NZ711375B2 - Methods for increasing efficacy of folr1 cancer therapy - Google Patents

Methods for increasing efficacy of folr1 cancer therapy Download PDF

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Publication number
NZ711375B2
NZ711375B2 NZ711375A NZ71137512A NZ711375B2 NZ 711375 B2 NZ711375 B2 NZ 711375B2 NZ 711375 A NZ711375 A NZ 711375A NZ 71137512 A NZ71137512 A NZ 71137512A NZ 711375 B2 NZ711375 B2 NZ 711375B2
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New Zealand
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folrl
tumor
antibody
staining
acid sequence
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NZ711375A
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NZ711375A (en
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Christina N Carrigan
Sharron Ladd
Gillian Payne
Kathleen R Whiteman
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Immunogen Inc
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Publication of NZ711375B2 publication Critical patent/NZ711375B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6857—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from lung cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells

Abstract

Disclosed is a method of identifying a tumour as sensitive to treatment with an anti-Folate Receptor 1 (FOLR1) antibody.

Description

METHODS FOR SING EFFICACY OF FOLR1 CANCER THERAPY CROSS-REFERENCE TO RELATED ATIONS This application is a divisional of New Zealand Application No. 615742, filed on March 2012, and is related to International Patent Application No. , filed on 30 March 2012 and claims priority from U.S. Provisional Application No. 61/471,007, filed on 1 April 2011; each of which is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION Field of the Invention The field of invention generally relates to increasing the efficacy of the treatment of cancers characterized by the overexpression of human folate receptor 1 (FOLR1).
More ically, the ion concerns more effective treatment of patients susceptible to or diagnosed with cancer, in which the tumor cells press FOLR1 as determined by a gene expression assay, with a FOLR1 nist, e.g., a FOLR1 immunoconjugate.
Background Art Cancer is one of the leading causes of death in the developed world, with over one million people diagnosed with cancer and 500,000 deaths per 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 me. There are more than 200 different types of cancer, four of which—breast, lung, colorectal, and prostate—account for over half of all new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26).
Folate or 1 (FOLR1), also known as Folate Receptor-alpha, or Folate Binding Protein, is an N-glycosylated protein expressed on the plasma membrane of cells. FOLR1 has a high affinity for folic acid and for l reduced folic acid derivatives. FOLR1 mediates delivery of the physiological folate, 5- methyltetrahydrofolate, to the interior of cells.
FOLR1 is overexpressed in a vast majority of ovarian cancers, as well as in many e, endometrial, pancreatic, renal, lung, and breast cancers, while the expression of FOLR1 on normal tissues is restricted to the apical membrane of epithelial cells in the kidney proximal tubules, alveolar pneumocytes of the lung, bladder, testes, choroid plexus, and thyroid an SD, et al., Cancer Res 52: 3396-3401 (1992); Antony AC, Annu Rev [TEXT CONTINUES ON PAGE 2] - 1a - Nutr 16: 501-521 ; Kalli KR, et a1. Gynecol Oncol 108: 619—626 (2008)). This expression pattern of FOLRl makes it a desirable target for FOLRl —directed cancer therapy.
Because ovarian cancer is typically asymptomatic until advanced stage, it is often diagnosed at a late stage and has poor prognosis when treated with currently ble procedures, typically chemotherapeutic drugs after al de-bulking (von Gruenigen V et al., Cancer 112: 2221-2227 (2008); Ayhan A et al., Am J Obstet Gynecol 196: 81 e81-86 (2007); Harry VN et 61]., Obstet Gynecol Surv 64: 548-560 ). Thus, there is a clear unmet l need for more effective therapeutics for ovarian cancers.
SUMMARY OF THE INVENTION The present invention is based on the discovery of a dynamic range of expression of FOLRI in tumor tissue and the discovery that tumors with increased levels of FOLRl expression are more responsive to treatment with anti-FOLRl antibodies or anti-FOLRl immunoconjugates. The present invention advantageously permits treatment of patients who have a greater likelihood of responding to treatment by administering therapeutic agents, i.e., anti-FOLRl antibodies or anti-FOLRl immunoconjugates, to patients who are found to have an increased expression level of FOLRl.
The present ion provides a method for identifying a subject pre-disposed to respond favorably to a Folate Receptor 1 (FOLRl)—targeting anti-cancer therapeutic, the method comprising detecting FOLRl expression in a tissue sample from the subject.
The present invention also provides a method for increasing the likelihood of effectiveness of a cancer treatment, the method comprising administering a therapeutically effective dose of a FOLRl-targeting anti-cancer eutic to a subject, n FOLRI expression in a tissue sample from the subject has been found to be sed.
The present invention also provides a method for predicting effectiveness of a low— dose cancer ent, the method comprising administering a therapeutically effective dose of a FOLRl—targeting anti-cancer therapeutic to a subject, n said subject has been found to have increased expression of FOLRl in a sample.
In one embodiment, the methods are directed to n carcinoma, non-small cell lung adenocarcinoma ding bronchioloalveolar carcinoma), renal carcinomas, and endometrial carcinomas.
In one embodiment, the extent and uniformity of FOLRI sion is detected by immunohistochemistry (IHC), flow cytometry, or nucleic acid hybridization. in r embodiment, the level of FOLRl expression is detected by immunohistochemistry. Non- limiting examples of IHC include IHC methods that distinguish between varying levels of FOLRI and calibrated IHC methods, such as those described herein. The FOLRl sion can be scored using an appropriate scoréng , including but not limited to the scoring methods bed herein. For example, FOLRI expression can be scored using a calibrated IHC method that includes a range of 0, 1, 2, 3, and 3+ for staining intensity with 0 being the lowest level of staining intensity and 3+ being the highest level of staining intensity. Alternatively or additionally, FOLRI sion can be scored using a ated IHC method that includes a staining uniformity that ranges from focal (<25% of cells stained), to heterogeneous (25-75% of cells stained), to homogenous (>75% of cells stained), where focal staining is the least uniform staining and homogeneous is the most uniform staining.
In a further embodiment, the FOLRI expression in a sample (e.g., a tumor tissue ) is ed and ed to one or more reference samples and the FOLRl expression in the tissue sample from a subject tumor, xenograft tumor, or cell line has a FOLRI specific score ating to extent and uniformity of expression as compared to the one or more reference samples. In various examples, a tissue sample or cell with a level 1, 2, 3 or 3+ FOLRl staining ity with a homogeneous staining pattern is considered to have increased FOLRl expression; a tissue sample or cell with a level 3 FOLRl staining intensity with heterogeneous or focal staining patterns is considered to have increased FOLRl expression. In another embodiment the FOLRI expression in a sample is measured and compared to one or more reference samples to identify a comparable level of ng.
In one embodiment, the reference sample has a pre-assigned IHC score and/or a pre- determined antigen per cell (or ABC) number and the n or ABC number for the sample tissue can be determined based on the comparison.
In one embodiment, the FOLRl expression in a sample (e.g., a tumor tissue sample) is measured and compared to one or more control samples and the FOLRl expression in the tissue sample from a subject tumor, xenograft tumor, or cell line has a FOLRl specific score correlating to extent and uniformity of expression as compared to the one or more control samples. In one embodiment, the FOLRl expression in the sample is compared to a negative control sample which demonstrates no or low detectable FOLRI expression. In another embodiment, the FGLR'E expression in the sample is compared to a ve control sample having sed FOLRl expressinn {level i, 2, 3. or 3+). In some embodin‘ien‘ts, the control samples include, but are not limited in a, SW2, SWéZO, T47D, IGRG‘V’J, 300,19 FRI, i-Ielga, or KB cells. In particular embodir‘nents, the control samples include cells or cell pellets from cells transfected with folate receptor (e.g., 300.19 FRl).
In one embodiment, the FOLRl-targeting anti-cancer therapeutic is a FOLRI immunoconjugate. In one embodiment, the immunoconjugate ses an anti-FOLRI dy, a linker, and a cytotoxin.
In a further ment, the anti-FOLRI antibody is huMOVl9. In another ment, the linker is selected from the group consisting of a cleavable linker, a non~ cleavable linker, a hydrophilic linker, and a dicarboxylic acid based linker. in another embodiment, the linker is selected from the group consisting: N—succinimidyl 4-(2— pyridyldithio)pentanoate (SPP) or N—succinimidyl yridyldithio)-2—sulfopentanoate (sulfo—SPP); inimidyl 4-(2-pyn'dyldithio)butanoate (SPDB) or N-succinimidyl 4-(2— pyridyldithio)-2—sulfobutanoate (sulfo-SPDB); N—succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC); N—sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC); N—succinimidyl(iodoacetyl)-aminobenzoate (SIAB); and N—succinimidyl—[(N-maleimidopropionamido)—tetraethyleneglycol] ester (NHS-PEG4-maleimide). In another embodiment, the linker is N—succinimidyl 4-(2- pyridyldithio)-2—sulfobutanoate (sulfo-SPDB). In r embodiment, the cytotoxic agent is selected from the group consisting of a maytansinoid, maytansinoid analog, benzodiazepine, taxoid, CC—1065, CC-1065 analog, mycin, duocarmycin analog, calicheamicin, dolastatin, dolastatin analog, auristatin, tomaymycin derivative, and leptomycin derivative 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 opyl)-maytansine or deacetyl-NZ-(4-mercapto—4-methyl— l - oxopentyl)—maytansine. In another embodiment, the xic agent is N(2')-deacetyl-N2— (4-mercaptomethyl-l-oxopentyl)—maytansine (DM4). In a further ment, the immunoconjugate comprises the antibody HUMOV19, sulfo-SPDB, and DM4 (IMGN853).
The invention is also directed to a kit for measuring FOLRI expression in a subject comprising a FOLRl detection reagent, and instructions for use. In one embodiment, the FOLRl detection reagent comprises a FOLRl binding peptide, protein or a molecular probe (i.e. nucleic acid). In another embodiment, the FOLRl detection reagent is an anti-FOLRI antibody. In another embodiment, the kit r ses a ary antibody which binds the anti-FOLRl dy. In one embodiment the antibody is included at a concentration of 0.5 to 7.5 ug/ml, desirably 0.9 to 3.8 +/- 0.5 ug/ml. In various embodiments, the antibody is included at a concentration of 1.0 +/— 0.5 ug/ml, 1.5 +/— 0.5 ug/ml, 1.9 +/- 0.5 ug/ml, 2.5 +/— 0.5 ug/ml, 3.0 +/- 0.5 ug/ml, 3.5 +/- 0.5 pig/ml, 3.8 +/- 0.5 ug/ml, or up to 4.2 ug/ml. In another embodiment, the antibody is ed in concentrated solution with instructions for ons to achieve a final concentration of 0.9 to 3.8 +/— 0.5 rig/ml. In another embodiment, the kit further comprises a detection reagent selected from the group consisting of: an enzyme, a fluorophore, a radioactive label, and a luminophore.
In another embodiment, the detection reagent is selected from the group consisting of: biotin, digo-xigenin, fluorescein, tritium, and ine.
The kit can also include instructions for detection and scoring of FOLRl expression.
The kit can also e control or reference samples. Non-limiting examples of control or reference samples include tissue samples, cell pellets or cells. The controi or reference samples may be derived from tissue culture cell lines (normal or tumor), normal tissue (normal control) or tumor tissues (positive control) samples. Exemplary cell lines include SW620, T47D, IGROV-l, HELA, KB, JEG-3 and cell lines stably or ently transfected with an expression vector that expresses FOLRl (e.g., 300.19FR1). Exemplary tissues that detection methods are may be used as normal reference tissues in the FOLRl expression described herein and include normal lung, salivary gland, and pancreas.
The invention is also directed to a method for identifying a cancer likely to respond to an anti-FOLRl antibody, or anti-FOLRl immunoconjugate comprising: (a) contacting a biological sample comprising cells from said cancer with an agent that binds FOLRl protein on the cell surface; (b)detecting g of said agent that binds FOLRl n on the cell surface of said biological sample of (a); (c) assigning a score to said binding of step (b), wherein said score is assigned based on ison to one or more reference samples; and (d) comparing said score in step (c) to the score of a reference tissue or cell, wherein a score for said cancer FOLRl level that is greater than the score for a normal or low P‘O'LRI sing reference sample or a score for said cancer FOLRl level that is equal to or greater than the score for a high FOLRl expressing reference sample fies said cancer as likely to respond to an anti—FOLRI antibody or anti-FOLRl immunoconjugate. In certain embodiments, the cancer is ovarian or lung cancer.
The invention is also ed to a method of identifying a tumor as sensitive to treatment with an anti-FOLRI antibody, or anti—FOLRI immunoconjugate, said method comprising: (a) measuring the level of FOLRI expression in a tumor tissue sample obtained from said tumor, wherein said ing comprises the use of a detection method that guishes between staining intensity or staining uniformity in a FOLRI expressing cancer sample as compared to staining intensity or staining uniformity in one or more reference samples; (b) determining a FOLRI staining intensity score for said tumor tissue ; and (c) ing the FOLRI ng intensity score determined in step (b) to a relative value determined by measuring FOLRI protein expression in at least one nce sample, wherein said at least one reference sample is a tissue, cell, or cell pellet sample which is not sensitive to treatment with an anti—FOLRI antibody, or anti-FOLRl immunoconjugate, and wherein a FOLRI staining intensity score for said sample determined in step (b) that is higher than said relative value identifies said tumor as being sensitive to treatment with an anti—FOLRl dy, or anti—FOLRI immunoconjugate. In certain embodiments, the detection method is performed manually or using an automated system. In one embodiment, the detection method is IHC. In another embodiment, the IHC is calibrated IHC that can distinguish different levels of FOLRl expression.
The invention is also directed to a method of optimizing a therapeutic regimen with an anti-FOLRI dy or an anti-FOLRI immunoconjugate for a subject having lung or ovarian cancer, said method comprising: (a) contacting said sample from said subject with an antibody that specifically binds cell surface FOLRI; (b) éng the binding of said antibody in (a) to said cell surface FOLRl in said sample using a detection method that can distinguish between staining intensity or staining uniformity in a FOLRI expressing cancer sample as compared to staining intensity or staining uniformity in one or more reference samples and assigning a staining score to said sample; and (c) administering a high dose of an anti-FOLRl immunoconjugate when the score in step (b) is less than or equal to the score for a normal or low FOLRl expressing reference sample or administering a low dose of an anti—FOLRI immunoconjugate when the score is r than the score for a normal or low FOLRl expressing reference sample.
The ion is also directed to a method of detecting the expression of cell surface FOLRl on cancer cells in a tumor tissue sample from a subject, said method comprising: (a) obtaining tumor tissue sample, wherein said cancer sample is formalin-fixed paraffin embedded; (b) ting said sample with an antibody that cally binds cell surface FOLR1; (c) measuring the binding of said antibody in (b) to said cell surface FOLR1 in said tumor tissue sample using a detection method that can distinguish between staining intensity or staining uniformity in a FOLR1 expressing cancer sample as compared to staining intensity or staining uniformity in one or more reference samples; and (d) assigning a FOLR1 expression score to said FOLR1 after comparing the level of cell surface FOLR1 staining intensity or staining uniformity in said tumor tissue sample to one or more reference samples.
The invention is also directed to a method of identifying a subject having a lung or ovarian cancer as likely to respond to a low dose anti-FOLR1 antibody or anti-FOLR1 immunoconjugate treatment regimen, said method comprising: (a) contacting a biological sample comprising cells from said n or lung cancer with an agent that binds cell surface FOLR1 protein; (b) detecting binding of said agent to said ical sample of (a); (c) assigning a score to said binding of step (b), wherein said score is assigned based on comparison to one or more reference samples; and (d) comparing said score in step (c) to the score of a reference tissue or cell, n a score for said ovarian or lung cancer FOLR1 level that is greater than the score for a normal or low FOLR1 expressing reference sample or a score for said ovarian or lung cancer FOLR1 level that is equal to or greater than the score for a high FOLR1 expressing reference sample identifies said ovarian or lung cancer as likely to d to a low dose anti-FOLR1 antibody or anti-FOLR1 conjugate. In certain embodiments, the method further comprises stering a therapeutically ive amount of a zed anti-FOLR1 antibody or an anti-FOLR1 conjugate to said subject. [0023a] Definitions of the specific embodiments of the ion as claimed herein follow. [0023b] According to a first embodiment of the ion, there is provided a method of identifying a tumor as sensitive to treatment with an anti-Folate Receptor 1 (FOLR1) antibody or antigen binding fragment thereof, the method comprising: (a) measuring the level of FOLR1 expression in a tumor tissue sample obtained from the tumor, wherein the measuring comprises the use of a detection method that distinguishes between ng intensity and ng uniformity in a FOLR1-expressing cancer sample as compared to staining intensity and staining uniformity in one or more reference samples; (b) determining a FOLR1 staining intensity score for the tumor tissue sample; and (c) comparing the FOLR1 staining intensity score determined in step (b) to a reference value determined by measuring FOLR1 protein expression in at least one reference sample, wherein the at least one reference sample is a , cell, or cell pellet sample which is not sensitive to treatment with an anti-FOLR1 antibody or antigen binding fragment f, wherein a FOLR1 staining ity score for the tumor tissue sample ined in step (b) that is higher than the reference value identifies the tumor as being sensitive to treatment with an anti-FOLR1 antibody or n binding fragment thereof, and wherein the anti-FOLR1 antibody or antigen binding fragment thereof for treatment comprises (i) a heavy chain (HC) CDR1 comprising the amino acid sequence GYFMN (SEQ ID NO:6); a HC CDR2 comprising the amino acid sequence RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a HC CDR3 comprising the amino acid sequence YDGSRAMDY (SEQ ID NO:8) and (ii) a light chain (LC) CDR1 comprising the amino acid sequence KASQSVSFAGTSLMH (SEQ ID NO:9); a LC CDR2 comprising the amino acid sequence RASNLEA (SEQ ID NO:10); and a LC CDR3 comprising the amino acid sequence QQSREYPYT (SEQ ID NO:11). [0023c] ing to a second embodiment of the invention, there is provided a method of identifying a tumor as sensitive to treatment with an anti-FOLR1 immunoconjugate, the method comprising: (a) measuring the level of FOLR1 sion in a tumor tissue sample obtained from the tumor, wherein the measuring comprises the use of a detection method that distinguishes between staining intensity and staining mity in a FOLR1 expressing cancer sample as compared to staining ity and staining uniformity in one or more reference samples; (b) ining a FOLR1 staining intensity score for the tumor tissue sample; and (c) comparing the FOLR1 staining intensity score determined in step (b) to a nce value determined by measuring FOLR1 protein expression in at least one reference sample, wherein the at least one reference sample is a tissue, cell, or cell pellet sample which is not sensitive to treatment with an anti-FOLR1 immunoconjugate, wherein a FOLR1 staining intensity score for the tumor tissue sample determined in step (b) that is higher than the reference value identifies the tumor as being sensitive to treatment with an anti-FOLR1 immunoconjugate, wherein the anti-FOLR1 immunoconjugate for treatment has the formula (A) – (L) – (C), wherein: (A) comprises an anti-FOLR1 antibody or antigen binding fragment thereof sing (i) a HC CDR1 comprising the amino acid sequence GYFMN (SEQ ID NO:6); a HC CDR2 comprising the amino acid sequence RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a HC CDR3 comprising the amino acid sequence MDY (SEQ ID NO:8) and (ii) a LC CDR1 comprising the amino acid sequence KASQSVSFAGTSLMH (SEQ ID NO:9); a LC CDR2 comprising the amino acid sequence RASNLEA (SEQ ID NO:10); and a LC CDR3 comprising the amino acid sequence QQSREYPYT (SEQ ID NO:11), (L) comprises a linker, (C) comprises a cytotoxic agent, and wherein (L) links (A) to (C).
BRIEF DESCRIPTIONS OF THE DRAWINGS Figure 1. Manual ng Method: Anti -FOLR1 antibodies detect FOLR1 sion in transfected cells. 300.19 cells were transfected with a polynucleotide that encodes human FOLR1. FOLR1 n expression was detected using the murine antibody BN3.2. Smith AE et al, Hybridoma (Larchmt). 2007 Oct;26(5):281 - Figure 2. Manual Staining Method: Anti-FOLR1 dies can distinguish different levels of FOLR1 expression. Antibody BN3.2 was used to detect FOLR1 expression in various xenograft cells. The limit of detection for the BN3.2 dy was approximately 4000 antibodies bound per cell (ABC).
[TEXT CONTINUES ON PAGE 8] Figure 3. Manual Staining Method: Anti-FOLRl antibodies can distinguish different levels of FOLRI expression in tissue samples. BN3.2 was used to detect FOLRl expression in both ovarian tumors (A), as well as non-small cell lung cancer tumors (B).
Figure 4. Manual Staining Method: Uniform FOLRl expression in ovarian and NSCLC tumors. FOLRl expression was high in many of the ovaréan carcinomas, as well as lung adenocarcinomas and bronchioloalveolar carcinomas tested. The ty of n carcinoma samples had the highest intensity staining in serous or endometrioid cells. In the NSCLC tumors, the highest ABC values were found in bronchioloalveolar carcinoma and papillary adenocarcinoma.
Figure 5. Manual Staining Method: FOLRl expression is generally confined to the membrane of NSCLC cells. High resolution copy revealed that the majority of FOLRl staining was restricted to the membrane in NSCLC tumors.
Figure 6. Manual Staining Method: FOLRl expression is lly d to the membrane of ovarian cancer cells. High resolution microscopy revealed that the majority of FOLRl staining was restricted to the ne in ovarian tumors.
Figure 7. In vivo efficacy of huMovl9-targeted conjugates in a KB xenograft model. FOLRl-targeting cleavable conjugate huMovl9-SPDB—DM4 (B) in comparison with non-FOLRl—targeting huC242-SPDB-DM4 (D), and eavable conjugate huMovl9-PEG4-Mal-DM4 (C) in comparison with non-targeting huC242-PEG4Mal-DM4 (B) were tested using an established xenograft model of KB cells implanted subcutaneous into SCID mice. Targeting of FOLRl by huMov19 resulted in significant ion in mean tumor volume.
Figure 8. Dose-response anti-tumor activity of IMGN853 treatment in OVCAR—3 human ovarian carcinoma xenografts. Mice were treated with a single intravenous ion of 3 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS.
Figure 9. Dose—response umor ty of IMGN853 treatment in lGROV—l human ovarian carcinoma xenografts. Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS.
Figure 10. Dose-response anti-tumor activity of IMGN853 treatment in OV-90 human ovarian carcinoma xenografts. Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS.
Figure 11. Dose-response anti—tumor ty of 3 treatment in SKOV—3 human ovarian carcinoma xenografts. Mice were treated with a single enous injection of 3 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS.
Figure 12. Dose-response anti-tumor activity of IMGN853 treatment in KB human cervical adenocarcinoma xenografts. Mice were treated with a single intravenous injection of IMGN853 at 1.0, 2.5 or 5.0 mg/kg. A control group of s received a single intravenous injection of PBS.
Figure 13. Automated Staining Methods: Representative Photographs and Histograms depicting FOLRl Expression in Cell Lines by IHC and Flow Cytometry.
SW620, T47D, Igrov-l, 300.19/FR1, HeLa, and KB cells were all scored for FOLRl staining intensity and uniformity. SWti30 and IGROV-l cells were scored 1—3 hetero, T47D was scored 1-2 hetero, HeLa was scored 2-3 hetero, while 300.19/FR1 and KB were scored 3 homo.
Figure 14. Automated Staining Methods: Representative FOLR] Staining in Serous Ovarian Cancer. Staining patterns demonstrating 3 homo, 2-3 homo, 2 homo, and 2 hetero staining are shown for tissue ns from serous ovarian cancer by IHC. [0038} Figure 15. Automated Staining Methods: Representative FOLRl Staining in Endometrioid Ovarian Cancer. Staining patterns demonstrating 3 homo, 2—3 homo, 3 focal, and 1-2 hetero staining are shown for tissue sections from endometroid cancer by IHC.
Figure 16. Automated Staining Methods: entative FOLRl Staining in NSCLC of the arcinoma Subtype (excluding ioloalveolar carcinomas).
Staining patterns demonstrating 3 homo, 2-3 homo, 2 hetero, 2 homo, and 1-2 hetero staining are shown for tissue sections from non-small cell lung cancer, adenocarcinoma subtype by IHC. {0040} Figure 17. Automated Staining Methods: Representative FOLRl Staining in trial Adenocarcinoma. Staining patterns demonstrating 3 hetero, 2 hetero, and 1 hetero staining are shown for tissue sections from endometrial adenocarcinoma by IHC. {0041} Figure 18. Automated Staining Methods: Representative FOLRl Staining in Renal Clear Cell Carcinoma. Staining patterns demonstrating 2 homo, 2 hetero, and l heteto staining are shown for tissue sections from renal cell cancer by IHC. tests; Figure 19. The cytotoxic aetivity of iMGNSiiB in vitro. Five Fifiil‘fiwpositive eeli lines (KB? iGROV—l, IE i~3, SKOVG and OVCAR.~3} and two FOLRiwnegative cell, tines iwa and SW2) were analyzed for their sensitivity to the cytotoxic effects of liviGN8530 Cells were d to il‘VKiNSSB {solid line) or to lMGNtiSB plus 0.5 nM unconingated iinMo'Vl9 (M9346A) (dashed iine) for 5 days, and the eeil survival was determined by WSlmg-based assay, Representative data are shown, The percent of surviving eelis was d t base it} iogaritlnn Of'ii‘itj concentration of EMGN853. rests} Figure 20. The sensitivity of‘tlie linQRl-positive cell lines to EMGNXSB versus the level of itiOLRi expression. Potency and specificity of EMGN853 was anaiyzed against FGLRl—positive cell lines with a wide range of FOLRl expression. Cell lines were incubated Win lMGN853 and KB, igi'oV—L and 3eg~3 were sneeifieaily sensitive to iMGNts‘Sfi While unconjugated huMoVIS} (M9346A) snowed decreased activity of the ate. Skew—3 and ()V‘earfi were not sensitiVe to EMGNSSS and unconjugaled huMovl?) A) did not change the activity of the conjugate. gss4t; Figure 21. Automated Staining Methods: n Carcinoma Xenograft etiieaey models stained for FOLRl. ng patients trating 1:3 hetero (Swat '3), i—3 homo (lgrov i), l~2 hetero (CV 90) and negative (SKOV 3:) are shown for tissue sections from ovarian cancer xenografts by Eli—1C. insist Figure ‘22. Automated Staining Methods: Mouse Xenograft Modeisi ng patterns for FOLRl in xei'iograi‘ts for NSCLC (A), Entionietriuni Carcinoma (B) and Cervical Carcinoma (C) Ceil Lines are shown. NSCLC samples demonstrated 23 homo or 2 homo staining, endometrinni oma demonstrated 2 hetero/3 focal staining, and cervical carcinoma demonstrated 3 homo staining. tests} ,li'ig‘are 23. Assay control tissues automated staining guide. Staining patterns for negative (esophagus ti} and positive control samples (salivary gland L2 hetero? lung 21 homo, pancreas 3 home) are shown as determined by automated EHC. {9347; Figure- 34. Tumor tissues automated staining guide. Representative staining patterns for ievei 3,, {eve}. 2, and level i ng are shown on control tissue as detennined by automated iii-iii. {sets} Figure 25. Turner tissues automated staining guide, Representative ng patterns for level 3?, level, 2, and Eevei l/negative staining are shown. on control tissue as determined by automated ii-lC.
DETAILED DESCRIPTION OF THE INVENTION {00419} The present inventien presides methods for increasing the efficacy of er the hood of response to the treatment of cancers characterised by the everexpressien of FOLRI. The present invention is based on the discovery of a dynamic range of expression of FOLRE in tumor tissue as compared to nerrnai tissue and the discovery that tumors with sed. Eevels nf FOLK} expression are more sive te treatment with anti—FOLK} antibodies or ‘OlgRi irnmunoconjugate *, We have else discovered differences in sensitity and detection of c ranges between ated and manual methods. Kits comprising one or more reagents useful fer practicing the methods of the invention are further provided 1. Definitions To facilitate an understanding of the present invention, a number of terms and phrases are defined below. {0051} The terms "human folate receptor 1" or "FOLRI", as used herein, refers to any native human FOLRI, unless otherwise indicated. The term "FOLRI" encompasses "full- length," unprocessed FOLR] as well as any form of FOLRl that results from processing within the cell. The term also asses naturally occurring variants of FOLRl, e.g., splice variants, allelic ts and ms. The FOLRI 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 FOLRI sequences include, but are not limited to NCBI reference s P15328, NP_001092242.1, AAX292681, AAXBH 19.}, Ni§>g057937.l, and NP_O57936.1, and those shown in SEQ iii) NOS: 1 and 2.
The term “increased expression" of FOLRI refers to a sample which contains elevated levels of FOLRl expression. In one example, the FOLRI expression is measured by IHC and given a staining intensity score or a staining uniformity score by comparison to controls (e.g., ated controls) exhibiting defined scores (e.g. an intensity score of 3 is given to the test sample if the intensity is comparable to the level 3 calibrated l or an intensity of 2 is given to the test sample if the intensity is comparable to the level 2 calibrated control). For example, a score of 1, 2, 3, or 3+ or greater by immunohistochemistry indicates an increased expression of FOLRI. A staining uniformity that is heterogeneous or homogeneous is also indicative of increased FOLRI expression. “11., The staining iretensity and ng uniformity scores can be used alone or in combination (e.g., 2 homo, 2 hetero, 3 homo, 3 hetero, etc.). In another example, an increase in FOLRI sion can be determined by detection of an increase of at least 2-fold, at least 3-fold, or at least 5-fold) relative to control values (e.g., expression level in a tissue or cell from a subject without cancer or with a cancer that does not have elevated FOLRI ).
A "reference sample" can be used to correlate and compare the results ed in the methods of the invention from a test sample. Reference samples can be cells (e.g., cell lines, cell pellets) or tissue. The FOLRl levels in the "reference sample" may be an absolute or relative amount, a range of amount, a minimum and/or maximum amount, a mean amount, and/or a median amount of FOLRI. The diagnostic methods of the invention involve a comparison between expression levels of FOLRI in a test sample and a "reference value." In some embodiments, the reference value is the expression level of the FOLRl in a reference sample. A reference value may be a predetermined value and may also be determined from reference samples (e.g., control biological samples) tested in parallel with the test samples. A nce value can be a single cut—off value, such as a median or mean or a range of , such as a confidence interval. Reference values can be established for various subgroups of duals, such as individuals predisposed to cancer, individuals having early or late stage cancer, male and/or female individuals, or individuals oing cancer therapy. Examples of normal reference samples or values and positive reference samples or values are described herein.
In some embodiments, the reference sample is a sample from a healthy tissue, in ular a corresponding tissue which is not affected by cancer. These types of reference samples are referred to as ve control samples. In other embodiments, the nce sample is a sample from a tumor tissue that expresses FOLRl. These types of reference samples are referred to as positive control samples. Positivie control samples can also be used as a comparative indicator for the uniformity (hetero versus homo) and/or degree (1, 2, 3, 3+) of ng ity, which correlates with the level of FOLRl expression. Positive control comparative samples are also referred to as calibrated reference samples which demonstrate a dynamic range of staining intensity or uniformity. As shown in Examples 1- 9, non FOLRl-expressing reference samples include human esophagus tissue; low FOLRI nce includes salivary gland (particularly the intercalated ducts) and lung (particularly respiratory epithelium) tissue; and high expressing tissue includes the pancreas cularly ductal cells). For cell lines, low expressors include, but are not limited to -12...
OVCAR3 and T471), moderate expressers include, but are not limited in SW‘tSZi), i lRUX/ll, 515.63, and high expressers include, but are not d to, KB and EGROVE. Particularly desirable positive high FOLK} nce is a cell line stably or transiently transt‘eeted with Folate Receptor 1 (e.g., 300.19/FR1). Appropriate positive and negative reference levels of FOLRl for a particular cancer, may be determined by measuring levels of FOLRl in one or more appropriate subjects, and such reference levels may be tailored to specific populations of subjects (e.g., a reference level may be age-matched so that comparisons may be made between FOLRl levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a n age . Such reference levels may also be tailored to specific techniques that are used to measure levels of FOLRl in biological samples (e.g., immunoassays, etc.), where the levels of FOLRl may differ based on the specific technique that is used.
The term "primary antibody" herein refers to an antibody that binds specifically to the target protein antigen in a tissue sample. A primary antibody is lly the first dy used in an histochemical (IHC) procedure. In one embodiment, the primary antibody is the only antibody used in an IHC procedure. The term "secondary antibody" herein refers to an antibody that binds specifically to a primary antibody, thereby forming a bridge n the primary antibody and a uent reagent, if any. The secondary antibody is generally the second antibody used in an immunohistochemical procedure. {9956} A "sample" or "biological sample" of the present invention is of biological origin, in specific embodiments, such as from eukaryotic organisms. In preferred embodiments, the sample is a human sample, but animal samples may also be used in the practice of the invention. Non-limiting sources of a sample for use in the present invention include solid tissue, biopsy aspirates, ascites, fluidic extracts, blood, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, , organs, cell cultures and/or cell e constituents, for example. The present invention is particularly useful for cancer samples which generally se solid tissue samples, or other bodily fluids such as ascites, where the amount of available material is small. The method can be used to examine an aspect of expression of FOLRl or a state of a sample, including, but not d to, comparing different types of cells or tissues, ing different pmental , and detecting or determining the presence and/or type of disease or abnormality.
For the purposes herein, a "section" of a tissue sample refers to a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis according to the present invention. In some cases, the selected portion or section of tissue comprises a homogeneous population of cells. In other cases, the selected portion comprises a region of tissue, e.g. the lumen as a non-limiting example. The ed n can be small as one cell or two cells, or could represent néany thousands of cells, for example. In most cases, the collection of cells is important, and while the ion has been bed for use in the detection of cellular components, the method may also be used for detecting non-cellular components of an sm (e.g. soluble components in the blood as a non— limiting example). {0058] By "correlate" or "correlating" is meant comparing, in any way, the performance and/0r results of a first analysis with the performance and/or results of a second analysis.
For example, one may use the results of a first analysis in carrying out the second analysis and/or one may use the results of a first analysis to determine whether a second analysis should be performed and/or one may compare the results of a first analysis with the results of a second analysis. In one embodiment, increased expression of FOLRl correlates with increased likelihood of effectiveness of a FOLRl-targeting anti-cancer therapy.
FREQ} The term "antibody" means an immunoglobulin molecule that izes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site Wéthin the le region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal dies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized dies, human dies, fusion proteins sing an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the d biological activity. An antibody can be of any the five major classes of immunoglobulins: EgA, IgD, IgE, IgG, and IgM, or sses (isotypes) thereof (e.g. IgGl, lgGZ, IgG3, lgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains ed to as alpha, delta, epsilon, gamma, and mu, resFectively. The different s of immunoglobulins have different and well known subunit structures and three~dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc. {0060} A ing" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds, such as FOLRl. In a certain ment blocking dies or antagonist antibodies substantially or tely t the biological activity of the antigen. bly, the biological activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%. {006;} The term "anti-FOLRI antibody" or "an antibody that binds to FOLRl" refers to an antibody that is capable of binding FOLRl with ent affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting FOLRl. The extent of binding of an anti-FOLRl antibody to an unrelated, non—FOLRl protein is less than about 10% of binding of the antibody to FOLRl as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to FOLRl has a dissociation constant (Kd) of :1 nM, 5100 nM, 510 nM, 51 nM, or 50.1 nM. Examples of anti-FOLRI antibodies are known in the art and are disclosed in US Appl. Pub. No. 2012/0009181, which is herein incorporated by reference.
The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', 2, and Fv fragments, linear antibodies, single chain antibodies, and multispecifrc dies formed from antibody fragments. {0063] A "monoclonal antibody" refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic inant, or epitope.
This is in contrast to polyclonal antibodies that typically e different antibodies directed against different antigenic determinants. The term lonal antibody” encompasses both intact and ength monoclonal dies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an n recognition site. Furthermore, ”monoclonal antibody" refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals. {90%} The term "epitope" or "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the n is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, s epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope lly es at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
"Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a moiecule (e.g., an dy) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to sic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., 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 antigen slowly and tend to dissociate readily, whereas high— affinity antibodies generally bind antigen faster and tend to remain bound longer. A varéety of s of measuring g affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.
"Or better" when used herein to refer to binding affinity refers to a stronger binding between a molecule and its binding partner. "Or better" when used herein refers to a stronger binding, represented by a smaller numerical Kd value. For example, an antibody which has an y for an n of "0.6 nM or better", the dy’s affinity for the antigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6 nM.
The phrase "substantially similar," or "substantially the same", as used herein, denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody of the invention and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference n the two values to be of little or no biological and/or tical significance within the context of the biological characteristics measured by said values (e.g., 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/comparator antibody. » {006813 A polypeptide, antibody, polynucleotide, , cell, or composition which is "isolated" is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in . isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antibody, polyaucleotide, vector, cell, or composition which is isolated is substantially pure.
As used herein, "substantially pure" refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
The term "immunoconjugate" or "conjugate" as used herein refers to a compound or a derivative thereof that is linked to a cell g agent (i.e., an anti-FOLRl antibody or fragment thereof) and is defined by a generic a: C-L-A, wherein C = cytotoxin, L = linker, and A — cell binding agent or anti-FOLRl antibody or antibody nt. lmmunoconjugates can also be defined by the generic formula in reverse order: A-L-C. [0%71] A "linker" is any chemical moiety that is capable of linking a compound, usually a drug, such as a maytansinoid, to a cell-binding agent such as an anti FOLRI antibody or a fragment thereof in a stable, covalent . Linkers can be tible to or be ntially resistant to nduced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include charged linkers, and hydrophilic forms thereof as described herein and know in the art.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
Examples of cancer e, but are not limited to, oma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non—small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic , glioblastoma, cervical cancer, n cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal , endometrial or e carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate , vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancers. {0073] "Tumor" and "neoplasm" refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre—cancerous lesions.
The terms "cancer cell," "tumor cell," and grammatical equivalents refer to the total population of cells deréved from a tumor or a pre—cancerous lesion, including both nontumorigenic cells, which comprise the bulk of the tumor cell tion, and tumorigenic stem cells (cancer stem cells). As used herein, the term “tumor cell” will be modified by the term “non—tumorigenic” when referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.
The term "subject" refers to any animal (e. g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms ct" and "patient" are used interchangeably herein in reference to a human subject. stration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which ns no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile.
An "effective amount" of an dy as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amoun can be determined empirically and in a routine , in relation to the stated purpose.
The term "therapeutically effective " refers to an amount of an antibody or other drug effective to "treat" a disease or disorder in a t or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of ing". To the extent the drug can prevent growth and/or kill ng cancer cells, it can be cytostatic and/or cytotoxic. In n ments, identification of -18<— increased FOLRl levels allows for administration of sed amounts of the FOLRl- targeting therapeutic to achieve the same therapeutic effect as seen with higher dosages. A "prophylactically effective amount" refers to an amount effective, at dosages and for s of time necessary, to achieve the desired prophylactic result. Typically but not arély, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
The term "respond favorably" generally refers to causing a beneficial state in a subject. With respect to cancer treatment, the term refers to providing a therapeutic effect on the subject. Positive therapeutic effects in cancer can be measured in a number of ways (See, W.A. Weber, J. Nucl. Med. 50:lS-IOS (2009)). For example, tumor growth inhibition, molecular marker sion, serum marker expression, and molecular imaging techniques can all be used to assess therapeutic y of an anti—cancer therapeutic. With to tumor growth inhibition, according to NCI standards, a T/C S 42% is the respect minimum level of anti-tumor activity. A T/C <10% is considered a high anti—tumor activity level, with T/C (%) = Median tumor volume of the d / Median tumor volume of the control x 100.
The word "label" when used herein refers to a detectable nd or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label can be able by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze al alteration of a substrate compound or composition which is detectable.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, are not limited to: alkyating agents, antimetabolites, spindle poison plant alkaloids, cytoxic/antitumor antibiotics, omerase inhibitors, dies, photosensitizers, and kinase tors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy.
Terms such as ing" or "treatment" or "to treat" or "alleviating" or “to alleviate” refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) lactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment e those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. in certain embodiments, a t is sueeessfitliy "treated” for cane-er according to the s of the present invention if the patient shows one or more of the following: ion in eaehexia, increase in survival time, elongation in time to tumor progression, reduction in tumor mass, reduction in tumor burden and/or a prolongation in time to tumor metastasis, time to tumor recurrence, tumor response, eompiete response, partiai response, stabie disease, progressive disease, progression tree survival (FPS), overali al (08), each as measured by standards set by the Nationai Cancer institute and the US. Food and Drug Administration for the approvai of new drugs. See Johnson et ai, (2003) 3, Che.
Oneoi. 21t?):i404~i41 t.
"Progression free survivai" (PPS), also ed to as or "Time to Tumor Progression" (YEP) indicates the length of time during and after treatment that the cancer does not Progression-free survival includes the amount of time patients have grow. experéenced a complete response or a partial response, as well as the amount of time patients have experienced stable disease.
"Disease free survival" (DFS) refers to the length of time during and after ent that the patient remains free of disease.
"Overall Survival" (OS) refers to a gation in life expectancy as compared to naive or untreated individuals or patients.
As used in the present disclosure and claims, the singular forms "a," "an," and "the" e plural forms unless the context clearly dictates otherwise.
It is understood that wherever embodiments are bed herein with the language "comprising," otherwise analogous embodiments described in terms of "consisting of‘ and/or "consisting essentially of" are also provided.
The term "and/or" as used in a phrase such as "A and/or B” herein is ed to e both "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the ing ments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). 11. Biological samples 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 alcohol immersion (Battifora and Kopinski, J. Histochem. _20e Cytechem. (ll-$86) 34:109.?) are also suitable. The s used may also be embedded in n. in one embodiment, the tissue samples are both lbrmalinafixed and paraffin-- embedded (PEPE). in another embodiment, the FFPE block is hematoxylin and ecsin d prior to selecting ene 01' more portions fer analysis in order to select specific areats) fer the FFPE core sample. s of preparéng tissue blocks from these particulate specimens have been used in previous EEC studies cf varicus pregnnstic factors, and/er is well. known to those of skill in the art (see, for example, Abbcndanzo et al, Am 3 Clin . @990 l‘vlay;93(5):698»702; Allred et al., Arch Surg. l99t) .Ean;l25(l):lll7—l3). {@591} Briefly, any intact organ or tissue may be cut into fairly small pieces and incubated in various es (eg. formalin, alcohol, etc.) for varying periods of time until the tissue is "fixed". The samples may be virtually any intact tissue ally removed from the body.
The s may be cut into reasonably small piece(s) that fit on the equipment routinely used in histopathology laboratories. The size of the cut pieces typically ranges from a few millimeters to a few centimeters.
III. Detection Antibody Conjugates The present invention further es antibodies against FOLRl, generally of the monoclonal type, that are linked to at least one agent to form a detection antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic it is conventional to link or ntly bind or complex at least one desired molecule or moiety.
Such a molecule or moiety may be, but is not limited to, at least one reporter molecule. A reporter molecule is defined as any moiety that may be ed using an assay. Non- limiting examples of reporter molecules that have been ated to antibodies include enzymes, radiolabels, haptens, cent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or s, such as biotin.
Any cell binding agent (e.g., an antibody or polypeptide) of sufficient selectivity, specificity or affinity may be employed as the basis for detection of the FOLRl polypeptide.
Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art. Sites for binding to biological active molecules in 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 -21“. variable domain is involved in antibody self—binding (Kang et al., 1988) and contains epitopes (idiotopes) recognized by anti-antibodies (Kohler et al., 1989).
Certain examples of protein binding (e.g., antibody) conjugates. are those ates in which the protein binding agent (e.g., antibody) is linked to a able label.
"Detectable labels" are compounds and/or ts 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 r quantified if desired. {0095} Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., US. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference). The imaging moieties used can be paramagnetic ions; ctive isotopes; fluorochromes; tectable substances; and/or X—ray imaging, for example.
Exemplary fluorescent labeis contemplated for use as protein binding (e.g., antibody) conjugates include Alexa 350, Alexa 430, Alexa 488, AMCA, BODIPY 630/650, BODIPY 5, BODIPY—FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Dylight 488, Fluorescein ocyanate, Green fluorescent protein (GFP), HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phycoerythrin, REG, Rhodamine Green, Rhodamine Red, tetramethyl rhodamine (TMR), Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, Texas Red, and derivatives of these labels (i.e halogenated analogues, modified with isothiocyanate or other linker for conjugating, etc), for example. An exemplary radiolabel is tritium. £13097} Protein binding (e.g., antibody) detection conjugates contemplated in the present invention e those for use in Vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. es of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen dase and/or glucose oxidase. Preferred ary binding ligands are biotin and/or avidin and avidin nds. The use of such labels is well known to those of skill in the art and are described, for example, in US.
Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.
Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983). In ular, 2- and 8—azido analogues of purine nucleotides have been used as site-directed photoprobes to fy nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8- azido nucleotides have also been used to map nucleotide binding domains of purified ns (Khatoon et al., 1989; King et al., 1989; and Dholakia et al., 1989) and may be used as antibody binding agents.
Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate x employing, for example, an organic chelating agent such a diethylenetréaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or hloro-3a-6d-diphenylglycouril-3 ed to the antibody (US. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).
Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Protein binding (e. g., antibody) conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In US. Pat. No. 4,938,948, g of breast tumors, for example, is achieved using monoclonal dies, and the detectable imaging es are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N—succinimidyl(4- hydroxyphenyl)—propionate.
In other embodiments, derivatization of immunoglobulins by selectively introducing sulfliydryl groups in the Fc region of an immunoglobulin using reaction conditions that do not alter the antibody ing site are contemplated. Antibody ates ed according to this methodo‘rogy are disclosed to exhibit improved longevity, specificity and sensitivity (US. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region, have also been disclosed in the literature nnessy et al., 1987).
In other embodiments of the invention, immunoglobulins are radiolabeled with nuclides such as tritium. In additional embodiments, nanogold particles (such as sizes from about 0.5 nm-40 nm) and/or Quantum Dots (Hayward, Calif.) are employed.
IV. Enzymes and Substrates (Chromagens) The use of substrates and tors is contemplated for ion of FOLRl, such as the ary embodiments provided below, for example. _23, adish 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 s, HRP and some ke activities can be inhibited by excess ate. The complex formed between HRP and excess hydrogen de is catalytically inactive and in the absence of an electron donor (e.g. genic substance) is reversibly inhibited. It is the excess hydrogen peroxide and the absence of an electron donor that brings about ing of endogenous HRP activities. {00104} When used in assays systems, HRP can also be used to convert a defined substrate into its activated chromagen, thus causing a color change. The HRP enzyme may be conjugated to an antibody, protein, peptide, polymer, or other molecule by a number of methods. Such methods are known in the art. Adding glutaraldehyde to a solution containing an admixture of HRP and antibody will result in more antibody molecules being conjugated to each other than to the enzyme. In the two-step procedure, HRP reacts with the bifunctional reagents first. In the second stage, only activated HRP is d with the antibody, resulting in much more nt labelling and no 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 substrate, for example. Conjugation with biotin also involves two steps, as biotin must first be derivatized to the yl-N— hydroxysuccinimide ester or to biotin hydrazide before it can be reacted with the epsilonamino groups of the HRP enzyme. ] 3,3'—Diaminobenzidine (DAB) is a substrate for enzymes such as HRP that produces a brown end product that is highly insoluble in alcohol and other organic solvents.
Oxidation of DAB also causes polymerization, resulting in the ability to react with osmium tetroxide, and thus increasing its staining intensity and electron density. Of the several metals and methods used to intensify the optical y of polymerized DAB, gold chloride in combination with silver sulfide appears to be the most successful. 3-Amino—9-ethylcarbazole (ABC) is a substrate for enzymes such as HRP. Upon oxidation, forms a rose-red end product that is alcohol soluble. Therefore, specimens processed with ABC must not be immersed in l or alcoholic solutions (e.g., Harris' hematoxylin). Instead, an aqueous counterstain and mounting medium should be used. ABC is unfortunately susceptible to further oxidation and, when exposed to excessive light, will fade in intensity. Storage in the dark is therefore recommended. 4-Chloro-1—naphthol (CN) is a substrate for enzymes such as HRP and precipitates as a blue end product. Because CN is soluble in alcohol and other organic solvents, the specimen must not be ated, exposed to alcoholic counterstains, or coverslipped with mounting media containing organic ts. Unlike DAB, CN tends to diffase from the site of itation. p-Phenylenediamine dihydrochloride/pyrocatechol (Hanker-Yates reagent) is a an electron donor substrate for enzymes such as HRP and gives a lack reaction product that is insoluble in alcohol and other c solvents. Like polymerized DAB, this reaction product can be osmicated. Varying results have been achieved with Hanker-Yates reagent in peroxidase techniques. [0100} Calf intestine alkaline phosphatase (AP) (molecular weight 100 kD) is an enzyme that removes (by hydrolysis) and transfers phosphate groups from organic esters by breaking the P-O bond; an intermediate enzyme-substrate bond is briefly . The chief metal activators for AP are Mg++, Mn++ and Ca++. {0101] AP had not been used extensively in immunohistochemistry until publication of the unlabeled alkaline phosphataseantialkaline phosphatase (APAAP) procedure. The soluble immune complexes utilized in this procedure have molecular weights of approximately 560 kD. The major advantage of the APAAP ure compared to the PAP technique is the lack of interference posed by endogenous peroxidase activity. Because of the potential distraction of endogenous peroxidase activity on PAP staining, the APAAP technique is recommended for use on blood and bone marrow smears. Endogenous alkaline atase activity from bone, kidney, liver and some white cells can be ted by the addition of l min-ft levamisole to the substrate solution, although 5 mM has been found to be more effective. Intestinal alkaline phosphatases are not adequately inhibited by levamisoie.
In the immunoalkaline phosphatase staining method, the enzyme hydrntyzes ol phosphate esters (substrate) to phenolic compounds and phosphates. The phenols couple to ess diazonium salts (chromogen) to produce insoluble, colored azo dyes.
Several different combinations of substrates and chromogens have been used successfully.
Naphthol AS-MX phosphate can be used in its acid form or as the sodium salt. The chromogens Fast Red TR and Fast Blue BB e a bright red or blue end product, respectively. Both are soluble in alcoholic and other organic solvents, so aqueous mounting media must be used. Fast Red TR is red when ng cell smears. {area} Additional exemplary substrates include naphthol AS—BI phosphate, ol AS- TR phosphate and 5—bromochloro-3—indoxyl phosphate (BCIP). Other possible chromogens include Fast Red LB, Fast Garnet GBC, Nitro Blue Tetrazolium (NBT) iodonitrotetrazolium Violet (INT), and derivatives of the structures, for example.
V. Immunodetection Methods In still further embodiments, the present invention concerns immunodetection methods for binding, purifying, removing, quantifying and/or otherwése lly ing biological components such as a ligand as contemplated by the present invention. The dies prepared in accordance with the present invention may be ed to detect wild-type and/or mutant ligand proteins, polypeptides and/or peptides. As described throughout the present application, the use of wild—type and/or mutant ligand c antibodies is contemplated. Some immunodetection methods e flow cytometry, enzyme linked imrnunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent useful immunodetection assay, and Western blot to mention a few. The steps of various methods have been described in the scientific literature, such as, e.g., tle M H and Ben-Zeev 0, Methods Mol Biol. 1999;109:215-37; Gulbis B and Galand P, Hum Pathol. 199% Dec;24(12):1271—85; and De Jager R et al., Semin Nucl Med. 1993 Apr;23(2):l65-79, each incorporated herein by reference.
In l, the immunobinding s include obtaining a sample suspected of comprising ligand n, polypeptide and/or peptide, and contacting the sample with a first ligand binding peptide (e.g., an anti-ligand antibody) in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes. in terms of antigen detection, the biological sample ed may be any sample that is suspected of comprising a wild—type or mutant ligand protein-specific antigen, such as a tissue section or specimen, a homogenized tissue extract, biopsy aspirates, a cell, separated and/or purified forms of any of the above wild-type or mutant FOLRl-containing compositions, or even any biological fluid that comes into contact with the tissue, including blood and/or serum, although tissue samples or ts are preferred.
Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the ion of immune complexes -26, (prémary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any ligand protein antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the prémary immune complexes to be detected.
In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous ches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. US. Patents concerning the use of such labels include US. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 149 and 4,366,241, each orated herein by nce. Of course, one may find additional advantages h the use of a secondary binding ligand such as a second antibody arid/or a biotin/avidin ligand binding ement, as is known in the art.
The anti—ligand antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding agent that has binding affinity for the dy. In these cases, the second binding agerét may be linked to a detectable label. The second binding agent is itself often an antibody, which may thus be termed a "secondary" antibody, or a polymer ion system. The primary immune complexes are contacted with the labeled, ary binding agent, or antibody/polymer detection system, under effective conditions and for a period of time ent to allow the formation of secondary immune xes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected. [0111: Further methods include the detection of primary immune complexes by a two-step approach. A second g agent, such as an antibody, that has g affinity for the dy 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 g affinity for the second antibody, again under effective conditions and for a .27~ period of time sufficient to ailow the formation of immune complexes {tertiary . compiexes). The third ligand or dy is iinked to a detectabie iabei, allowing detection of the tertiary immune complexes thus . This system may provide for signal. arripiitication if this is desired. it}: E2} In another embodiment, a biotinylated monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin. In that method the sample to be tested is first incubated in a solution comprising the first step antibody. If the target antigen is present, some of the antibody binds to the n to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then ed by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or mentary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution comprising the second step antibody against biotin. This second step antibody is labeled, as for example wéth an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a protein binding (e.g., antibody) conjugate can be produced that is macroscopically visible.
Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain on) methodology. The PCR method uses a DNA/biotin/streptavidin/antibody complex that is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with le primers with appropriate controls. In specific ments, the enormous amplification lity and specificity of PCR can be utilized to detect a single antigen molecule. Such ion may take place in real-time. For example, the use of tative real-time PCR is contemplated. {(3114} In the clinical diagnosis and/or monitoring of patients with various forms of disease, the detection of a FOLRI mutant, and/or an alteration in the levels of FOLRl, in comparison to the levels in a corresponding biological sample from a normal subject is indicative of a t with the disease. However, as is known to those of skill in the art, such a clinical diagnosis would not necessarily be made on the basis of this method in isolation. Those of skill in the art are very familiar with differentiating between significant differences in types and/or amounts of biomarkers, which represent a positive identification, and/or low level and/or ound changes of biomarkers. Indeed, background expression levels are often used to form a "cut-off" above which increased detection will be scored as significant and/or positive. 39115; In one embodiment, immunological detection (by immunohistochemistry) of FOLRI is scored for both intensity and uniformity (percent of stained cells — membrane only). Comparative scales for FOLRl expression for intensity correlate as 0 — Negative, 0- 1 — Moderate to Strong, — Moderate, 2-3 - Weak to Moderate, 2 - Very Weak, 1 — Weak, 1-2 3 — Strong. tatively, Score 0 represents that no membrane staining is observed in tumor cells. A Score 1 represents a faint/barely perceptible membrane staining in tumor cells. For Score 2, a moderate membrane staining is observed in tumor cells. Lastly, Score 3 or 3+ represents a moderate to strong membrane staining in the tumor cells. Those samples with 0 or 1 score for FOLRI expression may be characterized as not overexpressing FOLRl, whereas those samples with 2 or E scores may be terized as overexpressing FOLRI. Samples pressing FOLRl may also be rated by immunohistochemical scores corresponding to the number of copies of FOLRI les expressed per cell, and have been determined biochemically: 0=0-10,000 copies/cell, l=at least about 200,000 copies/cell, 2=at least about 500,000 copies/cell, and 3=at least about 2,000,000 copies/cell. Comparative scales for FOLRl percent cell membrane staining uniformity correlate as follows: 0 — Negative, Focal - 25- - <25%, heterogeneous (hetero) 75%, and homogeneous (homo) - >75%.
VI. Nucleic Acid Hybridization In situ hybridization is lly carréed out on cells or tissue sections fixed to slides. In situ hybridization may be performed by l conventional methodologies (See for e.g. Leitch et al. In situ Hybridization: a cal guide, Oxford BIOS Scientific Publishers, copy handbooks v. 27 (1994)). In one in situ procedure, fluorescent dyes (such as fluorescein isothiocyanate (FITC) that fluoresces green when d by an Argon ion laser) are used to label a nucleic acid ce probe that is complementary to a target nucleotide sequence in the cell. Each cell comprising the target nucleotide sequence will bind the labeled probe, producing a fluorescent signal upon exposure of the cells to a tight source of a wavelength riate for excitation of the specific fluorochrome used.
Various degrees of hybridization stringency can be employed. As the hybridization conditions become more stringent, a greater degree of complementarity is required between -29.. the probe and target to form and maintain a stable duplex. Stringency is increased by raising temperature, lowering salt concentration, or raising formamide concentration. Adding n sulfate or raising its tration may also increase the effective concentration of labeled probe to increase the rate of ization and ultimate signal intensity. After hybridization, slides are washed in a solution generally comprising reagents similar to those found in the hybridization solution with washing time varying from minutes to hours depending on required stringency. Longer or more stringent washes typically lower nonspecific background but run the risk of decreasing overall sensitivity. {aria} Probes used in nucleic hybrédization analysis may be either RNA or DNA oligonucleotides or polynucleotides and may contain not only naturally-occurring nucleotides but their analogs, like genin dCTP, biotin dcTP 7—azaguanosine, azidothymidine, inosine, or uridine, for example. Other useful probes include peptide probes and analogues thereof, branched gene DNA, peptidometics, peptide nucleic acid (PNA) and/or antibodies, for example. {911§§ Probes should have sufficient complementarity to 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 with the stringency of the hybridization medium and/or wash medium. Preferably, completely homologous probes are employed in the present invention, but s of skill in the art will readily iate that probes exhibiting lesser but sufficient homology can be used in the present invention (see for e.g. Sambrook, J., Fritsch, E. F., Maniatis, T., lar Cloning A Laboratory Manual, Cold Spring Harbor Press, (1989)). {@129} Probes may also be generated and chosen by several means including, but not d to, mapping by in situ ization, somatic cell hybrid panels, or spot blots of sorted chromosomes; chromosomal linkage is; or cloned and isolated from sorted chromosome libraries from human cell lines or somatic cell hybrids with human chromosomes, radiation c cell hybrids, issection of a chromosome region, or from yeast artificial somes (YACs) identified by PCR primers c for a unique chromosome locus or other suitable means like an adjacent YAC clone. Probes may be genomic DNA, cDNA, or RNA cloned in a d, phage, cosmid, YAC, Bacterial Artificial Chromosomes (BACS), viral vector, or any other suitable vector. Probes may be cloned or synthesized chemically by conventional methods. When cloned, the isolated probe nucleic acid nts are typically 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 e.g. Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning A tory Manual, Cold Spring Harbor Press, (1989)]. {$122} Probes are preferably labeled, such as with a fluorophor, for example. Examples of fluorophores e, but are not limited to, rare earth es (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, iferone, phycocrytherin, phycocyanin, or commercially available fluorophors such SPECTRUM ORANGETM and SPECTRUM GREENTM and/or derivatives of any one or more of the above. Multiple probes used in the assay may be labeled with more than one distinguishable fluorescent or pigment color. These color differences provide a means to identify the hybridization positions of specific probes. Moreover, probes that are not separated lly can be identified by a different color light or pigment resulting from mixing two other colors (e.g., light een=yellow) pigment (e.g., blue+yellow=green) or by using a filter set that passes only one color at a time.
Probes can be labeled directly or indirectly with the fluorophor, utilizing conventional methodology known to one with skill in the art.
VII. ion Kits and Compositions Also provided by the invention are kits for use in the practice of the present invention as disclosed herein. Such kits may se containers, each with one or more of the various reagents (typically in concentrated form) ed in the methods, including, for example, one or more binding agents (antibodies), already attached to a marker or optionally with reagents for coupling a binding agent to an antibody or nucleic acid molecule (as well as the marker itself); buffers, the appropriate nucleotide triphosphates (e.g. dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more sequence-specific or rate prémers for use in detection of nucleic acid molecules by amplification; and/or reagents and instrumentation for the isolation (optionally by microdissection) to t the practice of the ion. A label or indicator describing, or a set of instructions for use of, kit components in a ligand detection method of the present invention, will also be typically included, Where the instructions may be associated with a package insert and/or the packaging of the kit or the components thereof. -31...
In still further embodiments, the present invention concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies are generally used to detect wild—type and/or mutant proteins, polypeptides and/or peptides, the antibodies will preferably be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to a wild-type and/or mutant protein, polypeptide and/or peptide, and/or optionally, an immunodetection reagent and/or further optionally, a wild-type and/or mutant protein, polypeptide and/or peptide.
The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with and/or linked to the given dy. Detectable labels that are associated with and/or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary dies or polymers that have g y for the first antibody.
Further suitable immunodetection reagents for use in the present kits e the two—component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody or polymer that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and/0r all such labels may be suitably ed in connection wéth the present invention.
The kits may further comprise a suitably aliquoted composition of the wild-type and/or mutant protein, polypeptide and/or polypeptide, r labeled and/or unlabeled, as a n may be used to prepare standard curve for a detection assay. The kits may dy- or polymer-label ates either in fully conjugated form, in the form of intermediates, and/or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media and/or in lized form. {0123] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the antibody may be placed, and/0r preferably, ly aliquoted. The kits of the present invention will also typically include a means for containing the antibody, antigen, and/or any other reagent containers in close confinement for commercial sale. Such containers may e injection and/or blow- molded plastic containers into which the desired Vials are retained. gnzw The kits may further comprise one or more therapeutic agents for the treatment of cancer, such as a FOLRl immunoconjugate and/or a chemotherapeutic agent. -32.. terse; The kit may further comprise an a FOLRl detection reagent used to measure FOLRl expression in a t comprésing a FOLRl detection reagent, and instructions for use. In one embodiment, the FOLRl detection reagent comprises a FOLR] binding peptide, protein or a lar probe (i.e. nucleic acid). In another embodiment, the FOLRl detection reagent is an anti-FOLRl antibody. In r embodiment, the kit further comprises a secondary antibody which binds the OLRI antibody. In one embodiment the FOLRl- specific antibody is included at a concentration of 0.5 to 7.5 ug/ml, ably 0.9 to 3.8 +/— 0.5 ug/ml. In another embodiment, the dy is included at a concentration of 1.0 +/- 0.5 ug/ml, 1.5 +/— 0.5 ug/ml, 1.9 +/- 0.5 ug/ml, 2.5 +/- 0.5 ug/ml, 3.0 +/- 0.5 ug/ml, 3.5 +/— 0.5 ug/ml, 3.8 +/- 0.5 ug/ml, or up to 4.2 ug/nil. In another embodiment, the antibody is included in trated solution with instructions for ons to achieve a final concentration of 0.9 to 3.8 +/- 0.5 ug/ml. In another embodiment, the kit further comprises a detection reagent selected from the group consisting of: an enzyme, a fluorophore, a radioactive label, and a luminophore. In another embodiment, the detection reagent is selected from the group consisting of: biotin, digoxigenin, fluorescein, m, and ine. {$131} The kit can also include instructions for detection and scoring of FOLRl expression.
The kit can also include control or reference samples. miting examples of control or reference samples include cell pellets or tissue culture cell lines derived from normal (normal control) or tumor (positive control) samples. Exemplary cell lines include KB, NCI-H2llO, Igrov-l, 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 FOLRI, and cell lines stably or transiently transfected with an expression vector that expresses FOLRl. Additional es for positive control tissues can also be found in Examples 9- 11. The kit can also comprise a staining guide which Visually depicts positive and normal reference samples for staining intensity and uniformity. Such staining guides can have reference samples from normal lung, pancreas, and/or salivary gland, and stained tumors with standardized scores (e.g., ovarian, lung, renal, and endometrial s, as well as those described in the Examples and in Figures 23—25) VIII, FOLifilubinding agents Any dies that bind FOLRI can be used in the detection methods of the present invention. es oftherapeuticaily effective anti—FOLRI antibodies can be tbund in US Appl. Pub. No. US 2012/0009181 which is herein incorporated by reference. The fulllength amino acid (aa) and nucleotide (nt) sequences for FOLR1 are known in the art and also provided herein as represented by SEQ ID NOs: 1 and 2, respectively. A specifically useful antibody for detection of FOLR1 is the mouse monoclonal antihuFOLR1 clone BN3.2 (Leica # NCL-L-FRalpha). An example of a therapeutically effective OLR1 antibody is huMov19 (M9346A). The polypeptides of SEQ ID NOs: 3-5 comprise the variable domain of the heavy chain of huMov19 (M9346A), and the variable domain light chain version 1.00, the variable domain light chain version 1.60 of huMov19, respectively. The huMov19 (M9346A) antibody 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 9 (M9346A) antibody is encoded by the plasmids ted 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 having ATCC t nos. PTA-10772 and PTA-10773 or 10774.
Examples of FOLR1 immunoconjugates useful in the therapeutic methods of the invention are ed below.
IX. FOLR1 Immunoconjugates The present invention also includes methods for sing the efficacy of conjugates (also ed to herein as immunoconjugates), comprising the anti-FOLR1 dies, antibody fragments, functional equivalents, improved antibodies and their aspects as disclosed , linked or conjugated to a cytotoxin (drug) or g.
Exemplary FOLR1 immunoconjugates can be found in US Appl. Pub. No. US 009181, which is herein incorporated by reference. A particularly effective therapeutic immunoconjugate of the invention comprises the huMov19 antibody described above.
Suitable drugs or prodrugs are known in the art. In certain embodiments, drugs or prodrugs are xic agents. The cytotoxic agent used in the cytotoxic ate of the present invention can be any compound that results in the death of a cell, or induces cell death, or in some manner decreases cell viability, and includes, for example, maytansinoids and maytansinoid analogs, benzodiazepines, taxoids, CC-1065 and CC- 1065 analogs, duocarmycins and duocarmycin analogs, enediynes, such as calicheamicins, atin and dolastatin analogs including auristatins, tomaymycin derivatives, leptomycin derivatives, rexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, [TEXT CONTINUES ON PAGE 35] - 34a - melphaian, mitoh’tyciri C, mbucii and morphoiirio doxombicin, In certain embodiments, the cytotoxic agents are maytansinoids and sirioids analogs, {0135} The drug or prodrug cart, for exampie, be limited to the anti—FOLK antibody, such as hul‘vtoviéi, or fragment thereof through a disulfide bond. The iinirer molecule or crosslinking agent comprises a reactive chemical group that can react with the airtix-FOLRl antibody or tragn‘tent f, in certain embodiments, ve ai groups for reaction with the cell—binding agent are ,NLSticciitimidyi esters and. Neuifosuccinimidyi esters. onaliy the linker molecule comprises a reactive chemical group, in certain embodiments a opyridyl group that can react with the drug to form a disuitide bond. in certain embodiments, ticker molecules inciude, for example, ccinimidyi 3«{2~ pyridyidithio} propionate (SPDP) (see, cg, Cartsson et ai‘, Biochem. J, {173: 723337 )g A’T—SUCCiHitnidyi 4-«(2:pyridyidithiokutaiioate (SPDB) (see, eg US: Patent No. 4,563,304): i\7l~3ttccininiidyi 4*(2*pyridyidithioiflm-suifobutanoate (sulfo-SPDB) (see US Publication No. 20090274713) , inimidyl 4—(2—pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry number 341498-08—6), 2-iminothiolane, or succinic anhydride.
Antibody-maytansinoid conjugates with non-cleavable links can also be prepared.
Such crosslinkers are described in the art (see ThermoScientific Pierce Crosslinking Technical Handbook and US Patent Application Publication No. 2005/0169933) and include but are not d to, N—succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), inimidyl—4-(N-maleimidomethyl)-cyclohexane—l- carboxy—(6-amidocaproate), which is a “long chain” analog of SMCC (LC—SMCC), K- maleimidoundecanoic acid N—succinimidyl ester (KMUA), B-maleimidopropanoic acid N- succinimidyl ester (BMPS), y—maleimidobutyric acid N-succinimidyl ester (GMBS), 8- maleimidocaproic acid N—hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl—N- hydroxysuccinimide ester (MBS), N-(oc-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6—(B-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4—(p- idophenyl)~butyrate (SMPB), and N—(p-maleimidophenyl)isocyanate (PMPI), N- succinimidyl(iodoacetyl)—aminobenzoate (SIAB), N-succinimidyl iodoacetate (SEA), N— succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP). In certain embodiments, the antibody is modified with crosslinking reagents such as succinimidyl 4—(N-maleimidomethyl)—cyclohexane—l-carboxylate (SMCC), sulfo—SMCC, maleimidobenzoyl-N—hydroxysuccinimide ester (MBS), sulfo—MBS or succinimidyl- iodoacetate, as described in the literature, to introduce l-lO reactive groups (Yoshitake et a1, Eur. J. Biochem, 101:395-399 (1979); Hashida et al, J. Applied m., 56—63 (1984); and Liu et at, Biochem, 182690-697 ).
The present invention includes s wherein about 2 to about 8 drug molecules ("drug load"), for example, maytansinoid, are linked to an anti-FOLRl antibody or fragment thereof, the anti-tumor effect of the conjugate is much more efficacious as compared to a drug load of a lesser or higher number of drugs linked to the same cell binding agent.
"Drug load", as used herein, refers to the number of drug les (e.g., a maytansinoid) that can be attached to a cell binding agent (e.g., an anti-FOLRl antibody or fragment thereof). In one aspect the number of drug molecules that can be attached to a cell binding agent can average from about 2 to about 8 (e.g., 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, .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 n embodiments, the drug is N21 deacetyl—NT-(3—mercapto-l-oxopropyl)—maytansine (DMl) or N27—deacetyl—N2’-(4-mercapto- 4-methyl-l-oxopentyl) maytansine (DM4). Thus, in a certain embodiment, the antibody huMovl9 is conjugated to DMI or DM4. In another embodiment, the antibody FR—l-21 is conjugated to DMl or DM4. In r embodiment, the antibody FR-l-48 is conjugated to DMl or DM4. In another embodiment, the antibody FR—1-49 is conjugated to DMl or DM4. In r embodiment, the dy FR—l—57 is ated to DMl or DM4. In another embodiment, the antibody FR—l-65 is conjugated to DMl or DM4.
X. ation of FOLRl sion and therapeutic efficacy ’ In certain embodiments, the invention provides a method for identifying subjects with an increased likelihood for responding to FOLRl-targeting anti-cancer therapies. The invention is based, in part, on the discovery that elevated FOLRl expression levels correlates with efficacy of FOLR-l -targeting anti-cancer therapeutics.
Evaluation of patient samples and correlation to in vivo efficacy using xenograft models demonstrates the power of the expression analysis for selecting subjects more likely to respond to treatment. IHC provides a score for FOLRl expression on tumor cells: 0 (no expression) to 3+ (very high levels of expression). In vivo data using xenograft models demonstrates that samples scoring 1, 2, 3, or 3+ for FOLRl expression, preferably a score of 2, 3, or 3+, have an increased likelihood to respond to FOLR—l-targeted anti-cancer therapies at clinically-relevant doses of FOLRl conjugates (e.g., 5 mg/kg xenograft -36~ dose of a FOLRI immunoconjugate can approximate a 185 mg/m2 in patients). Thus, identification of individuals having an elevated FOLRl score would help identify those duals who might respond to a clinically relevant . As described in more detail below, sensitivity to FOLRI eutics correlated with FOLRl scoring of 2 or higher, especially with level 3 scoring. Moreover, expression of more uniform levels of FOLRl provides better correlation with therapeutic benefit. Thus, a homogeneous staining uniformity is preferred but combinations of increased staining intensity with heterogeneous staining mity are also indicative of increased FOLRl expression. For example, scores of greater than 2 hetero is a patient selection criterion for treatment with a FOLRl therapeutic agent. {(31%} FOLRl expression analysis also identifies patients in whom decreased levels of a FOLRl-targeting anti-cancer therapy ("low dose therapy") can be effective to cause anti- tumor responses. As is appreciated in the art, compounds are generally administered at the st dosage that achieves the desired eutic response. This is specifically important for therapeutics that cause clinical, and often undesired, side effects. The ability to recognize those subjects with elevated FOLRl expression levels allows for minimization of the dosage of the FOLR-l—targeting therapeutic, thus decreasing le side effects, while ining therapeutic efficacy.
{M41} As shown herein, FOLRl expression scores of 2 hetero or greater correlate with increased siveness to anti—FOLRI immunoconjugates. In certain embodiments, the increased responsiveness is cachexia, increase in survival time, elongation in time to tumor progression, reduction in tumor mass, reduction in tumor burden and/or a prolongation in time to tumor metastasis, time to tumor recurrerice, tumor response, complete se, l response, stable e, progressive disease, progression free al (PFS), or overall survival (OS). In certain embodiments, FOLRl expression scores of 2 hetero or greater correlate with increasing PFS, DFS, or OS.
Kits for use in the detection methods and correlation to reference/control s can comprise control (positive and/or negative) or reference samples. The positive control or positive reference samples can be derived from tissue culture cell lines, normal tissue tumor tissue. Positive and negative reference samples can be d from cell lines including SW620, T47D, IGROV-l, HeLa, KB, EEG-3, other tumor cell lines, and cell lines stably or transiently transfected with an expression vector that encodes FOLRl. Normal or turner tissue samples and tissue culture eeii Eines can also be used as a negative central reference s. Fer additienai sampies, see Examples 9—} I and Figures 22.3»25.
XI. Pharmaceutical compositions and therapeutic methods {@143} FOLRl-binding agents (including antibodies, immunoconjugates, and ptides) are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as the treatment of cancer. In certain embodiments, the agents are useful for inhibiting tumor growth, inducing differentiation, reducing tumor volume, and/or reducing the tumorigenicity of a tumor. The methods of use may be in vilro, ex vivo, or in viva methods. In certain embodiments, the FOLRl-binding agent or antibody or immunoconjugate, or polypeptide is an nist of the human FOLRI to which it binds. {$144} In n embodiments, the disease treated with the FOLRl—binding agent or antagonist (e.g., a huMovl9 antibody or immunoconjugate) is a cancer. In certain embodiments, the cancer is characterized by tumors expressing folate receptor 1 to which the FOLRl-binding agent (e.g., antibody) binds.
The present invention provides for s of treating cancer comprising stering a therapeuticaliy effective amount of a inding agent to a subject (e.g., a subject in need of treatment). In certain embodiments, the cancer is a cancer selected from the group consisting of colorectal , pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, brain cancer, kidney cancer, prostate cancer, gastrointestinal , melanoma, cervical cancer, bladder , glioblastoma, and head and neck . In n embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the subject is a human.
The present invention further provides methods for inhibiting tumor growth using the antibodies or other agents described herein. In certain ments, the method of inhibiting the tumor growth comprises contacting the cell with a FOLRl-binding agent (e.g., antibody) in Vitro. For example, an immortalized cell line or a cancer cell line that expresses FOLRI is cultured in medium to which is added the antibody or other agent to inhibit tumor growth. In some embodiments, tumor cells are isolated from a patient sample such as, for example, a tissue biopsy, pleural effusion, or blood sample and ed in medium to which is added an FOLRl -binding agent to inhibit tumor growth.
In some embodiments, the method of ting tumor growth comprises contacting the tumor or tumor cells with the FOLRl-binding agent (e.g., antibody) in Vivo. In certain embodiments, contacting a tumor or tumor cell with a FOLRl-binding agent is undertaken in an animal model. For e, FOLRl-binding agents can be administered to xenografts expressing one or more FOLRls that have been grown in immunocompromised mice (e.g.
NOD/SCID mice) to inhibit tumor growth. In some ments, the FOLRE—binding agent is administered at the same time or shortly after introduction of tumorigenic cells into the animal to prevent tumor growth. In some embodiments, the FOLRl-binding agent is administered as a eutic after the tumorigenic cells have grown to a specified size. {$148} In certain embodiments, the method of inhibiting tumor growth compréses administering to a subject an therapeutically effective amount of a FOLRl-binding agent.
In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or has had a tumor removed.
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 tumor. In certain embodiments, the tumor is an ovarian tumor. {915%} In certain embodiments, the invention es methods of ting tumor growth using low doses of a FOLRl-binding agent. The term "low dose" as used herein refers to the eutically effective dose of a FOLRl—binding agent which is less than the usual or the conventional dose required to produce the therapeutic effect.
Thus, in certain embodiments the inventions provides methods of treating cancer using huMovl9 antibody and immunoconjugates. In n embodiments, the huMovl9 conjugate is huMovl9-SPDB-DM4; huMovl9-sulfo-SPP-DM1; 9—SPF- DMI; or huMovl9-PEG4-Mal-DM4. In a certain embodiment, the huMovl9 immunconjuage is huMovl9-SPDB-DM4, which is also referred to as IMGN853. {$152} In certain embodiments, formulations are prepared for storage and use by combining a purified antibody or agent of the present invention with a pharrnaceutically acceptable vehicle (e.g. carrier, excipient) (Remington, The e and Practice of cy 20th Edition Mack hing, 2000). Suitable pharmaceutically acceptable vehicles include, but: are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and nine; preservatives (e.g. cyldimethylbenzyl ammonium chloride; hexamethonium chioride; benzalkonium chloride; benzethonium chloréde; phenol, butyl or benzyl alcohol; alkyl parahens, such as methyl or propyl paraben; cateehol; resorcinol; cyclohexanol; 3~pentanoh and ItiaCI‘t‘fiSQl/i; low molecular weight ptides (cg. less than about it} amino acid residues); proteins such as serum albumin, gelatin, or oglohulins; hydrophilie polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, niarniose, or destrins; chelating agents such as Eli)’l‘A; sugars such as sucrose, mannitol, ose or sorbitol; saltmt‘onning counter—ions such as sodium; metal complexes (cg. Lin—protein complexes); and nic surfactants such as 'l‘W'lEElEN or polyethylene glycol (PEG). {eras} The pharmaceutical itions of the present invention, can be stered in Administration can be topical any number ot‘ ways for either local or systen'iic treatment. {such as to mucous membranes including l and rectal delivery) such as transdermal patches, ointntents, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary (cg, by inhalation or insufflation of powders or aerosols, including by nehulizer; intratracheal, intranasal, epidermal and transdennal); oral; or parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial leg, hecai or intraventricnlar) administration. {@154} An antibody or iimnunoconiugate of the invention can be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having ancer properties. The second compound of the pharmaceutical combination tonnulation or dosing regimen preferably has complementary activities to the ADC of the combination such that they do not adversely affect each other.
Pharmaceutical itions comprising the FOLRl-hinding a;\4 ant and the second anti» cancer agent are also provided.
U} U! For the ent of the disease, the appropriate dosage of an antihody or agent of the present invention depends on the type of disease to he treated, the severity and course of the e, the responsiveness of the disease, whether the antibody or agent is administered for therapeutic or preventative purposes, previous therapy, patient’s clinical y, and so on all at the discretion of the treating physician. ”l‘he dy or agent can he administered one time or over a series oftreatinents lasting from several days to several months, or until a cure is effected or a tion of the disease state is achieved {eg reduction in turner sire). Optimal dosing schedules can he calculated from measurements of drug accumulation in the hotly of the patient and will vary depending on the relative potency of -49“ an individual antibody or agent. The administering physician can easily ine m dosages, dosing methodologies and repetition rates. In certain embodiments, dosage is from 0.0] ug to 100 mg per kg of body weight, and can be given once or more daily, weekly, monthly or yearly. In certain embodiments, the antibody or other FOLRl-binding agent is given once every two weeks or once every three weeks. In certain embodiments, the dosage of the antibody or other binding agent is from about 0.1 mg to about 20 mg per kg of body weight. The treating physician can te repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or s. {eissi The combination therapy can provide "synergy" and prove "synergistic", i.e. the effect ed when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect can be attained when the active ingredients are: (l) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g. by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered er. {3157} Embodiments of the present disclosure can be r defined by nce to the ing non-limiting examples, which describe in detail 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 t departing from the scope of the present sure.
EXAMPLES It is understood that the examples and embodiments described herein are for illustrative puposes only and that various modifications or changes in light thereof will be ted to persons skilled in the art and are to be included within the spirit and purview of this application.
The Folate Receptor-l (FOLRl) has been reported to be highly expressed in ovarian tumors and expressed at high to moderate levels in brain, , bladder, endometrioid, -41, lung, pancreatic, and renal carcinomas. However, the expression of FOLK} is limited in normal tissues and includes kidney, lung, d plexus, pancreas, breast, thyroid? ovary, te, and lung. {316%} Methods have been reported that quantify FOLRl using fresh frozen tissue nates. Whole ”tissue homogenates cannot distinguish cytoplasmic from membraneassoeiated expression and y frozen samples are not amenable in the clinical setting.
However, formalin fixed paraffin embedded {FFPE} samples can be archived for patients in the clinic.
Exempie i {($161} lininunolzistoehemieal staining of FQLRl in cell, samples - manual s Fornialin fixed paraffin embedded eeli s and tissues were used as test samples with the following staining reagents and eoni‘litions. lHC Antibodies FFPE assay conditions l‘ """ W} MMWW...“ l..16?,_ Amihie—M...“_ l"SEB§H_NNMM““ COHdlthI’lfi' ' mmmm“ e mouse onal anti-hiiFOLRl Antigen val Borg (Biocaré) PH clone BN32 (Leiea # Ni; E.-L- lRalpha) Avidin/Biotin; : Blocking Steps iimmnnoflen: Piokai‘yotie inant mwwipergggie protein corresponding :0 189 amino Test or Control 2.0 ug/mL aeia‘ls ofthe external domain ofthe folate Article We“ a135131014111ij ipfluem mammo2%‘ lCOfllI‘G} A i i‘horseserurnfi {giggles} (miner: Clone we eeozsii Secondary Antib9dy l 10 ng/rnL ‘fieeondaryAnilmd‘i’ ‘Detection System* i AVidin-Biotin- i ieBiotinVlated horse anti-mouse (Veetor, peroxidase- l ione ijis1&le> Complex; ABC (Vector Labs) i i DAB(DakoL > 5 min E 39162} E‘onnaiin—fixed paraffin-embedded (FFPE) patient. ovarian tumor biopsies and ovarian xenogral’i tumors were stained with the murine anti-FQLRi antibody elone BNEQ, (Leica Cat 4N(.ll-lRalplia) and a conmi nulls“(3‘1 fioni Conner. Following antigen retrieval in pH 9.5 buffer, slides were blocked with 2% horse serum plus avidin. Slides were washed in PBS, and incubated at reein temperature fer {if} minutes with the attti~FOlRl er eentrel mitigGl antibetiy, followed by 30 minutes with laintinylated anti—meme lgsfi and 4G minntee with avidinbiotinperoxidase eemplex to detect bound secendary antibeiyi incubation for 5 minutes with DAB (33 xfifibtjl’lv’lflllle tetralmtreehlerid.) resultedin the color signal. Slides were eeenterstained with hematexyiin Ri staining intensity and distrihution patterns were seated relative to central lgG staining {_nenepeeifie). Intensity was sceie01 en a scale of O 1103 (O-==no staining, 1 : weak, 2 = moderate and 3 strong) and distribution was scored as focal (<25% ei’ cells stained), heterogeneous (25-75% of cells stained) and homogeneous (>75% of cells stained).
FFPE samples were d from tumor micro arrays, as well as human tissue blocks from seven different , as outlined below.
EFF}? Test Samples _\\“\“\\“\“““ i“lltrnan Tenet Miere \nays DthllpilOfi Commerc1al Source 5 il“A“ 96__cores from ”3 types of cancer3—3_________________________________________________ eMued tumet BiomaxCat# MC961 eOt-ariantame} _6_4_ cores _M a1nCat# T8235725t i$38le _ 9Ie__s_ B10maxCat#L% eCeleimtal ma :85 ate cores B10maXCat#BC000110 “——“T‘“” ElemMeme sNILraher cial So ce LeBreast 'l'nmeru etlel ereetal Carei “““““““ CHTN {$1653 The FOLRl test article, marine anti~FOLRi atone BN32, was tested to determine binding speeiticit‘v tn the huFOLRl antigi.1}. Using tne reperted IHC staining methods FFPE sections of 30049 and 300—19 transfeeted with huFCfiLRi (3004(Ii/FO'LRL) cell pellets were stained and evaiuated fer FOUU. The FOLRl test article specifically stained 300~l9/FOLR1+ cells and retumed tie staining in 38049 cells (3 home and negative, -43, respectively). These s demonstrate that clone BN3.2 specifically targets the huFOLRl n. (Figure l).
The BN3.2 antibody was also used to detect FOLRl expression on tissue samples.
The immunoreactivity of each test and control article with tissues and cell pellets was determined by the consulting pathologist, Dr. David Dorfman. The cell pellet controls were first evaluated followed by tissue samples. For each tissue evaluated, a ption of the staining intensity and staining uniformity was reported. The staining intensity score and uniformity scales are described below. The final reported score for each tissue sample ted is the score of the test article minus the score of the respective l article.
The ABC level for each sample was estimated by comparing the staining score to the calibrated cell pellet ls.
InLeHSIty(amountofstaln) l Tlnil‘urmiiy (nmnberafstained cells} : \ \“i 0 ve \ : lwwweak ,__M_, olNegatwe 2 Moderate FOC31¢E<2V Strong Heterogeneous(hetero)E2575/ """" """""""""veryStronrlHomogeneous(homrww>75/ {0157} Antibodies bound per cell (ABC) values were determined for the FOLRl-positive tumor cell lines (KB, IGROVl, JEG3, and ) using anti-FOLRl-Phycoerythyn'n, BD brite Beads, and flow cytometry and were shown to have different ABC values.
(Figure 2). Staining conditions were optimized for FOLRl so that the cell pellets prepared from the FOLRl-positive tumor cell lines showed varying levels of staining intensity by IHC. The KB cell pellet exhibited very strong (3+) homogeneous staining with high intensity, the IGROVl cell pellet showed strong (3) staining, the JEG3 cell pellet showed moderate (2-3) heterogeneous staining, while staining of the OVCAR3 cell pellet showed low intensity (1—2) heterogeneous staining. The FOLRl staining intensity trends observed from the cell pellets correspond to the reported ABC values, where KB cells exhibited the highest ABC value of 1,700,000, IGROV—l cells exhibited the next highest ABC value of 0, JEG-3 exhibited a lower ABC value or 41,000 ABC, while OVCAR3 cells -44_ showed the lowest ABC: value of 4,000. The staining results and respective ABt‘IZ vaiues are iisted in the table below.
ABC values and respective staining results for huFOLRl on cell lines and respective cell pellets.
FQLR i KB 1,709,000 EGROV} 260,000 3 heme a'B'EGS 4i ,sus 2~3 hetero UV’CARE E 4,000 Additional cell lines, including Capan-l, Jar, Hec—l-A, Hec—l—B, lshikawa, NCI H292, BT474EEI, PA-l, OV—90, CaOv-4, CaOv—E, A2780, Ovcar-S, Ovcar—4, HCT—lS, 786-0, NCI H838, NCI H 522, NCl H2110, NCI H1734, NCI H228, and FU.OV-3 were tested and found to be FOLRl positive but FOLRl sion levels and sensitivity to the anti-FOLRl immunoconjugate activity varied. For reference es, cell lines having consistent FOLRl expression and ivity to anti—FOLRI immunoconjugates are preferred. {0168} Particularly important was that the lHC method was able to reliable ed FOLRl expression in ovarian carcinomas and non-small cell lung cancer ) tissue s.
As shown in Figure 3, FOLRl expression could reliably be detected in ovarian carcinoma and NSCLC samples scored 2 hetero to 3 homo. ABC values for these samples ranged from approximately 41,000 for samples scored 2 hetero, to greater than 260,000 for samples scored 3 homo. As shown in Figure 4, high staining intensity and mity of ng was also observed in ovarian carcinomas, lung adenocarcinomas, and bronchioloalveloar carcinomas. Furthermore, expression of FOLRI in NSCLC samples (Figure 5) and in ovarian carcinomas (Figure 6) were further found to be predominantly zed to the membrane in tissue samples. Expression was detected across multiple samples from the same, as well as different tumor samples. Interestingly, none of the samples from colorectal, breast, or small cell lung tumors were scored greater than 2 hetero.
In vivo efficacy ofhuMov19-PEG4Mal-DM4 and huMov19—SPDB-DM4 ates in comparison with similar non-targeting conjugates in a KB xenograft model FOLRl—targeting cleavable conjugate huMov19-SPDB—DM4 in comparison with rgeting huC242—SPDB-DM4, and non-cleavable conjugate huMov19-PEG4-Mal- DM4 in comparison with non—targeting —PEG4Mal-DM4 were tested using an established xenograft model of KB cells (very high FOLRl expression, 3+ homozygous by manual IHC) ted subcutaneous into SCID mice. Mice were randomized by body weight into treatment groups and treated either singly (SPDB ates) on day 3 post cell inoculation, or three times weekly on days 3, 10, and 17 post cell inoculation with 5 and 10 mg/kg of a conjugate, respectively. The median tumor volume of the different treatment groups is plotted in Figure 7. The treatments with either huMov19—SPDB-DM4, or huMov19-PEG4Mal-DM4 ed in a decrease in median tumor volume as compared to the PBS control, while the treatments with either of the respective non-targeting conjugate did not produce any significant effect.
Example 3 Dose-response anti—tumor activity of IMGN853 treatment in OVCAR-3 human n carcinoma xenografts {317%} The anti-tumor effect of IMGN853 was evaluated in an established subcutaneous xenograft model of ovarian carcinoma. SCID mice were inoculated with 3 ovarian carcinoma cells (1 x 107 cells/animal) injected subcutaneously into the right flank. When the tumors d about 100 m3 in size (21 days after tumor cell inoculation), the mice were randomly divided into four groups (6 animals per group). Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS. Tumor growth was monitored by measuring tumor size twice per week. Tumor size was calculated with the formula: length x width x height x 1/2.
IMGN853 was highly active against 3 tumors (IHC score of 3 homozygous using manual IHC methods) in terms of tumor growth inhibition (T/C = 0 %) at both the 2.5 and 5.0 mg/kg dose levels (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 -46.. mice and CR in 4/6 mice d with IMGN853 at the 2.5 mg/kg dose level. 3 was active at the 1.2 mg/kg dose level, resulting in a T/C of 18%, with 2/6 PR and 1/6 CR.
According to NCI standards the T/C values ranging from 10% to 42% are considered to be active, T/C of less than 10% are considered to be highly active.
Example 4 Dose-response anti—tumor ty of IMGN853 treatment in IGROV-l human ovarian carcinoma xenografts. {017.2} The anti—tumor effect of IMGN853 was ted in an established subcutaneous xenograft model of ovarian carcinoma. SCID mice were inoculated with IGROV-l ovarian carcinoma cells (1 X 107 cells/animal) injected subcutaneously into the right flank. When the tumors reached about 100 mm3 in size (7 days after tumor cell inoculation), the mice were randomly divided into four groups (6 animals per group). Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS. Tumor growth was monitored by measuring tumor size twice per week. Tumor size was ated with the formula: length X width x height X 1/2.
IMGN853 was highly active against IGROV-l tumors (IHC score of 3 homozygous by manual methods) at the 2.5 and 5.0 mg/kg dose levels, resulting in T/C values of 5% for both dose levels (Figure 9). There were partial tumor regressions in 5/6 and 6/6 mice in the 2.5 and 5.0 mg/kg groups, respectively. IMGN853 was inactive at the 1.2 mg/kg dose (T/C = 47%).
Example 5 Dose-response anti-tumor activity of IMGN853 treatment in OV-90 human ovarian oma xenografts.
The anti-tumor effect of IMGN853 was evaluated in an ished subcutaneous xenograft model of ovarian carcinoma. SCID mice were inoculated with OV-90 ovarian carcinoma cells (1 X 107 animal) injected aneously into the right flank. When the tumors reached about 100 m3 in size (13 days after tumor cell inoculation), the mice were randomly divided into four groups (6 animals per group). Mice were d with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of -47r animals received a singie intravenous injection of PBS. A, control group of animals received PBS administered intravenously a". the same schedule. Tumor growth was monitored lay measuring tumor size twice per week. or size was calculated with the formula: length x width x height X 1/3..
{M75} lMGNSSZS was active against OV~9C¥ tumors {ii-EC score of 3 lretero~homo by manual methods) at the 2,5 and 5.0 rag/kg dose ievels, resulting in T/C values of 36 and. 18%.», respectively (Figure l0). ‘l‘wo s had partial, tumor regressions in the SiO trig/leg the treatment groups. EMGNSSB was group; there were no other tumor regressions in any of inactive at the 1.2 mg/kg dose (T/C = 77%).
Exanqfieo Dose-response anti-tumor activity of IMGN853 ent in SKOV-3 human n carcinoma xenografts. [0176} 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) ed subcutaneously into the right flank. When the tumors reached about 100 mm3 in size (26 days after tumor cell inoculation), the mice were randomly divided into four groups (6 animals per group). Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS. Tumor growth was monitored by measuring tumor size twice per week. Tumor size was calculated with the formula: length X width X height x 1/2. lMGNSfiS was inactive against SKOV—B tumors (lllC score of 1—3 focal by manuai methods) at all , with growth of ihlilGNiéSfi—treated tumors paraileling the PBS control group (Figure ll). There was no data analysis performed; and the study was temiinated early based on the inactivity or? lMGNSSCi in this model. esponse antiutumor activity of llvltiiN853 treatment in KB human cervical arcinoma xenografts.
{M78} The antiwtumor effect of lMGNSSES was evaluated in an established aneous xeuograft cervical adenocarcinoma model. SCED mice were inoculated with KB cervical ,4gr adenocarcinoma cells (1 X 107 cells/animal) injected aneously into the right flank.
When the tumors reached about 100 mm3 in size (7 days after tumor cell inoculation), the mice were randomly divided into four groups (6 s per group). Mice were treated with a single intravenous injection of IMGN853 at 1.0, 2.5 or 5.0 mg/kg. A control group of animals ed a single intravenous injection of PBS. Tumor growth was monitored by measuring tumor size twice per week. Tumor size was calculated with the formula: length X width X height X 1/2.
IMGN853 was highly active against KB tumors in terms of tumor growth inhibition (T/C = 0 %) at both the 2.5 and 5.0 mg/kg dose levels (Figure 12). Six of six mice in the 5.0 mg/kg and five of siX mice in the 2.5 mg/kg treatment group had CR5, and remained tumor- free to 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 lmmunohistochemical staining of FOLRl in formalin fixed paraffin embedded (FFPE) samples— ted methods. {0180} The IHC staining assay uses IVD class I reagents including the Novocastra FOLRl antibody (Novocastra/Leica Cat # NCI-L-FRalpha, clone BN3.2) as the test article and the Leica Bond RX automated r. Bound test or control article were detected by incubation wéth the Leica Bond Refine detection system which includes a post primary reagent (rabbit anti—mouse IgG), followed by a polyneer reagent (goat anti—rabbit polymer) and 3,3- Diaminobenzidine tetrahydrochloride (DAB) gen. FFPE samples were d with the specified concentration(s) of primary antibody (prepared by diluting in Leica diluent FOLRl) as outlined below.
IHC Antibodies wFolate Rec$8}'Kififig‘ZfiBQSEEQEE/Leica‘E‘éiQiiiicil‘fffiiigifiii? Test article” Lot 159506), murine, clone BN3.2, liquid concentrate: 75 ug/mL IgGl an Coulter Cat.# 6602872, LotsZS7SPSO4-23 Control article” and 2S7SPSO4-26) stock concentrationzl mg/mL, murine, clone 2T8—2F5 _49“ FFPE Assay Method using Leica Bond RX Amman Time minutes 1333515112 ?“ -— w:Perokide(Refine mdooenous Peiexjidasc Blank kit component)m5m1nuteswwm fest Arti‘cie F:0LR1 at 1.9 ug/mL1n Leica 15 minutesuwm diluent Winn“‘“"llw"mm““V“;N‘m‘mmmmary,, Reag€n¥kRefine‘ “““““““““““““ ‘ E kit) POWEW E'Iriiiiiitégmj lVIlXedDAB kit)“ W10mlnutesmm: :(JouriterstammwHematoxylm (Refine kit) "5mhiutes WM {$181} All stained samples were evaluated and scored. Control samples were first evaluated followed by test samples (whole sections and individual cores from the TMAs).
For each tumor tissue or cell pellet evaluated, a description of the staining intensity and respective proportion of tumor cells stained was reported. Membrane associated staining was recorded for every sample. When duplicate scores were evaluated from one patient, only the higher score was included in the analysis. If the score described only cytoplasmic staining then the final score was reported as zero (0). Intensity and uniformity were given to each sample as described in the table outlined below. Staining ity and distribution patterns were scored relative to control IgG staining pecific). Intensity was scored on t=== moderate and 3 E strong) and distrébution a scale of 0 to 3 (0 = no staining, 1 = weak, 2 was scored as focal (<25% of cells d), heterogeneous (25-75% of cells stained) and homogeneous (>75% of cells stained). In normal tissue, only the defined substructures were ted when calculating intensity and proportion.
IHC Scoring System ting of Intensity and Uniformity Scales Intensrty (brlghtness of stain) f intensity wed Intens1ty Category Ity Reported ‘ l O Negatlve {H Very Weak intensity (brivhmess QfSEain) RRoak to lviodercrte 2 Moderate 2—3 Moderateto Strong E9182} FFPE tumor samples were derived from tumor micro arrays, as well as human tissue blocks from seven ent tumors, as outlined below.
FFPE Test Samples: TMAs Anatomic Site Number of Total # of Cores per Patients Patient E Kidney Pantomics KIC1501 R-KID- 2 69 122711-1 1 Pantomics i LUC1501 EP-T-ARR-LNG— E 2 70 122711 1 E a “‘“EW” W ‘ 777777 Tristar 69571059/TA1249 E-PTARR-OVA- E ‘ E122711 1 : -5 RR-OVA- E 122111 1 a E OVClSOl EP-T—ARR—OVA- E 12271 l- l ...... WE.” “WWW...“ ...m E;...........».... 6957109l/TA1322 E-PT-ARR-OVA- E010912- l l ......"-—_ I .......«......“ ,, m““W E Uterus (Endo— Pantomics EMClSOl PT-ARR-EME- Emetrium) _WEWWMMM1227ll—l.. +._m“......“.....
Various Pantomics MTUZESTW P—T-ARR— E I 12271l—mlVVVVVVVVVV w...;..;.;(._;«.‘........... ...7...7....r.r....n..»““““an.muu...................._ «SE, FFPE Test Samples: Whole Sections .“xx“.“wunn“NV“...VVVVVV V.VVWVV_V_V__.“\“»““ Organ iCode Source Diagnosis (per Source Documentation) Ovary Tanflmijflu 1 Unknown endometroid adenocarcinoma ,V;;,,,,JV bJ genex g endometrord adenocarcinoma ,;,,,,;.;V,,, LN CHTN arcinoma, mixed with features of endomehod serous and clear W... VVVVVVVVVVVV“Wm 4 adenocarcmoma, high grade w/ mixed papillary, serous, g endometrloidandclear cell areas 5 i; serous papillaryadenocarcinoma Vme‘mmmcm“VVVVVWMM”...“me SBI‘OUS adenocarcinoma VVVVVrVr.r.r_r_““»»»‘“‘mme.
Proteogenex +.,,,,,,,,, ....................mm ‘ genex serous papillary adenocarcinoma 1 I1 VVVVVVVVVVVVVV.VVVsccVVVVVtcccccccccccVVVVV.aaass.......5cccccrr..............4 adenocarcmoma, poorly entiated adenocarcinoma, acinar welldifferentiatedwith bronchioloalveolar featuresa““Wuug.s........VVVVVVVV.VVVVVVVVV—r..““‘»»““““ml adenocarcmoma, mucinous features adenocarcinoma adenocarcinoma l «A. adenocarcinoma (bronchioloalveolar) carcinoma adenocarcmoma adenocarcinoma, moderately differentiated, with clear 3 cell features : l a t < t i ..s...‘.V..VVVVVVVVVVVVVVV.V~‘ccccc‘cceec.ccccVVVVaaaa....a......ss_..rr..rrrr‘.............t..VVVVVVVV_____Vv;; Cells (tumor cells or transfected cells) were formalin fixed and paraffin embedded (FFPE). FFPE cell pellet samples shown to exhibit varying ranges of FOLRI sion by flow cytometry and normal human tissues were used in this study to characterize ve and negative controls and for is of specificity. The cell pellets exhibiting varying levels of FOLRl and the respective scores are reported below. There is a poor correlation between staining scores and the respective FOLRl expression levels (antibodies bound per cell, ABC, determined by calibrated flow cytometry) in the cell pellets. For example a score of 1—3 hetero is given for the SW620 and l exhibiting 40,098 and 565,481 ABC values, respectively. Additionally, Hela cells showing an ABC value of 1.5 million resulted in a score of 2-3 hetero while 300.19/FR1 exhibiting 830,003 ABC returned a higher score of 3 homo.
Final Scores for Celi Pellets at a Test e Concentration of 1.9 ug/mL § {39.31 Line aABC Value rrrrrr “1:“Staining Score »“““ Wu““u....n~“~“vw_»__rrrrrrrrrr w—v—_ § 3 83137621} 411,888 I-3 hetero T471) l -2 hetero —(nun........“WW“.“way.“u.»»»‘ “.Tmfié Wuuuu.«....VVwwwww«Whe‘““»»»“‘Wu—— _ 1URUV 1 L565,481 1-3 hetero g 30019/ FRl 830003 3homo E HBEA E1 500587 tero KB "14,000,000 3homo aThe reported ABC Valueis anaverageofant1bod1es boundpercell1n thecellpopulation and“ was determined as follows: a concentration of 1.0 X 108M of anti—FOLRl ~PE (1 :1) was used ine ABC values on the respective cell line using flow cytometry s and Quantibrite TM Beads (BD Biosciences).
- The flow cytometry histograms represent the distrébution of cells versus the number of anti-FOLRl bound per cell (FOLRl expression level). Both the histograms and respective IHC staining results indicate that each of these cells lines contain a heterogeneous tion of cells having a broad range of FOLRl expression. The exception is the 300.19/FR1 cell line showing botl: a m flow cytometry histogram and IHC staining score. This data suggests that cell lines each expressing a more m level of FOLRl may provide a better correlation between ABC values and respective staining scores. Although the assay demonstrated positive staining in all FOLRl-positive cell pellet controls, there is a poor ation between staining scores and the respective FOLRl expression levels from most of these cell pellets. Therefore, cell pellets from this group could not be identified as high-, medium—, and low-expressing controls. Representative photographs and rams depictirzg FOLRl expression in cell lines by IHC and flow cytometry are shown in FIGURE 13. {111353 To determine assay conditions, a range of dilutions of test and control article were tested to select conditions that exhibit an appropriate level of sensitivity. Experiments were -53.. performed on a panel of FFPE samples including FOLRl-positive cell s and a TMA consisting of FOLRl positive and negative normal tissues (adrenal (cortex/medulla), breast (ducts and s/connective ), fallopian tube (surface epithelium/muscle wall), kidney (tubules/ glomeruli), lung (type I/Il pneumocytes/interalveolar connective tissues), pancreas (ducts/islets of Langerhans), salivary gland (ducts/stroma), skin (eccrine /epidermis), stomach (surface epithelium/submucosa)), and whole sections of tumor s (10 ovarian tumor samples and 10 lung tumor samples). Each sample was stained with a serial dilution of test article concentrations (0.25, 0.5, 0.9, 1.9, 3.8, and 7.5 ug/mL) or control article concentration of 1.9 ug/mL or 9.8 ug/mL. The relative staining intensities for each dilution were compared for each sample to identify the optimal dilution. The criteria for optimal dilution was a dilution which 1) caused no background staining in samples stained with isotype control 2) caused no ng in negative tissue controls stained with test article and 3) differentiated between varying levels of membrane-associated FOLRl expression among test samples representing the indication of interest (ovarian tumor, endometrial tumor, NSCLC tumor, and kidney tumor FFPE tissues). Of the five dilutions of test article evaluated, the tration of 1.9 ug/mL showed the best dynamic automated protocols (Bake and Dewax range in staining results using the Leica Bond RX Protocol, HIER using the ER2 for 20 minutes protocol, and the staining protocol IHC F- With Extra Rinses).
Example 9 Identification and terization of controls that characterize the dynamic range of the assayautomated ng methods Quality controls: Human normal salivary gland, lung and pancreas were identified as positive tissue ls to be ed in each assay to verify that the staining procedure performed as expected. Human normal esophagus was identified as a negative control.
These controls were characterized as follows: in order to establish ls which cover the dynamic range of the assay, a tissue microarray (TMA) consisting of several FOLRl ve and negative normal tissue s expected to exhibit the dynamic range of the assay was used as an assay verification control during the optimization and validation phases. Four normal tissues with identified structures in this TMA were identified as suitable assay controls as follows: respiratory epithelium of normal human lung (score of 2 homo); ducts of normal human pancreas (score of " homo apical); intercalated ducts of normal human salivary gland {score of l~2 hetero); and normal human esophagus (score of 0) Over a total of 5; assay runs the identified le assay controls from this TMA gave identical results. These results indicate that the selected ls give consistent results and span the dynamic range of the assay. ures in Normal ’if'issues identified as Controls that Span the Dynamic Range of the Assay ENtirin;E iii}Structure StainingScoremiim Staining Scorefiest‘i E'G:gen : (controi article) article) EEsophagiix kill stmctnre; 0 ive) Efiiiicganm; livaryGland lotei‘calated ducts NEW0 (negative) WE i~2 here i WWW RespiratoryEpitheimvo‘iElhomo NPantria:---~WEDncts (E) {negati“3)meMMwE”honiowapitai [\rpicaE staining is definedas polarized nonuinfmm membrane staining e 10 Perfom‘iance analysis oftite automated staining method. {$187} The intended use of this assay is to specifically detect FOLRl reproducibly and with the appropriate sensitivity to differentiate varying levels and varying mity of membrane—associated FOLRI expression (optimal dynamic range) in ovarian, endometrial, NSCLC, and kidney FFPE tumor tissues. Therefore, specificity, reproducibility, and sensitivity were considered as mance ia.
The specificity and sensitivity of the study assay was evaluated by comparison of normal tissue ng with the study assay to previously reported results. Staining s frorn this study were compared with corresponding ng results from Scorer et al 2010 (A Fall Immunohistochemical Evaluation of a Novel Monoclonal Antibody to Folate or alpha. The Novocastra Journal of Histopathology, REAGENTS: 2010(3):8-12, describing the same antibody clone BN3.2) with FFPE normal tissue and from the Tissue Cross Reactivity (TCR) study using IMGN853 (huMovl9 (M9E46A) antibody) on fresh. frozen normal tissue (ImmunoGen Report lMH28—OO3). Comparison of the staining results from each method indicate that the three assays showed generally similar normal tissue staining profiles with differing relative sensitivities, with the Scorer assay being least sensitive, the study assay (IMH28-011) having intermediate sensitivity, and the TCR study method being the most sensitive. Some structures showed positive staining in the two more ive methods (study assay and TCR assay) only. There were no examples of positive staining in the least ive assay used by Scorer that were not also positive in the study assay and TCR method. These results demonstrate that the specificity and sensitivity of the study assay is appropriate for the evaluation of FOLRl sion in normal tissues.
The specificity and sensitivity of the study assay was further characterized by staining and evaluating a panel of tumor TMAs consisting of ovarian, endometrial, NSCLC, and kidney tumors (a sample set representative of the assay’s intended clinical use).
Positive staining was tently localized to the tumor tissue with normal adjacent tissue components including , blood vessels, lymphocytes and normal organ tissue staining negative or positive as expected. For each subtype of either ovarian carcinoma or NSCLC, the distribution of staining scores among TMAs from the different vendors showed a similar distribution of scores ting this method is not sensitive to various fixation and processing conditions. Because the distribution patterns were similar among the TMAS, the data from the different arrays was combined and scores were categorized. A summary of these scores for tumor subtypes that contained 20 or more samples per subtype are listed in the following tables. As summarized in these tables, a dynamic range of scores is noted for each tumor type and indicates that this assay shows the appropriate sensitivity to distinguish varying levels and varying uniformity of membrane—associated FOLRl expression in ovarian, endometrial, NSCLC, and kidney FFPE tumor tissues. Representative photos of serous n, endometroid ovarian, NSCLC, endometrial carcinoma, and renal clear cell carcinoma are ed in s 14-18. Additional representatitive photos useful, for example, in a staining guide or diagnostic kit, are shown in Figures 23—25. These studies indicate the assay is specific and has the appropriate sensitivity for use as a diagnostic or companion diagnostic reagent.
Summary of Staining Scores for Predominant es of Ovarian Tumors Subg"“p““;""" “E‘Silifiglvumser 1 We) ~56- Eff} RS l)Focal staining patterns were excluded Summary of Staining Scores for NSCLC Tumors Sample Number §Tota1 23 722 hetero"A heteroa = L Fugitiww iAdeho- A11b s 47 20 icarcmoma $100) (25) . (58) Spec1fied 7 Bronchiolo- ~~ (100)(29) ;alveolar ! i (71) w 7777777777 gm“...”EmrrrrrrrrrrL “71; Squamous All 74 l 100 1 5 oma i in )5;()W: ( ) ) Focal stainlng patterns were excluded All adenocarcinoma s were included except the specified bronchioloalveolar carcinoma samples _ 57 Summary of ng Scores for Adenocarcinoma ofthe Endometrium and Clear Cell Tumors of the Kidney WW, “uh...“‘e Tumor/Subflpe i Sample Number 1:%) i Exec“..‘.\w.m.n.}..~m......m..V“w.wV__vmv.r.““““ee» m“ i mm...“tT."nmfimmzzzrfiteeee i a Total E 23 _2 2 1 1-3 focal k 3 Any Negative i E heteroa heteroa “ : Positivity l : ‘ We.“a»... .w...wmvw.‘wr .ze..““‘““»“““ ._._.......... wfivw eeeeecee‘“ i Endometnum/ 58 5 3 E 30 10 40 18 i Aden0_ u..u“~§un~~finwwwmea »»»»»“““““ E un“......u“ui,u.“w“vVV__-;;;=A.x»§\»»\\\.“W (100) (9) 40) E (52) (69) (31) carcinoma a {gvvvvvv,_ w ,,,,,, t 3 Cell (100) (0) E (68) (18) (85) (15) WWW “WW“ FoEai“;i;1‘Hng§aEEfi wereieileiiiied The precision of the study assay was igated by evaluating intra—run and inter- run reproducibility of the assay using three FFPE tumor tissue samples of ovarian, NSCLC, or kidney tumor where each sample exhibits either a high, medium, or low score. For intra~ run reproducibility, nine slides each containing a section of lung, ovarian, and renal tumor were placed at nine random locations on the Leica Bond RX. For inter-run reproducibility, three slides containing sections from the same sample were d on three different days.
All slides from both intra-run and inter—run reproducibility experiments were evaluated and showed equivalent staining results for each respective sample: lung tumor (high: 3 homo), ovarian tumor (medium: 2 hetero), and renal tumor (low: 1-2 hetero). This data demonstrated ucibility across tissue types with low, medium and high level of expression.
Example 1 l A FOLRl expression score ofZ 2 geneous by IHC is a patient selection criterion for treatment with IMGN853.
The levels of FOLRl-expression in tumor cell lines were determined using the an antibody-l)E conjugate 4-PE) and the QuantiBRlTE system. Three ovarian carcinoma cell lines (Igrov—l, Skov-3 and Ovear-3), a choriocarcinoma cell line leg-3 and a cervical carcinoma cell line KB were included in the study. In order to obtain reliable ABC values, the binding experiments with an antibody—PE conjugate should be performed at a -58— saturating concentration (concentration, at which all available binding sites are occupied by the conjugate). To determine such concentration for the -PE conjugate, we performed binding experiments on a panel of FOLRl—positive cell lines with various FOLRl expression. The cells were incubated with a wide concentration, range of FRl—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 FACSCalibur flow cytometer. At a concentration of 1x10'8 M the conjugate saturated cell e binding sites on all tested cell lines lgrov-l, Jeg-3, Skov-3, Ovcar-3, and KB. In subsequent binding ABC-experiments -PE conjugate was used at concentration of 1x10"8 M. Each sample was analyzed in triplicates; several independent experéments were performed on each cell line. The highest expression was found on KB cells with the approximate ABC value of 4,000,000i300,000, followed by Igrov-l and Jeg-3 cell lines with the ABC values of 400,000i85,000 and 150,000i75,000, respectively. Two cell lines, Skov—3 and Ovcar—3, had low FOLRl expression, 20,000i10,000 and 7,00014,000 ABC, respectively. A cant experimentto-experément variation of ABC values was observed for Jeg—3 cells, where the ABC—values varied from 40,000 to 300,000. This varéability likely reflected some biological properties of the cell line rather than the assay ility, since ABC values obtained for the other analyzed cell lines were much less variable (see table below).
“Celi‘line l i ABC Experiment--toexPehEIehi‘QQIii {Mean i ‘53» it)” The highest ABC The lowest ABC , ,1 i 1 ered registered KB 00 3:300000,4 4,500,000““ WW3800000W ,, ‘i‘ Igrov-l % 400,000j:85000 5 480,000 0 Jeg-3 150,000 i:75,000, 14 imwwm 40w000w Skov—3“ r “3: 10,000 2m 10,OOOWWWWa SDStandarddev1ation n- number ofindependent experiments ABC values were determined by a FACS based assay with FR1-24 PE—labeled antibody and QuantiBRITE system. Mear: i Standard Deviation (SD) was calculated for independent experiments.
E8192} Potency and specificity of 1MN853wwas ed against FOLRE ~positive teii lines with a wide range of FOUR} expression (the ABC values of the cell lines are provided above) in addition FOL-R1negative ceiii lines Namaiwa and SWZ were included in the experiments. IMGN853 was highly cytotoxic against cells with high FOLRl expression KB (4,000,000i300,000 ABC), Igrov—l (400,000zt85,000 ABC) and Jeg—3 (150900171000 ABC), with the ICso values of 0.10i0.01 nM, 0.50i0.07 nM and 1.00i0.05 nM, respectively. The cell-killing activity against all three cell lines was FOLRl-dependent, since an excess of unmodified 9 (M9346A) antibody (0.5 uM) ly decreased potency of the conjugate to the typical non-specific levels (from 10 to 20~fold). 1MGN853 was only marginally active against the low FOLRl expressors Skov-3 and Ovcar-3 cells (20,000::10,000 and 7,000i4,000 ABC, tively), and against FOLRl-negative cells Namalwa and SW2, with the IC50 values greater than 2 nM. The cytotoxic activity of IMGN853 against these cell lines was low and not FOLRl-dependent, as blocking with huMovl9 (M9346A) did not affect it. See Figures 19 and 20.
FFPE samples prepared from mouse aft tumor models were evaluated for FOLRl positivity using the zed and validated assay described above. No staining was seen in tumor cells of any xenograft samples stained with control article. FFPE mouse xenograft tissues derived from the ing cell lines showed the following staining patterns: lgrov-l, KB, and 110 showed neous staining patterns with level 3 intensity; Ishikawa and Ovcar 3 showed heterogeneous staining patterns with level 3 intensity; LXFA737 showed homogeneous staining patterns with level 2 ity; OV-90 showed heterogenous patterns with level 2 intensity; and SKOV3 was negative.
Representative photos of tumor xenografts are provided in Figures 21 and 22. -60— E\ParentalN Disease‘ EFlnalbcore "SEEMS; Cell Line E Indication E Category or Tumor E IGROV—I EOvarian cancerE I —3 homo E 3 homo E l-3homo 7 E E1-3 homo Ishikawaum‘EndometnumE2-3hetero 7777777777 E3hetero cancer 1 2‘hetero/3 focal E E 2 hetero/3 focal i E2 hetero/3 focal , , “ “E‘éFVmical E 3 homo Ehomo cancer E 3WW mm we 1'2 hdfiiB'Ziiiiii........ 2 homo NCI— 2—3 homo” EE‘homo H”Ovarian OV-90 cancer 1-2 hetero 3:16:30 """"""WghetemE OVCAR3 63mm cancer 1333% hetero “ E Ovarian cancer Negatlve Negat‘Eémi Negatlve i A staining threshold (22 heterogeneous) requires both a minimal level of expression (staining ity) and m distribution of staining ntage of tumor cells expressing . Pre-clinical data es justification for this threshold in ovarian carcinoma. Mouse xenograft tumor samples with IHC scores of 2 2 heterogeneous exhibit sensitivity to IMGN 853 in vivo. FFPE samples prepared from mouse xenograft ovarian tumor models were evaluated for FOLRl positivity using the optimized and validated assay described above. Two n carcinoma xenograft models OVCAR-3, and IGROV— I showed a heterogeneous or homogeneous staining pattern with level 3 ity. The xenograft model derived from OV-9O ovarian carcinoma cells showed a heterogeneous ng pattern with level 2 intensity; the Skov-3 ovarian carcinoma model was FOLRI- negative. IMGN853 was highly active in the two ovarian models with level 3 FOLRI intensity and active in the OV—9O model with level 2 FOLRI intensity. No activity was observed in the SKOV-3 model. Xenograft models were also evaluated for other diseseas ~61— indications including lung, endometrium, and cervical tumors and, although correlations were ed between activity and FOLRl ng scores, additional samples must be tested. {@195} The sensitivity of ovarian tumor aft models to IMGN853 versus the Eevei of FOLRI expression Xenograft In vivo activity (5 mg/kg of Intensity score, distribution IMGN853, single dose) w, Mew...“ ,, OVCAR3 Highly active E - 3 heterogeneous E “mmmm... ,, u...mummw{momenmtme E IGROV-l Highly active E 3 homogeneous OV-90 Active 2 heterogeneous E SKOV-3 s Inactive Negative E = : The sensitivity of other tumor xenograft models to IMGN853 versus the level of FOLRl expression In vivo activity (5 Eng/kg 0f Intensity score, E IMGN853, single dose) distribution E Highly active 3 homogeneous lshikawa Eindometrium Inactive 2 homogeneous E E: ooooo WWW KB Eilervical Highly active 3 homogeneous E :“WWMMMWWHW All publications, patents, patent applications, intemet sites, and accession numbers/database sequences (including both polynucleotide and polypeptide ces) cited herein are hereby orated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, et site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.
- SEQEENCES SEQ ID NQt’i — huxtnan foiate mr 1 MAQRLMTI‘QLLLLLV“WAX/WGEESAQTR{AWAR'I‘ELLNVCMNAKBHKEKPGPEZEQKLHEQCR PWRKNACCS’ENTSQBAHKD‘VSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNE...GE’Wifi QQVDQSWRKERVLNVPLCKEEIXIEQWWEDCRTSYTCKSNWHKGWNW/"1‘86ENKCAVGA ACQPFHFY}???PTVLC’NEIWTHSYKVSNYSR‘Ci‘SG-KCEQMWFDRKQGNPNEEVAKE1‘Yl’fL/XAl‘s/i SGAGPWAA‘WPF1,,I,,S’3L,.AE.,MILLWLLS SEQ ID N02 ~ human fi‘fiate receptor 1 nudeic acid Sequsnce atggctcagcggatgacaacacagc;tgGigdeem:tagtgigggtggctgtagtaggggaggctcagacaaggattgcatgggccaggact gagcttcicaaigtctgcatgaacgccaagcaccacaaggaaaageGag,gccccgaggacaagttgcatgagcagtgEcgamctggagga agaatgectge‘igttctavmaaaaccagecaggaagcccataaggatgtttfict3cata‘iatagaficaactggaaccactgtggagagatggca. cctgcctgcaaacggcamcatcc21ggacacctgcctctacgagtgctcccacaacfiggggccctggatccagcaggtggatcagagctgg cgcaaagagcgggtactgaacgtgcccctgigcaaagaggactgtgagcaatggtgggaagafigtatgcacc‘tcctacacctgcaagagcaa ctggcacaagggctggaactggacttcagggtttaacaagtgcgcagigggagctgcctgccaacctficcatfictacficcccacacccactg caatgaaatctggactcacit.etacaaggtcagcaactacagccgagg{Iagtggccgctgcatccagatgtggficgacccagccca gggcaacaccaatgaggaggtggcgaggm:iatgctgcagccatgagtggggctgggccctgggcagcctggcc{itcctgcttagcctgg ccciaatgctgctgtggctgctcagc SEQ 11} N03 — huMOViQ VHC QVQLVQSGAE‘VVKPGASVKESCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFY NQKFQGKA‘E‘LTVDKSSNTAHMELLSLXIT‘SEDEAVYYCTRYDGSKAMDYWC:QG'I‘TVTVSS SEQ 113N024 ~ huMevw VLCV} .00 DEVLTQSPLSLAVSLGQFAHSCKASQSVSPA Ts’l‘S LMH'WYHQKPGQQPRLLIYRASNLEA(3V PDRFSGSGSKTDETLN 3 SP VELA'EEEILFAxtXT‘x’YCQQSREYPYTFGGGI‘KJLLEEIKR SEQ ID NOIS ~ huMQv19 O DI V1.30:8 3?1.;S’LAVSLGQPAHSCKASQSVSFAG’E‘SLMHWYHQKPGQQPRIZJiEJYKASNLEAGV PDRFSGSGSK3715)FTLHSPVEAEDAATYYCQQSREY}?YI'FGGGTKLEEKR A global biosciencc nonprofit organization dedicated to biological standards and biodiversity IP, Licensingiand-Serviccs 10801 University Boulevard -2209 USA Mammals, ngim'azo 1 Telephone: (800) 638—6597 facsimile: (703)334—2932 Internet: hrrpzjlwwwmtoaorg min-Wm“ mwwmmawW'tmmw gs»sexesemsamxwwwwswsiswiuessua-memmmsmmwm:_';‘m\wtt\3\v;§s;\§saatmmemwmOF DEPOSIT BUDAPEST RESTRICTED CERTIFICATEINTERNATIONALRECOGNITION OF BUDAPEST TREATY ON THE OF PATENT URE FOR THEPURPOSES THE DEPOSIT OF MICROORGANISMSINTERNATIONAL FORM TO. RULE 7.3 ‘ DEPOSIT ISSUED PURSUANT RECEIPT IN THE CASE OF AN ORIGINAL 10.2 ISSUED PURSUANT TO RULE AND lTY STATEMENT in connection received your deposit of seeds/Strain(s)lstrain'(s) Coilection (ATCC®) has The American Type Culture is provided to fulfill Patent Officerequirements.
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Claims (47)

WHAT IS CLAIMED IS:
1. A method of identifying a tumor as sensitive to treatment the method comprising: 1 (FOLRl) antibody or antigen binding fragment thereof, tissue sample obtained from the (a) ing the level of FOLRl expression in a tumor wherein the measuring comprises the use of a detection method that tumor, distinguishes between ng intensity and staining uniformity in a FOLRl- and staining uniformity in expressing cancer sample as compared to staining intensity one or more nce samples; tissue sample; and (b) determining a FOLRl staining intensity score for the tumor reference (c) comparing the FOLRl staining intensity score ined in step (b) to a value determined by measuring FOLRl protein expression in at least one reference is a tissue, cell, or cell pellet sample sample, wherein the at least one reference sample or antigen binding which is not sensitive to treatment with an anti-FOLRl antibody fragment thereof, determined in wherein a FOLRl staining intensity score for the tumor tissue sample step (b) that is higher than the reference value identifies the tumor as being sensitive binding fragment thereof, and treatment with an OLRl antibody or antigen for treatment wherein the anti-FOLRl dy or antigen g fragment thereof the amino acid sequence GYFMN (SEQ comprises (i) a heavy chain (HC) CDRl comprising the amino acid sequence RIHPYDGDTFYNQKFQG ID NO:6); a HC CDR2 comprising the amino acid sequence YDGSRAMDY (SEQ (SEQ ID NO:7); and a HC CDR3 comprising (LC) CDRl sing the amino acid chain sequence ID NO:8) and (ii) a light the amino acid sequence KASQSVSFAGTSLMH (SEQ ID N029); a LC CDR2 comprising RASNLEA (SEQ ID ; and a LC CDR3 comprising the amino acid sequence QQSREYPYT (SEQ ID NO] 1). or antigen binding
2. The method of claim 1, wherein the anti—FOLRl antibody amino acid HC variable domain comprising the nt thereof for treatment comprises (i) a the amino acid ce NO:3 and (ii) a LC variable domain comprising sequence of SEQ ID of SEQ ID N024 or 5. or binding
3. The method of claim 2, wherein the anti-FOLRl antibody antigen acid sequence fragment thereof for treatment ses (i) a HC comprising the same amino -68— d by the plasmid ted with the American as the amino acid sequence of the HC Type Culture Collection (ATCC) as PTA—10772 (ii) a LC comprising the same amino encoded by the d deposited with acid sequence as the amino acid sequence of the LC the ATCC as PTA-10773 or PTA—10774. with anti—FOLRI
4. A method of identifying as sensitive to treatment an a tumor immunoconjugate, the method comprising: tissue sample obtained from the (a) measuring the level of FOLRl expression in a tumor measuring ses the use of a detection method that tumor, n the intensity and staining mity in distinguishes between staining a FOLRI and staining uniformity in expressing cancer sample as compared to staining intensity one or more reference samples; the tumor tissue sample; and (b) determining a FOLRl staining intensity score for reference (c) comparing the FOLRI ng intensity score determined in step (b) to a value determined by measuring FOLRI protein expression in at least one reference is a tissue, cell, or cell pellet sample sample, wherein the at least one reference sample anti-FOLRI immunoconjugate, which is not ive to treatment with an determined in wherein a FOLRI staining intensity score for the tumor tissue sample identifies the tumor as being sensitive to step (b) that is higher than the reference value treatment with an anti—FOLRI immunoconjugate, for treatment has the formula (A) ~ (L) — wherein the anti-FOLRI immunoconjugate (C), wherein: thereof (A) comprises an anti—FOLRI antibody or antigen binding fragment amino acid sequence GYFMN (SEQ ID NO:6); a comprising (i) a HC CDRl comprising the RIHPYDGDTFYNQKFQG (SEQ ID NO:7); HC CDR2 comprising the amino acid sequence (SEQ ID N021 1), LC CDR3 comprising the amino acid sequence QQSREYPYT (L) comprises a linker, (C) ses a cytotoxic agent, and wherein (L) links (A) to (C).
5. The method of claim 4, wherein the anti-FOLRl antibody or antigen binding HC variable domain comprising the fragment thereof of the immunoconjugate comprises (i) a amino amino acid sequence of SEQ ID N023 and (ii) a LC variable domain comprising the acid sequence of SEQ ID NO:4 or 5. method of claim 5, wherein the anti—FOLRl antibody or antigen binding
6. The (i) amino a HC comprising the same fragment thereof of the immunoconjugate comprises HC encoded by the plasmid deposited with acid sequence as the amino acid ce of the acid sequence as the the ATCC as PTA—10772 and (ii) a LC sing the same amino PTA— the plasmid deposited with the ATCC as amino acid sequence of the LC encoded by 10773 or PTA-10774. wherein the linker is selected from the group
7. The method of any one of claims 4—6, a dicarboxylic acid based consisting of: a hydrophilic linker, and a eavable linker, linker. of: N- linker is selected from the group consisting
8. The method of claim 7, wherein the (SMCC); N—sulfosuccinimidyl 4- succinimidyl 4—(maleimidomethyl) cyclohexanecarboxylate (maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC); N-succinimidyl—4—(iodoacetyl)- aminobenzoate (STAB); and N-succinimidyl—[(N-maleimidopropionamido)— tetraethyleneglycol] ester (NHS-PEG4—maleimide). wherein the linker is a cleavable linker.
9. The method of any one of claims 4—6,
10. The method of claim 9, wherein the ble linker is selected from the group consisting of: N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP); N—succinimidyl 4-(2- pyridyldithio)~2~sulfopentanoate (sulfo—SPP); inimidyl 4-(2-pyridyldithio)butanoate (sulfo—SPDB). (SPDB); and N-succinimidyl 4-(2—pyridyldithio)sulfobutanoate linker is N—succinimidyl 4—(2-
11. The method of claim 10, n the cleavable pyridyldithio)sulfobutanoate -SPDB). wherein the cytotoxic agent is selected from
12. The method of any one of claims 4-11, analog, iazepine, taxoid, CC— the group consisting of: a sinoid, maytansinoid dolastatin, 1065, CC-1065 analog, duocarmycin, duocarmycin analog, calicheamicin, and leptomycin derivative or a prodrug dolastatin analog, atin, tomaymycin derivative, of the cytotoxic agent. is a maytansinoid.
13. The method of claim 12, wherein the cytotoxic agent wherein the agent is N(2’)-deacetyl-N(2')-(3-
14. The method of claim 13, cytotoxic mercapto—l—oxopropyl)—maytansine (DMl) or N(2')—deacetyl-N(2')—(4-mercapto-4—methyl—1— tyl)-maytansine (DM4).
15. The method of claim 13, wherein the linker is N-succinimidyl 4-(2—pyridyldithio) sulfobutanoate (sulfo-SPDB), and wherein the cytotoxic agent is N(2')—deacety1—N(2')-(4- mercapto-4—methyl- l -oxopentyl)—maytansine (DM4). binding or
The method of claim 13, wherein the anti—FOLRl antibody antigen amino comprises (i) a HC comprising the same fragment f of the immunoconjugate with of the HC encoded by the plasmid deposited acid ce as the amino acid sequence amino acid sequence as the the ATCC as PTA—10772 and (ii) a LC comprising the same ATCC as PTA- the plasmid deposited with the amino acid sequence of the LC encoded by 774, wherein the linker is N—succinimidyl 4—(2—pyridyldithio) 10773 or the agent is N(2‘)~deacetyl—N(2')-(4- sulfobutanoate (sulfo—SPDB), and n cytotoxic mercapto—4-methyl—l —oxopentyl)-maytansine (DM4). is performed
17. The method of any one of claims 1-16, wherein the detection method is performed
18. The method of any one of claims 1—16, wherein the detection method using an automated . of claims 1—18, n the detection method is
19. The method of any one immunohistochemistry (IHC). is calibrated IHC that can distinguish
20. The method of claim 19, wherein the IHC different levels of FOLRl expression, _ 71 the tumor tissue sample is a formalin
21. The method of claim 19 or claim 20, wherein fixed paraffin embedded sample.
22. The method of any one of claims 1-21, wherein the detection method produces a for samples having weak FOLRl expression, moderate FOLRl range of staining intensity sion, or strong FOLRl expression. has a
23. The method of any one of claims 19-21, wherein the tumor tissue sample FOLRl expression by IHC. ng intensity score of l or greater for tissue sample has a staining uniformity
24. The method of claim 23, wherein the tumor for FOLRl expression that is heterogeneous. tissue sample has a staining uniformity
25. The method of claim 23, wherein the tumor for FOLRl expression that is homogenous. tissue wherein 25-75% of cells of the tumor
26. The method of any one of claims 19—21, of l or greater. sample have a staining intensity score of cells of the
27. The method of any one of claims 19-21, wherein greater than 75% score of l or greater. tumor tissue sample have a staining intensity has a
28. The method of any one of claims 19-21, wherein the tumor tissue sample for FOLRl sion by IHC. staining ity score of 2 or greater the tumor tissue sample has a ng uniformity
29. The method of claim 28, wherein for FOLRl expression that is heterogeneous. the tumor tissue sample has a staining uniformity
30. The method of claim 28, wherein for FOLRl expression that is homogenous. tissue wherein 25—75% of cells of the tumor
31. The method of any one of claims 19—21, of 2 or greater. sample have a staining intensity score of the
32. The method of any one of claims 19-21, n greater than 75% of cells score of 2 or greater. tumor tissue sample have a staining intensity 19~21, wherein the tumor tissue sample has a
33. The method of any one of claims by IHC. staining intensity score of 1, 2, 3, or 3+ for FOLRl expression tissue sample has a staining uniformity
34. The method of claim 33, wherein the tumor for FOLRl expression that is heterogeneous. tissue sample has a staining uniformity
35. The method of claim 33, wherein the tumor for FOLRl expression that is homogeneous.
36. The method of any one of claims 1-35, wherein the level of FOLRI expression is fragment thereof that Specifically measured using a detection antibody or antigen binding binds FOLRl. detection antibody or n binding fragment
37. The method of claim 36, wherein the the amino acid sequence GYFMN (SEQ ID thereof comprises (i) a HC CDRl comprising acid sequence GDTFYNQKFQG (SEQ NO:6); a HC CDR2 comprising the amino the amino acid sequence YDGSRAMDY (SEQ ID ID N027); and a HC CDR3 comprising FAGTSLMH N028) and (ii) a LC CDRl comprising the amino acid sequence KASQSVS the amino acid sequence RASNLEA (SEQ ID (SEQ ID N019); a LC CDR2 comprising N010); and a LC CDR3 comprising the amino acid sequence QQSREYPYT (SEQ ID detection dy or antigen binding fragment
38. The method of claim 36, n the the amino acid sequence of SEQ ID thereof comprises (i) a HC variable domain comprising N0:4 or the amino acid sequence of SEQ ID N0:3 and (ii) a LC le domain comprising detection antibody or antigen binding fragment
39. The method of claim 38, wherein the acid thereof comprises (i) same amino acid sequence as the amino a HC comprising the ATCC as PTA-10772 and (ii) encoded by the plasmid deposited with the sequence of the HC a LC comprising the same amino acid sequence as the amino acid sequence of the LC encoded by the plasmid deposited with the ATCC as 773 or PTA~10774.
40. The method of claim 36, wherein the ion antibody or antigen g fragment thereofis BN3.2.
41. The method of any one of claims 36—40, wherein the detection antibody or antigen binding fragment thereof comprises a detection reagent.
42. The method of claim 41, wherein the detection reagent is selected from the group and a luminophore. consisting of: an enzyme, a phore, a radioactive label, is ed from the group
43. The method of claim 42, wherein the detection reagent ine. consisting of: biotin, digoxigenin, fluorescein, tritium, and concentration of the detection
44. The method of any one of claims 36-43, wherein the about 3.8 ug/ml. antibody or antigen binding fragment thereof is about 0.9 to is an endometrial tumor.
45. The method of any one of claims 1-44, wherein the tumor is an ovarian tumor.
46. The method of any one of claims 1—44, wherein the tumor is a tumor of the
47. The method of any one of claims 1—44, wherein the tumor peritoneum. ImmunoGen, Inc. By the patent attorneys for the applicant CULLENS
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