antibody or nucleic acid molecule (as well as the marker itself), buffers, the appropriate nucleotide triphosphates (for example, dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP and UTP ), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more specific sequence or degenerate primers for use in the detection of nucleic acid molecules by amplification, and / or reagents and instrumentation for isolation (optionally by microdissection) to support the practice of the invention. A marker or indicator that describes, or a set of instructions for use, kit components in the binder detection method of the present invention, will also typically be included, where the instructions can be associated with a package insert and / or the kit packaging or the components thereof.
[000133] In still other embodiments, the present invention relates to immunodetection kits for use with the immunodetection methods described above. Since antibodies are generally used to detect wild-type and / or mutant proteins, polypeptides and / or peptides, the antibodies are preferably included in the kit. The immunodetection kits will thus comprise, in appropriate containers, a first antibody that binds to a protein, polypeptide and / or peptide of wild type and / or mutant and / or optionally, an immunodetection reagent and / or optionally, a protein, polypeptide and / or peptide of wild type and / or mutant.
[000134] The kit's immunodetection reagents can take any of a variety of forms, including those detectable markers that are associated with and / or bound to a given antibody. Detectable markers that are associated with and / or linked to a secondary linker are also contemplated. Examples of secondary ligands are those secondary antibodies or polymers that have binding affinity for the first antibody.
[000135] Additional suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has a binding affinity for the first antibody, together with a third antibody or a polymer that has a binding affinity for the second antibody, the third antibody being linked to a detectable marker. As noted above, a number of examples of labels are known in the art / or all of these labels can be appropriately employed within the scope of the present invention.
[000136] The kit may further comprise an appropriate aliquoted composition of the wild-type and / or mutant protein, polypeptide and / or polypeptide, whether labeled and / or unlabeled, that can be used to prepare a standard curve for a detection assay . The kits can contain labeled conjugates of antibody or polymer, in fully conjugated form, in the form of intermediates, and / or as separate fractions to be conjugated by the user of the kit. The kit components can be packaged in an aqueous medium and / or in lyophilized form.
[000137] The container means of the kits generally include at least one vial, test tube, flask, bottle, syringe and / or other container means, in which the antibody can be placed, and / or, preferably, appropriately aliquoted . The kits of the present invention also typically include a means for containing the antibody, antigen, and / or any other reagent containers in strict containment for commercial sale. Such containers may include blow and / or injection molded plastic containers in which the desired vials are retained.
[000138] The kits may further comprise one or more therapeutic agents for the treatment of cancer, such as an FOLR1 immunoconjugate and / or a chemotherapeutic agent.
[000139] The kit may further comprise a FOLR1 detection reagent used to measure expression of FOLR1 in an individual comprising a FOLR1 detection reagent, and instructions for use. In one embodiment, the FOLR1 detection reagent comprises a FOLR1 binding peptide, protein or molecular probe (i.e., nucleic acid). In another embodiment, the FOLR1 detection reagent is an anti-FOLR1 antibody. In another embodiment, the kit further comprises a secondary antibody that binds to the anti-FOLR1 antibody. In one embodiment, the specific anti-FOLR1 antibody is included in a concentration of 0.5 to 7.5 μg / mL, preferably 0.9 to 3.8 +/- 0.5 μg / mL. In another embodiment, the antibody is included at a concentration of 1.0 +/- 0.5 μg / mL, 1.5 +/- 0.5 ng / mL, 1.9 +/- 0.5 μg / mL , 2.5 +/- 0.5 μg / mL, 3.0 + / 0.5 μg / mL, 3.5 +/- 0.5 μg / mL, 3.8 +/- 0.5 μg / mL, or up to 4.2 μg / mL. In another embodiment, the antibody is included in a concentrated solution, with instructions for dilutions to reach a final concentration of 0.9 to 3.8 +/- 0.5 μg / mL. In another embodiment, the kit also comprises a detection reagent selected from the group consisting of: an enzyme, a fluorophore, a radioactive marker, a luminophore. In another modality, the detection reagent is selected from the group consisting of: biotin, digoxigenin, fluorescein, tritium and rhodamine.
[000140] The kit can also include instructions for detecting and scoring FOLR1 expression. The kit can also include control or reference samples. Non-limiting examples of control or reference samples include cell pellets or tissue culture cell lines derived from normal (normal control) or tumor (positive control) samples. Examples of cell lines include KB, NCI-H2110, Igrov-1, Ishikawa, Jeg-3, Skov-3, Hela, T47D, Caco2, SW620, OAW28, HCC827, Ovcar-8, and Ovcar-3, Ov-90, other tumor cell lines known to express FOLR1, and cell lines stably or transiently transfected with an expression vector that expresses FOLR1. Additional examples for positive control tissues can also be found in Examples 9-11. The kit may also comprise a staining guide that visually describes normal and positive reference samples for staining intensity and uniformity. These staining guides may have reference samples of normal lung, pancreas and / or salivary glands, and tumors stained to standard degrees (for example, ovarian, lung, renal, endometrial cancers, as well as those described in Examples and in Figures 23 -25). VIII. FOLR1 liaison officers
[000141] Any antibodies that bind to FOLR1 can be used in the detection methods of the present invention. Examples of therapeutically effective anti-FOLR1 antibodies can be found in US Order Publication 2012/0009181, which is incorporated herein by reference. The full length amino acid (aa) and nucleotide (nt) sequences for FOLR1 are known in the art and also provided herein as represented by SEQ IDNOs: 1 and 2, respectively. A specifically useful antibody for the detection of FOLR1 is the anti-huFOLR1 mouse monoclonal antibody. BN3.2 (Leica # NCL-L-FRalpha). An example of a therapeutically efficient anti-FOLR1 antibody is huMov19 (M9346A). The polypeptides of SEQ ID NOs: 3-5 comprise the huMov19 heavy chain variable domain (M9346A), and the light chain variable domain of version 1.00, the light chain variable domain of huMov19 version 1.60, respectively. The huMov19 antibody (M9346A) comprises: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO: 6); a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (SEQ ID NO: 7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO: 8); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO: 9); a light chain CDR2 comprising RASNLEA (SEQ ID NO: 10); and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO: 11). In certain embodiments, the huMov19 antibody (M9346A) is encoded by plasmids deposited with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, VA 20110 on April 7, 2010 under the terms of the Budapest Treaty and ATCC deposit. No. PTA-10772 and PTA-10773 or 10774. Examples of FOLR1 immunoconjugates useful in the therapeutic methods of the invention are provided below. IX.Imunoconjugates FOLR1
[000142] The present invention also includes methods for increasing the effectiveness of conjugates (also referred to herein as immunoconjugates), comprising anti-FOLR1 antibodies, antibody fragments, functional equivalents, improved antibodies and their aspects, as disclosed herein, linked or conjugated to a cytotoxin (drug), or prodrug. Examples of FOLR1 immunoconjugates can be found in US Order Publication 2012/0009181, which is incorporated herein by reference. A particularly efficient therapeutic immunoconjugate of the invention comprises the huMov19 antibody described above.
[000143] Appropriate drugs or pro-drugs are known in the art. In certain embodiments, drugs or prodrugs are cytotoxic agents. The cytotoxic agent used in the cytotoxic conjugate of the present invention can be any compound that results in cell death, or induces cell death, or in some way decreases cell viability, and includes, for example, maytansinoids and maytansinoid analogs, benzodiazepines, taxoids , CC-1065 and CC1065 analogue, duocarmicin, duocarmicin analogs, enedins, such as calicheamicins, dolastatin and dolastatin analogs, including auristatins derived from tomamycin, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vincristine , melphalan, mitomycin C, chlorambucil and morpholino doxorubicin. In certain embodiments, cytotoxic agents are maytansinoids and maytansinoid analogs.
[000144] The drug or prodrug can, for example, be linked to the anti-FOLR1 antibody, such as huMov19, or a fragment thereof via a disulfide bond. The binding molecule or cross-linking agent comprises a reactive chemical group that can react with the anti-FOLR1 antibody or fragment thereof. In certain embodiments, the chemical groups reactive for reaction with the cell binding agent are N-succinimidyl esters and N-sulfosuccinimidyl esters. In addition, the linker molecule comprises a reactive chemical group, in certain embodiments a dithiopyridyl group that can react with the drug to form a disulfide bond. In certain embodiments, linker molecules include, for example, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) (see, for example, Carlsson et al., Biochem J., 173: 723-737 (1978)) N-succinimidyl 4- (2-pyridyldithio) -butanoate (SPDB) (see, for example, US Patent 4,563,304), N-succinimidyl 4- (2-pyridyldithium) -2-sulfobutanoate (sulfo-SPDB) (see Publication US 20090274713), N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP), (see, for example, CAS registration number 341498-08-6), 2-iminothiolane or acetylsuccinic anhydride.
[000145] Conjugates of maytansinoid antibodies with non-cleavable bonds can also be prepared. Such cross-linking agents are described in the art (see ThermoScientific Pierce Crosslinking Technical Handbook and US Patent Application 2005/0169933) and include, among others, N-succinimidil4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidil-4- ( N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidocaproate), which is an SMCC "long chain" analogue (LC-SMCC), K-maleimidoundecanoic acid N-succinimidyl ester (KMUA), N- β-maleimidopropanoic acid succinimidyl (BMPs), y-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), ester-maleimidobenzoyl-N-hydroxysuccinimide (MB) (a-maleimidoacetoxy) -succinimide (AMAS), succinimidyl-6- (β-maleimidopropionamido) hexanoate (SMPH), N-succinimidil4- (p-maleimidophenyl) -butyrate (SMPB), eN- (p-maleimidophenyl) isocyanate (PMP ), N-succinimidyl-4- (iodoacetyl) - aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-suc cinimidil 3- (bromoacetamido) propionate (SBAP). In certain embodiments, the antibody is modified with cross-linking reagents, such as succinimidyl 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC), sulfo-SMCC, maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS or succinimidyl iodoacetate, as described in the literature, to introduce 110 reactive groups (Yoshitake et al., Eur. J. Biochem, 101: 395-399 (1979) ,. Hashida et al., J. Applied Biochem, 56-63 (1984 ) and Liu et al., Biochem, 18: 690-697 (1979)).
[000146] The present invention includes aspects characterized by the fact that about 2 to 8 molecules of drug ("drug load"), for example, maytansinoid, are linked to an anti-FOLR1 antibody or a fragment thereof, the effect The conjugate's antitumor agent is much more effective compared to a drug load of a smaller or greater number of drugs related to the same cell-binding agent. "Drug load", as used herein, refers to the number of drug molecules (for example, a maytansinoid) that can be attached to a cell binding agent (for example, an anti-FOLR1 antibody or fragment the same). In one aspect, the number of drug molecules that can be associated with a cell-binding agent can average from about 2 to about 8 (for example, 1.9, 2,0,2,1, 2,2,2,3,2,4,2,5,2,6,2,7,2,8, 2,9,3,0,3,1,3,2,3,3,3, 4,3,5,3,6,3,7,3,8,3,9,4,0,4,1,4,2,4,3,4,4, 4,5,4,6, 4,7,4,8,4,9,5,0,5,1,5,2,5,3,5,4,5,5,5,6,5,7,5,8,5, 9,6,0, 6,1,6,2,6,3,6,4,6,5,6,6,6,7,6,8,6,9,7,0,7,1, 7,2,7,3,7,4,7,5,7,6, 7,7, 7,8, 7,9, 8,0, 8,1). In certain embodiments, the drug is N2'-deacetyl- N2 '- (3-mercapto-1-oxopropyl) -maitansine (DM1) or N2'-deacetyl-N2' - (4-mercapto-4-methyl-1-oxopentyl ) maytansine (DM4). Thus, in a given embodiment, the huMov19 antibody is conjugated to DM1 or DM4. In another embodiment, the FR-1-21 antibody is conjugated to DM1 or DM4. In another embodiment, the FR-1-48 antibody is conjugated to DM1 or DM4. In another embodiment, the FR-1-49 antibody is conjugated to DM1 or DM4. In another embodiment, the FR-1- 57 antibody is conjugated to DM1 or DM4. In another embodiment, the FR-1-65 antibody is conjugated to DM1 or DM4. X. Correlation of FOLR1 expression and therapeutic efficacy
[000147] In certain embodiments, the invention provides a method for identifying individuals most likely to respond to anticancer therapies targeting FOLR1. The invention is based, in part, on the discovery that high levels of FOLR1 expression correlate with the effectiveness of anti-cancer drugs targeting FOLR1.
[000148] Evaluation of patient samples and correlation with in vivo efficacy using xenograft models demonstrate the power of expression analysis of selection of individuals most likely to respond to treatment. IHC provides a degree for expression of FOLR1 in tumor cells: 0 (no expression) to 3+ (very high levels of expression). In vivo data using xenograft models demonstrate that samples with FOLR1 expression level of 1, 2, 3, or 3+, preferably a 2, 3 or 3+ count, are more likely to respond to therapies anticancer drugs targeting FOLR-1 with clinically relevant doses of FOLR1 immunoconjugates (eg, 5 mg / kg xenograft dose of an FOLR1 immunoconjugate can approximate 185 mg / m2 in patients). Thus, identifying individuals who have a high degree of FOLR1 would help to identify people who can respond to a clinically relevant dose. As described in more detail below, sensitivity to FOLR1 therapies correlated with a FOLR1 grade of 2 or more, especially with grade 3. In addition, the expression of more uniform levels of FOLR1 provides a better match with the therapeutic benefit. Thus, homogeneous staining uniformity is preferable, but combinations of increased staining intensity with heterogeneous staining uniformity are also indicative of increased FOLR1 expression. For example, a score of more than 2 hetero is a criterion for patient selection for treatment with a therapeutic agent FOLR1.
[000149] FOLR1 expression analysis also identifies patients in which decreased levels of anti-cancer therapy directed at FOLR1 ("low dose therapy") can be effective in causing antitumor responses. As is known in the art, the compounds are generally administered in the lowest dose that achieves the desired therapeutic response. This is especially important for drugs that cause adverse, and often undesirable, clinical effects. The ability to recognize these individuals with high levels of FOLR1 expression allows for the minimization of the dosage of the drug directed to FOLR-1, thus reducing possible side effects, while maintaining therapeutic efficacy.
[000150] As shown here, the degree of FOLR1 expression of 2 hetero or greater correlates with the increased responsiveness to anti-FOLR1 immunoconjugates. In certain modalities, the increase in response capacity is cachexia, the increase in survival time, the extension in time for tumor progression, reduction in tumor mass, reduction in tumor size and / or an extension of time for tumor metastasis, tumor recurrence time, tumor response, complete response, partial response, stable disease, progressive disease, progression-free survival (PFS), or overall survival (OS). In certain modalities, the degree of FOLR1 expression of 2 hetero or greater correlates with the increase in PFS, DFS or OS.
[000151] The kits for use in the detection and correlation methods for reference / control samples can comprise control (positive and / or negative), or reference samples. Positive control or positive reference samples can be derived from tissue culture lines, normal tissue or tumor tissue. Positive and negative reference samples can be derived from cell lines, including SW620, T47D, IGROV-1, HeLa, KB, JEG-3, other tumor cell lines, and from lines stably or transiently transfected with an expression vector that encodes FOLR1. Samples of normal or tumor tissues and tissue culture lines can also be used as negative control reference samples. For additional examples, see Examples 9-11 and Numbers 23-25. XI. Pharmaceutical compositions and therapeutic methods
[000152] FOLR1 binding agents (including antibodies, immunoconjugates and polypeptides) are useful in a variety of applications, including, but not limited to, therapeutic treatment methods, such as cancer treatment. In certain embodiments, the agents are useful for inhibiting tumor growth, inducing differentiation, reducing the volume of the tumor, and / or reducing the tumorigenicity of a tumor. The methods of use can be in vitro, ex vivo, or in vivo methods. In certain embodiments, the binding agent or antibody or immunoconjugate FOLR1, or polypeptide is an antagonist of the human FOLR1 to which it binds.
[000153] In certain embodiments, the disease treated with the binding agent or antagonist FOLR1 (for example, an antibody or huMov19 immunoconjugate) is a cancer. In certain embodiments, cancer is characterized by tumors that express a folate receptor 1 to which the FOLR1-binding agent (for example, an antibody) binds.
[000154] The present invention provides methods of treating cancer, comprising administering a therapeutically effective amount of a FOLR1 binding agent to an individual (e.g., an individual in need of treatment). In certain modalities, cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, brain cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, and head and neck cancer. In certain modalities, cancer is cancer of the ovary. In certain modalities, cancer is lung cancer. In certain modalities, the individual is a human being.
[000155] The present invention further provides methods for inhibiting tumor growth using the antibodies or other agents described herein. In certain embodiments, the method for inhibiting tumor growth comprises contacting the cell with a FOLR1-binding agent (e.g., antibody) in vitro. For example, an immortalized cell line or a cancer cell line that expresses FOLR1 is grown in the medium that the antibody has been added to, or another agent to inhibit tumor growth. In some embodiments, tumor cells are isolated from a patient sample, such as a biopsy, pleural effusion, or the blood sample in the tissue and cultured in the middle of which a FOLR1 binding agent is added to inhibit the tumor growth.
[000156] In some embodiments, the method of inhibiting tumor growth comprises contacting the tumor or tumor cells with the FOLR1-binding agent (e.g., antibody) in vivo. In certain embodiments, contact of a tumor cell or tumor with a FOLR1 binding agent is performed in an animal model. For example, FOLR1 binding agents can be administered to xenografts that express one or more FOLR1s that have been cultured in immune compromised mice (for example, NOD / SCID mice) to inhibit tumor growth. In some embodiments, the FOLR1 binding agent is administered at the same time or shortly after the introduction of tumorigenic cells into the animal to prevent tumor growth. In some embodiments, the FOLR1 binding agent is administered as a therapeutic agent after the tumorigenic cells have grown to a specified size.
[000157] In certain embodiments, the method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of a FOLR1 binding agent. In certain modalities, the individual is a human being. In certain embodiments, the individual has a tumor or has had a tumor removed.
[000158] In certain embodiments, the tumor is a tumor selected from the group consisting of brain tumor, colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor , gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma and head and neck tumors. In certain embodiments, the tumor is an ovarian tumor.
[000159] In certain embodiments, the invention provides methods for inhibiting tumor growth using low doses of a FOLR1 binding agent. The term "low dose" as used herein refers to the therapeutically efficient dose of an FOLR1 binding agent that is less than usual, or the conventional dose required to produce the therapeutic effect.
[000160] Thus, in certain embodiments, the inventions provide methods of treating cancer using huMov antibodies and immunoconjugates19. In certain embodiments, the huMov19 immunoconjugate is huMov19-SPDB-DM4; huMov19-sulfo-SPP-DM1; huMov19-SPP-DM1, or huMov19-PEG4-Mal-DM4. In a given embodiment, the huMov19 immunoconjugate is huMov19-SPDB-DM4, which is also referred to as IMGN853.
[000161] In certain embodiments, formulations are prepared for storage and use by combining a purified antibody or agent of the present invention with a pharmaceutically acceptable carrier (e.g. carrier, excipient) (Remington, The Science and Practice of Pharmacy 20th edition Mack Publishing, 2000). Suitable pharmaceutically acceptable vehicles include, but are not limited to, non-toxic buffers such as phosphate, citrate and other organic acids, salts such as sodium chloride, antioxidants, including ascorbic acid and methionine; preservatives (eg, octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben; catechol, resorcinol, cyclohexanol, 3-pentanol , and m-cresol), low molecular weight polypeptides (for example, less than about 10 amino acid residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates, such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars like sucrose, mannitol, trehalose or sorbitol; counterion salt formers such as sodium; metal complexes (for example, Zn-protein complexes), and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG).
[000162] The pharmaceutical compositions of the present invention can be administered in any number of routes for local or systemic treatment. Administration can be topical (for example, to mucous membranes, including vaginal and rectal release) such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary (for example, by inhalation or by insufflation of dust or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), orally, or parenterally, including intravenous, intraarterial, subcutaneous administration, by injection or infusion intraperitoneal or intramuscular, or intracranial administration (for example, intrathecal or intraventricular).
[000163] An antibody or immunoconjugate of the invention can be combined in a pharmaceutical combination formulation, or dosage regimen as combination therapy, with a second compound having anti-cancer properties. The second compound of the pharmaceutical combination formulation or dosage regimen preferably has activities complementary to the combination's ADC so that they do not adversely affect each other. Pharmaceutical compositions comprising the FOLR1 binding agent and the second anti-cancer agent are also provided.
[000164] For the treatment of the disease, the appropriate dosage of an antibody or an agent of the present invention depends on the type of disease to be treated, the severity and course of the disease, the ability to respond to the disease, whether the antibody or agent it is administered for preventive or therapeutic purposes, prior therapy, the patient's medical history, and the like, all at the discretion of the attending physician. The antibody or agent can be administered once or over a series of treatments lasting from several days to several months, or until a cure is achieved, or a decrease in the disease state is achieved (for example, reduced tumor size). Optimal delivery schedules can be calculated from measurements of drug accumulation in the patient's body and can vary depending on the relative potency of a particular antibody, or agent. The attending physician can easily determine the ideal dosages, dosing methodologies and repetition rates. In certain modalities, the dosage is 0.01 μg to 100 mg per kg of body weight, and can be given once or more daily, weekly, monthly or annually. In certain embodiments, the antibody or other FOLR1 binding agent is administered once every two weeks or once every three weeks. In certain embodiments, the dosage of the antibody or other FOLR1 binding agent is from about 0.1 mg to about 20 mg per kg of body weight. The attending physician can estimate repeat dose rates based on measured residence times and drug concentrations in body fluids or tissues.
[000165] Combination therapy can provide "synergy" and be "synergistic", that is, the effect obtained when the active ingredients used together is greater than the sum of the effects that results from the use of the compounds separately. A synergistic effect can be achieved when the active ingredients are: (1) co-formulated and administered or released simultaneously in a single-dose combined formulation, (2) alternately or in parallel, as separate formulations, or (3) by some other regime. When released in alternation therapy, a synergistic effect can be achieved when the compounds are administered or released sequentially, for example, by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, that is, in series, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
[000166] Modalities of the present disclosure can be further defined with reference to the following non-limiting examples, which describe in detail the preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure. EXAMPLES
[000167] It is understood that the examples and modalities described herein are for illustrative purposes only and that various modifications or alterations in the light of them will be suggested to those skilled in the art and are to be included within the spirit and scope of this application.
[000168] Folate Receptor 1 (FOLR1) has been reported to be highly expressed in ovarian tumors and expressed in high to moderate levels in the brain, breast, bladder, endometrioid, lung, pancreas, and renal carcinomas. However, FOLR1 expression is limited in normal tissues, and includes kidney, lung, choroid plexus, pancreas, breast, thyroid, ovary, prostate and lung.
[000169] Methods that quantify FOLR1 are reported using fresh frozen tissue homogenates. Whole tissue homogenates cannot distinguish cytoplasmic expression from that associated with the membrane and freshly frozen samples are not amenable to clinical practice. However, samples fixed in paraffin embedded formalin (FFPE) can be archived for patients at the clinic. Example 1
[000170] Immunohistochemical staining of FOLR1 in cell samples - manual methods of cell pellets and tissues fixed in formalin and embedded in paraffin were used as test samples with the following reagents and staining conditions.

[000171] Ovarian tumor biopsies from patients fixed in formalin and embedded in paraffin (FFPE) and ovarian tumor xenograft were stained with murine anti-FOLR1 antibody clone BN3.2 (Leica, Cat # NCL-L-FRalpha ) and Coulter's muIgG1 control. After antigen recovery in pH 9.5 buffer, the slides were blocked with 2% horse serum plus avidin. The slides were washed in PBS, and incubated at room temperature for 60 minutes with anti-FOLR1 antibody or muIgG1 control antibody, followed by 30 minutes with biotinylated mouse anti-IgG and 40 minutes with avidin-biotin-peroxidase complex to detect bound secondary antibodies. Incubation for 5 minutes with DAB (3,3-diaminobenzidine tetrahydrochloride) resulted in a color signal. The slides were contrasted with hematoxylin.
[000172] FOLR1 staining intensity and distribution patterns were scored with respect to IgG staining control (non-specific). The intensity was scored on a scale of 0 to 3 (0 = no staining, 1 = weak, 2 = moderate and 3 = strong) and the distribution was scored as focal (<25% of stained cells), heterogeneous (25-75 % of stained cells) and homogeneous (> 75% of stained cells).
[000173] FFPE samples were obtained from tumor microarrays, as well as from human tissue blocks from seven different tumors, as described below. FFPE Test Samples


[000174] The FOLR1 test article, clone BN3.2 anti-murine FOLR1, was tested to determine specificity of binding to the huFOLR1 antigen. Using the reported IHC staining methods, sections of FFPE from 300-19 and 300-19 cell pellets transfected with huFOLR1 (300-10 / FOLR1) were stained and evaluated for FOLR1. The test article FOLR1 specifically stained 300-19 / FOLR1 + cells and returned without any staining in 300-19 cells (3 homo and negative, respectively). These results demonstrate that the BN3.2 clone specifically targets the huFOLR1 antigen. (Figure 1).
[000175] The BN3.2 antibody was also used to detect expression of FOLR1 in tissue samples. The immunoreactivity of each test and control article with tissues and cell pellets was determined by the consultant pathologist, Dr. David Dorfman. Control cell pellets were first evaluated followed by tissue samples. For each evaluated tissue, a description of the intensity of staining and uniformity of staining was evaluated. The degree of staining intensity and homogeneity scales is described below. The final grade reported for each tissue sample evaluated is the grade of the test article minus the score of the respective control article. The ABC level, for each sample, was calculated by comparing the degree of staining with calibrated control cell pellets.

[000176] Cell-bound antibody (ABC) values were determined for FOLR1 positive tumor cell lines (KB, IGROV1, JEG3 and OVCAR3), using anti-FOLR1-Phycoerythrin, BD Quantibrite Beads, and flow cytometry and were demonstrated have different ABC values. (Figure 2). Staining conditions were improved for FOLR1 so that cell pellets prepared from FOLR1 positive tumor cell lines showed different levels of IHC staining intensity. The KB cell pellet exhibited very strong homogeneous staining (3+) with high intensity, the IGROV1 cell pellet showed strong staining (3), the JEG3 cell pellet showed moderate heterogeneous staining (2-3), while the cell pellet OVCAR3 showed low intensity heterogeneous staining (1-2). The trends in FOLR1 staining intensity observed from cell pellets correspond to reported ABC values, where KB cells exhibited the highest ABC value 1,700,000, IGROV-1 cells exhibited the next highest ABC value 260,000 , JEG-3 exhibited a lower value of ABC or ABC 41,000, while OVCAR3 cells had the lowest ABC value of 4,000. The staining results and the respective ABC values are listed in the table below. ABC values and respective staining results for parahuFOLR1 in cell lines and respective cell pellets

[000177] Additional cell lines, including Capan-1, Jar, Hec-1-A, Hec-1-B, Ishikawa, NCI H292, BT474EEI, PA-1, OV-90, CaOv-4, CaOv-3, A2780 , Ovcar-5, Ovcar-4, HCT-15,786-O, NCIH838, NCI H 522, NCI H2110, NCI H1734, NCI H228 and FU.OV-3 were tested and considered FOLR1 positive but FOLR1 expression levels and sensitivity for the anti-FOLR1 immunoconjugate activity varied. For reference purposes, cell lines having consistent FOLR1 expression and sensitivity to anti-FOLR1 immunoconjugates are preferred.
[000178] Particularly important was that the IHC method was able to reliably detect the expression of FOLR1 in tissue samples from ovarian carcinoma and non-small cell lung adenocarcinoma (NSCLC). As shown in Figure 3, FOLR1 expression can be reliably detected in ovarian carcinoma and NSCLC samples with grade 2 hetero to 3 homo. ABC values for these samples ranged from approximately 41,000 for hetero grade 2 samples, to over 260,000 for homo grade 3 samples. As shown in Figure 4, high staining intensity and staining uniformity were also observed in ovarian carcinoma, lung adenocarcinoma and alveolar bronchioles carcinoma. In addition, the expression of FOLR1 in NSCLC samples (Figure 5) and in ovarian carcinoma (Figure 6) was still found to be predominantly localized to the membrane in tissue samples. The expression was detected in several samples of the same, as well as in different tumor samples. Interestingly, none of the samples of colorectal, breast, or small cell lung tumors had a degree greater than 2 hetero. Example 2
[000179] The in vivo efficacy of huMov19-PEG4Mal-DM4 and huMov19-SPDB-DM4 conjugates compared to similar untargeted conjugates in a KB xenograft model.
[000180] FOLR1 cleavable targeting huMov19-SPDB-DM4 compared to huC242-SPDB-DM4 untargeted, and non-cleavable huMov19-PEG4-Mal-DM4 conjugate, compared to huC242-PEG4Mal-DM4 were tested using a established xenotransplantation model of KB cells (very high expression of FOLR1, 3+ homozygous by manual IHC) implanted subcutaneously in SCID mice. Mice were randomized by body weight into treatment groups and treated alone (SPDB conjugates) on day 3 after cell inoculation, or three times a week, on days 3, 10 and 17 after cell inoculation with 5 and 10 mg / kg of a conjugate, respectively. The average tumor volume of the different treatment groups is shown in Figure 7. Treatments with huMov19-SPDB-DM4, or huMov19-PEG4Mal-DM4 resulted in a decrease in the volume of the average tumor compared to the PBS control, while treatments with or of the respective non-targeted conjugate did not produce any significant effect. Example 3
[000181] Dose-response antitumor activity of IMGN853 treatment in human ovarian carcinoma xenografts OVCAR-3.
[000182] The antitumor effect of IMGN853 was evaluated in an established subcutaneous xenograft model of ovarian carcinoma. SCID mice were inoculated with OVCAR-3 ovarian carcinoma cells (1 x 107 cells / animal) injected subcutaneously in the right flank. When the tumors reached about 100 mm3 in size (21 days after inoculation of the tumor cell), the mice were randomly divided into four groups (6 animals per group). The mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg / kg. A group of control animals received a single intravenous injection of PBS. Tumor growth was monitored by measuring the size of the tumor twice a week. The size of the tumor was calculated using the formula: length x width x height x 1A
[000183] IMGN853 was highly active against OVCAR-3 tumors (grade IHC of 3 homozygous using manual IHC methods), in terms of inhibiting tumor growth (T / C = 0%) at both doses 2.5 and 5 , 0 mg / kg (Figure 8). There were complete tumor regressions (CR) in 6/6 mice treated with IMGN853 at 5.0 mg / kg. There were partial tumor regressions (PR) in 6/6 mice and CR in 4/6 mice treated with IMGN853 at the dose level of 2.5 mg / kg. IMGN853 was active at a dose of 1.2 mg / kg, resulting in an 18% T / C, with 2/6 PR and 1/6 CR. According to NCI standards, T / C values ranging from 10% to 42% are considered to be active, T / C less than 10% are considered to be highly active. Example 4
[000184] Dose-response antitumor activity of IMGN853 treatment in IGROV-1 human ovarian carcinoma xenografts.
[000185] The antitumor effect of IMGN853 was evaluated in an established subcutaneous xenograft model of ovarian carcinoma. SCID mice were inoculated with IGROV-1 ovarian carcinoma cells (1 x 107 cells / animal) injected subcutaneously on the right flank. When the tumors reached about 100 mm3 in size (7 days after inoculation of the tumor cell), the mice were randomly divided into four groups (6 animals per group). The mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg / kg. A group of control animals received a single intravenous injection of PBS. Tumor growth was monitored by measuring the size of the tumor twice a week. The size of the tumor was calculated using the formula: length x width x height x 1A
[000186] IMGN853 was highly active against IGROV-1 tumors (grade IHC of 3 homozygous by manual methods) at dose levels of 2.5 and 5.0 mg / kg, resulting in T / C values of 5% for both dose levels (Figure 9). There were partial regressions of tumors, in 5/6 and 6/6 mice in the groups of 2.5 and 5.0 mg / kg, respectively. IMGN853 was inactive at a dose of 1.2 mg / kg (T / C = 47%). Example 5
[000187] Dose-response antitumor activity of IMGN853 treatment in human ovarian carcinoma xenografts OV-90.
[000188] The antitumor effect of IMGN853 was evaluated in an established subcutaneous xenograft model of ovarian carcinoma. SCID mice were inoculated with ovarian carcinoma cells OV-90 (1 x 107 cells / animal) injected subcutaneously in the right flank. When the tumors reached about 100 mm3 in size (13 days after tumor cell inoculation), the mice were randomly divided into four groups (6 animals per group). The mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg / kg. A group of control animals received a single intravenous injection of PBS. A group of control animals received PBS administered intravenously at the same time. Tumor growth was monitored by measuring the size of the tumor twice a week. The size of the tumor was calculated using the formula: length x width x height x 1A
[000189] IMGN853 was active against OV-90 tumors (grade IHC of 3 hetero-homo by manual methods) at dose levels of 2.5 and 5.0 mg / kg, resulting in T / C values of 36 and 18% , respectively (Figure 10). Two animals showed partial tumor regression in the 5.0 mg / kg group; there were no other tumor regressions in either treatment group. IMGN853 was inactive at a dose of 1.2 mg / kg (T / C = 77%). Example 6
[000190] Dose-response antitumor activity of IMGN853 treatment in SKOV-3 human ovarian carcinoma xenografts.
[000191] The anti-tumor effect of IMGN853 was evaluated in an established subcutaneous xenograft model of ovarian carcinoma. SCID mice were inoculated with SKOV-3 ovarian carcinoma cells (1 x 107 cells / animal) injected subcutaneously on the right flank. When the tumors reached about 100 mm3 in size (26 days after inoculation of the tumor cell), the mice were randomly divided into four groups (6 animals per group). The mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg / kg. A group of control animals received a single intravenous injection of PBS. Tumor growth was monitored by measuring the size of the tumor twice a week. The size of the tumor was calculated using the formula: length x width x height x 1A
[000192] IMGN853 was inactive against SKOV-3 tumors (IHC grade 1-3 focal by manual methods) at all doses, with growth of tumors treated with IMGN853 in parallel to the PBS control group (Figure 11). No data analysis was performed and the study was stopped early based on the inactivity of IMGN853 in this model. Example 7
[000193] Dose-response antitumor activity of IMGN853 treatment in KB human cervical adenocarcinoma xenografts.
[000194] The antitumor effect of IMGN853 was evaluated in an established subcutaneous xenograft model of cervical adenocarcinoma. SCID mice were inoculated with KB cervical adenocarcinoma cells (1 x 107 cells / animal) injected subcutaneously in the right flank. When the tumors reached about 100 mm3 in size (7 days after inoculation of the tumor cell), the mice were randomly divided into four groups (6 animals per group). The mice were treated with a single intravenous injection of IMGN853 at 1.0, 2.5 or 5.0 mg / kg. A group of control animals received a single intravenous injection of PBS. Tumor growth was monitored by measuring the size of the tumor twice a week. The size of the tumor was calculated using the formula: length x width x height x 1A
[000195] IMGN853 was highly active against KB tumors in terms of inhibiting tumor growth (T / C = 0%), at both doses of 2.5 and 5.0 mg / kg (Figure 12). Six of the six mice in the 5.0 mg / kg treatment group and five of the six mice with 2.5 mg / kg had CRs and remained tumor-free at the end of the study (day 120). The 1.0 mg / kg dose was active, resulting in a T / C of 37%, but there were no partial or complete regressions. Example 8
[000196] Immunohistochemical staining in FOLR1 samples fixed in formalin and embedded in paraffin (FFPE) - automated methods.
[000197] The IHC stain assay uses IVD class I reagents, including the FOLR1 Novocastra antibody (Novocastra / Leica Cat # NCI-L-FRalpha, clone BN3.2), as the test article and Leica Bond RX automatic stain. Limit test or control article was detected by incubation with the Leica Bond Refine detection system, which includes a post primary reagent (rabbit anti-mouse IgG) followed by a polymer reagent (goat anti-rabbit polymer) and tetrachlorhydrate chromogen. of 3,3-diaminobenzidine (DAB). FFPE samples were stained with the specified concentration (s) of primary antibody (prepared by diluting FOLR1 in Leica diluent) as described below.
FFPE Assay Method using Leica Bond RX


[000198] All stained samples were evaluated and scored. Control samples were evaluated first followed by test samples (entire sections and individual nuclei of the TMAs). For each tumor tissue or cell pellet evaluated, a description of the staining intensity and respective proportion of stained tumor cells was reported. The color associated with the membrane was recorded for each sample. When duplicate grades were assessed for a patient, only the highest grade was included in the analysis. If the grade described only cytoplasmic staining, then the final result was reported as zero (0). The intensity and uniformity were given to each of the samples, as described in the table outlined below. Patterns of staining intensity and distribution were scored in relation to IgG staining control (nonspecific). The intensity was scored on a scale of 0 to 3 (0 = no staining, 1 = weak 2 = moderate and 3 = strong) and the distribution was scored as focal (<25% of stained cells), heterogeneous (25-75% of stained cells) and homogeneous (> 75% of stained cells). In normal tissue, only the defined substructures were evaluated to calculate the intensity and proportion.IHC Degree System Consisting of Intensity and Uniformity Scales


[000199] FFPE tumor samples were derived from tumor microarrays, as well as from tissue blocks from seven different human tumors, as outlined below.FFPE Test Samples: TMAs
FFPE Test Samples: Entire Sections

[000200] The cells (tumor cells or transfected cells) were fixed in formalin and embedded in paraffin (FFPE). FFPE from cell pellet samples exhibited different ranges of FOLR1 expression by flow cytometry and normal human tissues were used in this study to characterize positive and negative controls and to analyze specificity. Cell pellets that exhibit different levels of FOLR1 and their results are shown below. There is a weak correlation between the degrees of staining and the respective levels of expression of FOLR1 (antibodies bound by cell, ABC, determined by calibrated flow cytometry) in cell pellets. For example, a straight grade of 13 is given for SW620 and IGROV-1 showing values of ABC 40,098 and 565,481, respectively. In addition, HeLa cells showing an ABC value of 1.5 million resulted in a 23 degree hetero while 300.19 / FR1 exhibiting ABC of 830.003 returned to the highest degree of 3 homo. 1.9 mg / mL test
a The reported ABC value is an average of cell-bound antibodies in the cell population, and was determined as follows: a 1.0 x 10-8 M concentration of anti-FOLR1-PE (1: 1) was used to determine the ABC values in the respective cell lines using flow cytometry and Quantibrite ™ Beads (BD Biosciences) methods.
[000201] Flow cytometry histograms represent the distribution of cells versus the number of anti-FOLR1 bound per cell (FOLR1 expression level). Both histograms and respective IHC staining results indicate that each of these cell lines contains a heterogeneous population of cells that have a wide range of FOLR1 expression. The exception is the cell line 300.19 / FR1 showing a histogram of uniform flow cytometry and IHC degree of staining. These data suggest that cell lines expressing a more uniform level of FOLR1 may provide a better correlation between ABC values and the respective degrees of staining. Although the assay demonstrated positive staining in all positive FOLR1 control cell pellets, there is a weak correlation between the degrees of staining and the respective FOLR1 expression levels of most of these cell pellets. Therefore, cell pellets in this group could not be identified as being high, medium and low expression controls. Representative photographs and histograms that describe FOLR1 expression in cell lines by IHC and flow cytometry are shown in Figure 13.
[000202] To determine test conditions, a series of test and control article dilutions were tested to select conditions that have an appropriate level of sensitivity. The experiments were carried out on a panel of FFPE samples including cell pellets positive for FOLR1 and a TMA consisting of normal positive and negative tissues of FOLR1 (adrenal (cortex / medulla), breast (ducts and lobes / connective tissue), fallopian tube ( surface epithelium / muscle wall), kidney (tubules / glomeruli), lung (pneumocytes type I / II / interalveolar connective tissues), pancreas (ducts / islets of Langherans), salivary gland (ducts / stroma), skin (eccrine glands / epidermis), stomach (surface / submucosa epithelium)) and entire sections of tumor tissues (10 ovarian tumor samples and 10 lung tumor samples). Each sample was stained with a serial dilution of the test article (concentrations of 0.25, 0.5, 0.9, 1.9, 3.8, and 7.5 μg / mL) or the control article of concentration of 1.9 μg / mL or 3.8 μg / mL. The relative staining intensities of each dilution were compared for each sample to identify the optimal dilution. The criteria for optimal dilution were a dilution that 1) did not cause background staining in samples stained with the isotype control 2) did not cause any staining in the negative control tissue stained with the test article and 3) a distinction between different levels of expression associated with the FOLR1 membrane among the test samples representing the indication of interest (ovarian tumor, endometrial tumor, NSCLC tumor, in the kidneys and FFPE tumor tissues). Of the five dilutions of the test article evaluated, the concentration of 1.9 μg / mL showed the best dynamic range in the staining results using Leica Bond RX automated protocols (Bake and Dewax Protocol, HIER using ER2 for 20 minutes of protocol and the staining protocol IHC F-With Extra Rinses). Example 9
[000203] Identification and characterization of controls that characterize the dynamic range of automated test staining methods.
[000204] Quality controls: Normal human salivary gland, lung and pancreas, were identified as positive control tissues to be used in each assay to verify that the staining procedure performs as expected. Normal human esophagus has been identified as a negative control. These controls were characterized as follows: in order to establish controls that cover the dynamic range of the assay, a tissue microarray (TMA), which consists of several samples of normal FOLR1 tissues that are expected to exhibit the expected dynamic range to expose the The dynamic range of the assay was used as a verification control assay during the validation and improvement phases. Four normal tissues with structures identified in this TMA were identified as appropriate test controls as follows: respiratory epithelium of the normal human lung (grade 2 homo); ducts of the normal human pancreas (grade 3 apical homo); intercalary ducts of normal human salivary gland (grade 1-2 hetero) and normal human esophagus (grade 0). Over a total of four test runs, the appropriate control test identified from this TMA gave identical results. These results indicate that the selected controls give consistent results and cover the dynamic range of the assay. Structures in Normal Tissues identified as Controls that Cover the Dynamic Range of the Assay
Apical staining is defined as polarized non-uniform membrane staining Example 10
[000205] Analysis of the automated decolorization method.
[000206] The intended use of this assay is to specifically detect FOLR1 in a reproducible manner and with the appropriate sensitivity to differentiate different levels and varying the uniformity of the membrane associated with the expression of FOLR1 (optimal dynamic range) in FFPE tumor tissues of ovary, endometrium, NSCLC and kidney. Therefore, specificity, reproducibility and sensitivity were considered as performance criteria.
[000207] The specificity and sensitivity of the study assay were assessed by comparing the staining of normal tissues with the study assay with previously reported results. Staining results from this study were compared with the corresponding staining results by Scorer et al. 2010 (A Full Immunohistochemical Evaluation of a Novel Monoclonal Antibody to Folate Receptor - alpha. The Novocastra Journal of Histopathology, REAGENTS: 2010 (3): 8-12, describing the same BN3.2 antibody clone) with normal FFPE and study tissue Tissue Cross Reactivity (TCR), using IMGN853 (antibody huMov19 (M9346A)) in fresh normal frozen tissue (ImmunoGen Report IMH28-003). The comparison of the staining results for each method indicates that the three assays showed staining profiles of normal tissues, generally similar with different relative sensitivities, with the Scorer assay being less sensitive, the research assay (IMH28-011) having a sensitivity intermediate, and the TCR study method being the most sensitive. Some structures showed positive staining in the two most sensitive methods (study trial and TCR trial) only. There were no examples of positive staining in the less sensitive assay used by Scorer that were also not positive in the study trial and TCR method. These results demonstrate that the specificity and sensitivity of the study assay are appropriate for the evaluation of FOLR1 expression in normal tissues.
[000208] The specificity and sensitivity of the study trial were further characterized by staining and evaluation of a tumor TMA panel consisting of ovarian, endometrial, NSCLC and kidney tumors (a representative sample of the set is intended for clinical use of the trial ). Positive staining was consistently localized to the tumor tissue with adjacent normal tissue components, including stroma, blood vessels, normal lymphocytes, and negative or positive stained organ tissues, as expected. For each subtype of ovarian carcinoma or NSCLC, the distribution of the degrees of staining between TMAs from different suppliers showed a similar distribution of degrees suggesting that this method is not sensitive to various conditions of fixation and processing. As the distribution patterns were similar between the TMAs, the data from the different arrangements were combined and the counts were categorized. A summary of these scores for tumor subtypes that contained 20 or more samples per subtype is listed in the following tables. As summarized in these tables, a dynamic range of degrees is noted for each tumor type, and indicates that this assay demonstrates the appropriate sensitivity for distinguishing different levels and varying the membrane uniformity associated with FOLR1 expression in FFPE tissues from ovarian tumors, endometrial, NSCLC, and kidney. Representative photos of serous ovary, endomyroid ovary, NSCLC, endometrial carcinoma and clear cell renal carcinoma are provided in Figures 14-18. Additional representative photos useful, for example, in a staining guide or a diagnostic kit, are shown in Figures 23-25. These studies indicate that the assay is specific and has the appropriate sensitivity for use in diagnosis or accompanying diagnostic reagent.Summary of Degrees of Staining for SubtypesPredominant Ovarian Tumors
Focal staining patterns were excludedSummary of Degrees of Staining for NSCLC Tumors
i) Focal staining patterns were excluded All samples of adenocarcinoma were included except specimens of specified bronchioloalveolar carcinomaSummary of Staining Degrees for Endometrial Adenocarcinoma and Clear Kidney Cell Tumors
Focal staining patterns were excluded
[000209] The accuracy of the study assay was investigated by evaluating intra-assay and inter-assay reproducibility using three FFPE samples of ovarian tissue tumors, NSCLC, or kidney tumor, where each sample exhibited high, medium, or low degree. For intra-assay reproducibility, nine slides, each containing a lung, ovarian and kidney tumor cut, were placed at nine random locations on the Leica Bond RX. For inter-assay reproducibility, three slides containing sections from the same sample were stained on three different days. All slides from both the intra-assay and inter-assay reproducibility experiments were evaluated and showed equivalent staining results for each respective sample: lung tumor (high: 3 homo), ovarian tumor (average: 2 hetero) and tumor renal (low: 1-2 hetero). These data demonstrate reproducibility in all types of tissues with low, medium and high levels of expression. Example 11
[000210] A degree of FOLR1 expression of> 2 heterogeneous by IHC is a patient selection criterion for treatment with IMGN853.
[000211] FOLR1 expression levels in tumor cell lines were determined using an antibody-PE conjugate (FR1-24-PE) and the QuantiBRITE system. Three ovarian carcinoma cell lines (Igrov-1, Skov-3 and Ovcar-3), a Jeg-3 choriocarcinoma cell line and a KB cervical carcinoma cell line were included in the study. In order to obtain reliable ABC values, binding experiments on an antibody-PE conjugate must be performed at a saturation concentration (concentration at which all available binding sites are occupied by the conjugate). To determine this concentration for the FR1-24-PE conjugate, experiments were carried out to link a panel of positive FOLR1 cell lines with various FOLR1 expressions. The cells were incubated with a wide range of concentrations of the FR1-24-PE conjugate for two hours on ice, washed with FACS buffer (PBS with 1% BSA), fixed with 1% formaldehyde in PBS and analyzed on a cytometer. FACSCalibur flow. At a concentration of 1x10-8 M, the conjugate saturated all cell surface binding sites in all tested cell lines Igrov-1, 3-Jeg, Skov-3, Ovcar-3, and KB. In subsequent ABC binding experiments, the FR1-24-PE conjugate was used at a concentration of 1x10-8 M. Each sample was analyzed in triplicates; several independent experiments were carried out on each cell line. The highest expression was found in KB cells with the approximate ABC value of 4,000,000 ± 300,000, followed by the Igrov-1 and Jeg-3 cell lines with the ABC values of 400,000 ± 85,000 and 150,000 ± 75,000, respectively. Two cell lines, Skov-3 and Ovcar-3, had low expression of FOLR1, 20,000 ± 10,000 and 7,000 ± 4,000 ABC, respectively. There was a significant variation in ABC values from experiment to experiment for Jeg-3 cells, where the ABC values varied between 40,000 and 300,000. This variability possibly reflects some more biological properties of the cell line differently from the assay variability, since the ABC values obtained for the other analyzed cell lines were much less variable (see table below).
i) SD - standard deviation; n - number of independent experiments
[000212] ABC values were determined by a FACS-based assay with the FR1-24 antibody labeled with PE and the QuantiBRITE system. The mean ± standard deviation (SD) was calculated for independent experiments.
[000213] The potency and specificity of IMGN853 were analyzed against positive FOLR1 cell lines with a wide range of FOLR1 expression (ABC values of cell lines are provided above). In addition, Namalwa and SW2 negative FOLR1 cell lines were included in the experiments. IMGN853 was highly cytotoxic against cells with high expression of FOLR1 KB (4,000,000 ± 300,000 ABC), Igrov-1 (400,000 ± 85,000 ABC) and Jeg-3 (150,000 ± 75,000 ABC), with IC50 values of 0.10 ± 0.01 nM, 0.50 ± 0.07 nM and 1.00 ± 0.05 nM, respectively. The cell killing activity against all three cell lines was dependent on FOLR1, as an excess of unmodified huMov19 (M9346A) antibody (0.5 μM) markedly decreased the potency of the conjugate with typical nonspecific levels (from 10 to 20 times). IMGN853 was only marginally active against Skov-3 and Ovcar-3 cells that have low FOLR1 expression (20,000 ± 10,000 and 7,000 ± 4,000 ABC, respectively), and against FOLR1 Namalwa and SW2 negative cells, with higher IC50 values at 2 nM. The cytotoxic activity of IMGN853 against these cell lines was low and not dependent on FOLR1, since blocking with huMov19 (M9346A) did not affect the activity. See Figures 19 and 20.
[000214] FFPE samples prepared from mouse tumor xenograft models were evaluated for FOLR1 positivity using the improved and validated assay described above. The absence of staining was observed in tumor cells from any xenograft samples stained with the control article. The mouse xenograft FFPE tissue resulting from the following cell lines showed the following staining patterns: Igrov-1, KB, and NCI-H2110 showed homogeneous staining patterns, with intensity level 3; Ishikawa and Ovcar 3 presented heterogeneous staining patterns, with an intensity level of 3; LXFA737 showed homogeneous staining patterns with intensity level 2; OV-90 showed heterogeneous patterns with intensity level 2 and SKOV3 was negative. Representative images of tumor xenografts are provided in Figures 21 and 22.


[000215] A staining threshold (> 2 heterogeneous), requires a minimum level of expression (staining intensity) and minimum staining distribution (percentage of tumor cells that express FOLR1). Pre-clinical data provide justification for this limit in ovarian carcinoma. Mouse xenograft tumor samples with grades IHC> 2 exhibited heterogeneous sensitivity to exposure to IMGN853 in vivo. FFPE samples prepared from mouse ovarian tumor xenograft models were evaluated for FOLR1 positivity using the improved and validated assay described above. Two models of ovarian carcinoma xenograft OVCAR-3 and IGROV-1 showed a heterogeneous or homogeneous staining pattern, with intensity level 3. The xenograft model derived from OV-90 ovarian carcinoma cells showed a pattern of heterogeneous staining, with intensity level 2, the Skov 3 ovarian carcinoma model was negative for FOLR1. IMGN853 was highly active in the two ovarian models with FOLR1 intensity level 3 and active in the OV-90 model with FOLR1 intensity level 2. No activity was observed in the SKOV-3 model. Xenograft models have also been evaluated for other indications of the disease, including lung, endometrial tumors, and cervical tumors, and although correlations have been detected between FOLR1 activity and degree of staining, additional samples should be tested. The sensitivity of deovarian tumor xenograft models for IMGN853 versus the level of expression of FOLR1
The sensitivity of other IMGN853 tumor xenograft models versus the FOLR1 expression level

[000216] All publications, patents, patent applications, 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, website, or database access / sequence number were specifically and individually indicated to be incorporated as a reference.

权利要求:
Claims (29)
[0001]
1. Use of a Folate 1 antireceptor immunoconjugate (FOLR1), characterized by the fact that it is for the manufacture of a drug for cancer therapy in an individual, in which a cancer sample from an individual exhibits increased expression of FOLR1 using a detection method that uses an anti-FOLR1 detection antibody or an antigen-binding fragment thereof to distinguish between the intensity of staining and uniformity of staining in a cancer sample expressing FOLR1, compared to the intensity of staining and the uniformity of staining in one or more reference sample (s), in which the anti-FOLR1 immunoconjugate has the formula (A) - (L) - (C), in which: (A) comprises an anti-FOLR1 antibody or binding fragment the antigen thereof comprising: a heavy chain (HC) CDR1 comprising GYFMN (SEQ ID NO: 6); a CDR2 of HC comprising RIHPYDGDTFYNQKFQG (SEQ ID NO: 7); and an HC CDR3 comprising YDGSRAMDY (SEQ ID NO: 8), and a light chain CDR1 (LC) comprising KASQSVSFAGTSLMH (SEQ ID NO: 9); an LC CDR2 comprising RASNLEA (SEQ ID NO: 10); and an LC CDR3 comprising QQSREYPYT (SEQ ID NO: 11), (L) comprises a cleavable linker, (C) comprises a maytansinoid or analog thereof, and where (L) links (A) to (C).
[0002]
2. Use according to claim 1, characterized in that the detection antibody or antigen-binding fragment thereof comprises an HC CDR1 comprising GYFMN (SEQ ID NO: 6); a CDR2 of HC comprising RIHPYDGDTFYNQKFQG (SEQ ID NO: 7); and an HC CDR3 comprising YDGSRAMDY (SEQ ID NO: 8); and an LC CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO: 9); an LC CDR2 comprising RASNLEA (SEQ ID NO: 10); and an LC CDR3 comprising QQSREYPYT (SEQ ID NO: 11).
[0003]
Use according to claim 1 or 2, characterized in that the detection antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 4 or 5.
[0004]
Use according to claim 3, characterized in that the detection antibody or antigen-binding fragment thereof comprises: (i) a heavy chain comprising the same amino acid sequence as the heavy chain amino acid sequence encoded by the plasmid deposited with the American Type Culture Collection (ATCC) as PTA-10772, and (ii) a light chain comprising the same amino acid sequence as the amino acid sequence of the light chain encoded by the plasmid deposited at the ATCC as PTA-10773 or PTA-10774.
[0005]
Use according to any one of claims 1 to 4, characterized in that the detection antibody or antigen-binding fragment thereof further comprises a detection reagent.
[0006]
6. Use according to claim 5, characterized by the fact that the detection reagent is selected from the group consisting of: an enzyme, a fluorophore, a radioactive marker, a luminophore, biotin, digoxigenin, fluorescein, tritium, and rhodamine.
[0007]
7. Use according to any one of claims 1 to 6, characterized by the fact that the detection method produces a range of staining intensity for samples having low expression of FOLR1, intermediate expression of FOLR1 or high expression of FOLR1.
[0008]
8. Use according to any one of claims 1 to 7, characterized by the fact that the detection method is immunohistochemistry (IHC) and an FOLR1 score for the cancer sample is determined from the IHC.
[0009]
9.Use, according to claim 8, characterized by the fact that the FOLR1 score is at least equal to 2.
[0010]
10.Use, according to claim 8, characterized by the fact that at least 25% of the cells in the cancer sample have an FOLR1 score of at least 2.
[0011]
11.Use, according to claim 8, characterized by the fact that 25 to 75% of the cells in the cancer sample have an FOLR1 score of at least 2.
[0012]
12.Use, according to claim 8, characterized by the fact that more than 75% of the cells in the cancer sample have an FOLR1 score of at least 2.
[0013]
13.Use, according to claim 8, characterized by the fact that the FOLR1 score is at least equal to 3.
[0014]
14.Use, according to claim 8, characterized by the fact that at least 25% of the cells in the cancer sample have an FOLR1 score of at least 3.
[0015]
15.Use, according to claim 8, characterized by the fact that 25 to 75% of the cells in the cancer sample have an FOLR1 score of at least 3.
[0016]
16.Use, according to claim 8, characterized by the fact that more than 75% of the cells in the cancer sample have an FOLR1 score of at least 3.
[0017]
17. Use, according to claim 8, characterized by the fact that the cancer sample has a uniform color for the expression of FOLR1 which is heterogeneous.
[0018]
18. Use, according to claim 8, characterized by the fact that the cancer sample has a uniform color for the expression of FOLR1 which is homogeneous.
[0019]
19. Use according to any of claims 8 to 18, characterized by the fact that the IHC is performed manually.
[0020]
20. Use according to any of claims 8 to 19, characterized by the fact that the IHC is performed using an automated system.
[0021]
21. Use according to any one of claims 1 to 20, characterized by the fact that the cancer is selected from the group consisting of uterine cancer, non-small cell lung cancer, peritoneum cancer and ovarian cancer.
[0022]
22. Use according to any one of claims 1 to 20, characterized by the fact that the cancer is endometrial cancer.
[0023]
23. Use according to any one of claims 1 to 22, characterized in that the cleavable linker is selected from the group consisting of: N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP) or N-succinimidyl 4 - (2-pyridyldithio) -2-sulfopentanoate (sulfo-SPP), N-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB), and N-succinimidyl 4- (2- pyridyldithio) -2-sulfobutanoate (sulfo-SPDB) ).
[0024]
24. Use according to claim 23, characterized by the fact that the cleavable linker is N-succinimidyl 4- (2-pyridyldithio) -2-sulfobutanoate (sulfo-SPDB).
[0025]
25. Use according to any one of claims 1 to 24, characterized in that the maytansinoid or analog thereof is N2'-deacetyl-N2 '- (3-mercapto-1-oxopropyl) -maitansine (DM1) or N2'-deacetyl-N2 '- (4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4).
[0026]
26. Use according to any one of claims 1 to 25, characterized in that (A) comprises an anti-FOLR1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 4 or 5.
[0027]
27. Use according to claim 26, characterized in that (A) comprises an anti-FOLR1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO : 3 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 5.
[0028]
28. Use according to claim 26, characterized in that (A) comprises an anti-FOLR1 antibody comprising: (i) a heavy chain comprising the same amino acid sequence as the amino acid sequence of the heavy chain encoded by the plasmid deposited in the ATCC as PTA-10772, and (ii) a light chain comprising the same amino acid sequence as the amino acid sequence of the light chain encoded by the plasmid deposited in the ATCC as PTA-10773 or PTA-10774.
[0029]
29. Use according to claim 28, characterized by the fact that (L) comprises the cleavable linker N-succinimidyl 4- (2-pyridyldithio) -2-sulfobutanoate (sulfo-SPDB), and (C) comprises maytansinoid N2 '-deacetyl-N2' - (4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4).

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

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

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2019-03-19| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-18| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-04-07| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-01-05| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-16| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 30/03/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161471007P| true| 2011-04-01|2011-04-01|

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US61/471,007|2011-04-01|

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PCT/US2012/031544|WO2012135675A2|2011-04-01|2012-03-30|Methods for increasing efficacy of folr1 cancer therapy|

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